2024年09月, 北海道大学医学部, 2023年度エクセレントティーチャー最優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2024年09月, 北海道大学医学部, 2023年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2023年09月, 北海道大学医学部, 2022年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2022年08月, 北海道大学医学部, 2021年度エクセレントティーチャー最優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2022年08月, 北海道大学医学部, 2021年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2021年08月, 北海道大学医学部, 2020年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2020年11月, 北海道大学医学部, 2019年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2020年11月, 北海道大学医学部, 2019年度エクセレントティーチャー最優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2019年08月, 北海道大学医学部, 2018年度エクセレントティーチャー優秀賞　　　　　　　　　　　　　　　
堀之内孝広

2016年02月, 国立大学法人 北海道大学, 2015年度 北海道大学教育総長賞奨励賞　　　　　　　　　　　　　　　
堀之内孝広

2015年07月, 北海道大学医学部, 2014年度北海道大学医学部特別賞（最優秀教員賞連続受賞）　　　　　　　　　　　　　　　
堀之内孝広

2015年03月, 国立大学法人 北海道大学, 2014年度 北海道大学教育総長賞奨励賞　　　　　　　　　　　　　　　
堀之内孝広

2014年07月, 北海道大学医学部, 2013年度北海道大学医学部最優秀教員賞　　　　　　　　　　　　　　　
堀之内孝広

2008年06月, アクテリオン ファーマシューティカルズ ジャパン, 第3回 アクテリオン アカデミア プライズ　　　　　　　　　　　　　　　
エンドセリン系由来の循環器疾患に関与するイオン輸送システムの機能制御機構とその病態生理的意義の解明
堀之内 孝広

Cytotoxic effects of the cigarette smoke extract of heated tobacco products on human oral squamous cell carcinoma: the role of reactive oxygen species and CaMKK2.　　　　　　　　　　　　　　　
Nagao Kagemichi, Masanari Umemura, Soichiro Ishikawa, Yu Iida, Shota Takayasu, Akane Nagasako, Rina Nakakaji, Taisuke Akimoto, Makoto Ohtake, Takahiro Horinouchi, Tetsuya Yamamoto, Yoshihiro Ishikawa
The Journal of Physiological Sciences, 74, 35, 2024年06月, ［査読有り］
英語, 研究論文（学術雑誌）

Mechanism of cytotoxicity induced by the cigarette smoke extract (CSE) of heated tobacco products in vascular smooth muscle cells: A comparative study of the cytotoxic effects of CSE and the ferroptosis inducer, erastin
Takahiro Horinouchi, Yuichi Mazaki, Soichi Miwa
Journal of Pharmacological Sciences, 154, 86, 96, Elsevier BV, 2023年12月27日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 36604331

LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity.
Yuichi Mazaki, Haruka Handa, Yoshizuki Fumoto, Takahiro Horinouchi, Yasuhito Onodera
Cell communication and signaling : CCS, 21, 1, 300, 300, 2023年10月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells. METHODS: Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2. RESULTS: fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP. CONCLUSIONS: LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract.

Effects of cigarette smoke extract derived from heated tobacco products on the proliferation of lung cancer stem cells.
Hirata N, Horinouchi T, Kanda Y
Toxicology Reports, 9, 1273, 1280, 2022年06月, ［査読有り］
英語, 研究論文（学術雑誌）

メラノーマ細胞においてAnnexin A2のリン酸化はエンドセリン-1刺激によるAKTの活性化に関与する
真崎 雄一, 東 恒仁, 堀之内 孝広, 三輪 聡一
日本薬理学会年会要旨集, 95, 1-P-085, 公益社団法人 日本薬理学会, 2022年
日本語, Overexpression of endothelin (ET)-1 and endothelin receptors (ETRs) are associated with human cancer malignancy. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase pathway, phosphatidylinositol 3-kinase pathway, and protein kinase C pathway, through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain unclear. We previously reported that ETRs interacted with annexin A2, which is overexpressed in various cancers, and annexin A2 silencing suppressed ERK activation upon ET-1 stimulation in human umbilical vein cell line, EA.hy926 cells. Here we examined roles of annexin A2 in ET-1 signaling pathway of melanoma cells. In melanoma cells, ET-1 stimulation activated ATK, and phosphorylated serin residues of annexin A2. Annexin A2 silencing suppressed activation of AKT upon ET-1 stimulation. In addition, we found that serine residues mutant of annexin A2 suppressed activation of AKT. Our results suggested that phosphorylation of annexin A2 plays important roles in AKT activation of melanoma cells upon ET-1 stimulation.

加熱式たばこ及び紙巻きたばこの主流煙ガス相水抽出物に含まれるカルボニル化合物は血管内皮機能障害を引き起こす
堀之内 孝広, 真崎 雄一, 三輪 聡一
日本薬理学会年会要旨集, 95, 2-P-143, 公益社団法人 日本薬理学会, 2022年
日本語, The gas phase extract of mainstream smoke of combustion cigarettes includes many carbonyl compounds, which increase oxidative stress. The present study examined whether carbonyl compounds in heated cigarette-derived smoke extract (hCSE) and burned cigarette-derived smoke extract (bCSE) cause a reduction in endothelial nitric oxide synthase (eNOS) activity. Three types of heat-not-burn cigarettes (Ploom S, glo, and IQOS) and a combustion cigarette (hi-lite) were used to generate cigarette smoke at different heating or combustion temperatures [Ploom S (200°C), glo (240°C), IQOS (300–350°C), and hi-lite (770−870℃)]. The amounts of carbonyl compounds in hCSE/bCSE were assessed by measuring the carbonylation level of Ca²⁺-sensing receptor (CaSR) that promotes the phosphorylation of eNOS at Ser¹¹⁷⁷, which positively regulates eNOS activity. Although CaSR-mediated phosphorylation of eNOS were unaffected by hCSE from Ploom S and glo, hCSE/bCSE from IQOS and hi-lite reduced the eNOS phosphorylation. hCSE/bCSE from the cigarettes, except for that from Ploom S, facilitated carbonylation of CaSR with different potencies (rank order: glo < IQOS < hi-lite). The reduction of eNOS phosphorylation and the carbonylation of CaSR induced by hCSE/bCSE from IQOS and hi-lite were inhibited by treatment with mainstream smoke using a Carboxen-572 cartridge to scavenge carbonyl compounds. These results suggest that an increase in the heating or combustion temperature leads to an increase in the generation of carbonyl compounds, which cause endothelial dysfunction characterized by a reduction in eNOS activity.

加熱式たばこ主流煙の化学分析と生体影響評価　　　　　　　　　　　　　　　
堀之内孝広, 小林純子, 東恭平, 三輪聡一
室内環境, 24, 2, 125, 133, 2021年08月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］, ［国内誌］
日本語, 研究論文（学術雑誌）

Comparison of cytotoxicity of cigarette smoke extract derived from heat-not-burn and combustion cigarettes in human vascular endothelial cells
Takahiro Horinouchi, Soichi Miwa
Journal of Pharmacological Sciences, 147, 223, 233, Elsevier BV, 2021年07月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

メラノーマ細胞において、Annexin A2は、エンドセリン-1の刺激によるAKTの活性化に関与する
真崎 雄一, 東 恒仁, 堀之内 孝広, 三輪 聡一
日本薬理学会年会要旨集, 94, 3-P1-30, 公益社団法人 日本薬理学会, 2021年
日本語, Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). The overexpression of endothelin (ET)-1 or ETRs is related to malignancy of human cancer, although ET-1 was originally identified as an endothelium-derived vasocontractile peptide. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Previously, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers, and annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation in human umbilical vein cell line, EA.hy926 cells. Here we examine roles of annexin A2 in ET-1 signaling pathway of melanoma cells. ET-1 stimulation induces ATK activation in melanoma cells. On the other hand, annexin A2 silencing suppressed activation of AKT upon ET-1 stimulation. These results suggested that interaction of ETRs and annexin A2 play important roles in ET-1 signaling pathway of melanoma cells.

加熱式たばこ及び紙巻きたばこの主流煙ガス相水抽出物がヒト血管内皮細胞に及ぼす影響
堀之内 孝広, 真崎 雄一, 三輪 聡一
日本薬理学会年会要旨集, 94, 1-P2-10, 公益社団法人 日本薬理学会, 2021年
日本語, The present study established a method to prepare heated tobacco-derivedcigarette smoke extract (hCSE) and burned cigarette-derived CSE (bCSE), andcompared their effects on human umbilical vein endothelial EA.hy926 cells. Threetypes of tobacco heating devices (Ploom S, glo, and IQOS) were used in order togenerate cigarette smoke at different heating temperatures. hCSE and bCSE wereprepared using the puffing regimen parameters of the CORESTA approach (55 mLpuff volume, 3 sec puff duration, and 1 puff every 30 sec). Tar phase (nicotineand tar) of cigarette smoke was removed by passing through a Cambridge glassfiber filter. The rank order of amounts of nicotine and tar trapped on theCambridge filter was Ploom S (6 ± 1 mg/91 puffs) < glo (16 ± 1 mg/91 puffs)< IQOS (56 ± 3 mg/91 puffs) < hi-lite (228 ± 2 mg/91 puffs). Crude hCSEand bCSE caused a decrease in mitochondrial metabolic activity and theexpression level of endothelial nitric oxide synthase, with the rank order ofpotency as follows: Ploom S < glo < IQOS < hi-lite. The reduction inmitochondrial metabolic activity was diminished by removing nicotine and tarfrom cigarette smoke with Cambridge filter. These results indicated that highercytotoxicity of cigarette smoke to vascular endothelial cells were correlatedwith higher heating/burning temperatures [Ploom S (200℃) < glo (240℃)

Fibrinolysis-resistant carbonylated fibrin detected in thrombi attached to the vascular wall of abdominal aortic aneurysms.
Yuko Suzuki, Hiroki Tanaka, Takahiro Horinouchi, Hideto Sano, Naoki Honkura, Naoki Unno, Soichi Miwa, Tetsumei Urano
Scientific reports, 10, 1, 20728, 20728, 2020年11月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we investigated how carbonylation of fibrinogen by acrolein modified its indispensable function to enhance fibrinolysis after being converted to fibrin and contributed to generating a fibrinolysis-resistant fibrin clot. Acrolein-treated fibrinogen was subjected to tissue plasminogen activator-induced fibrinolysis assay and the effect of lysine residue carbonylation in fibrinogen on fibrinolysis was analyzed. The acrolein-treated fibrinogen-derived fibrin clot appeared more resistant to fibrinolysis and the N-acetyl 3-formyl-3,4-dehydropiperidino (FDP)-Lysine levels in the lysed solution were positively correlated with the duration of clot lysis. The lysine analog 6-amino hexanoic acid (6AHA), which mimics the C-terminal lysine of fibrin, was carbonylated and its enhancing effect on Glu1-plasminogen activation was evaluated. After incubation with acrolein, 6AHA was converted to N-acetyl FDP-6AHA, losing its ability to enhance Glu1-plasminogen activation. These results suggest that fibrinogen carbonylation by acrolein to generate N-acetyl FDP-Lysine resulted in the generation of fibrinolysis-resistant fibrin by attenuating the C-terminal lysine-dependent activation of the Glu1-plasminogen. In abdominal aortic aneurysms, fibrin(ogen) containing the acrolein adduct N-acetyl FDP-Lysine was detected in the vascular wall-attached thrombi. These results suggest that this mechanism is likely involved in the modification of fibrinolysis-resistant thrombi and to their persistence for a long period.

Cigarette Smoke Extract and Its Cytotoxic Factor Acrolein Inhibit Nitric Oxide Production in Human Vascular Endothelial Cells.
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Biological & pharmaceutical bulletin, 43, 11, 1804, 1809, 2020年11月01日, ［査読有り］, ［筆頭著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Acrolein (ACR), a highly reactive α,β-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

Extracellular Ca2+ promotes nitric oxide production via Ca2+-sensing receptor-Gq/11 protein-endothelial nitric oxide synthase signaling in human vascular endothelial cells.
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Journal of pharmacological sciences, 143, 4, 315, 319, 2020年08月, ［査読有り］, ［筆頭著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, This study examined the possible involvement of Ca2+-sensing receptor (CaSR) in nitric oxide (NO) production in human vascular endothelial cells. Extracellular Ca2+ elevated the intracellular Ca2+ concentration, the endothelial NO synthase (eNOS) phosphorylation level, and NO release from the cells. These responses were inhibited by a CaSR antagonist and a Gq/11 protein inhibitor. Application of an endothelial cell suspension induced vasorelaxation in isolated rat thoracic aorta precontracted by phenylephrine. Adding an NO scavenger to the organ bath abolished this vasorelaxation response. These results suggest that extracellular Ca2+ promotes NO generation via CaSR- and Gq/11 protein-mediated eNOS activation.

エンドセリン-1の刺激によるERKの活性化にAnnexin A2は関与する
真崎 雄一, 東 恒仁, 堀之内 孝広, 三輪 聡一
日本薬理学会年会要旨集, 93, 3-P-361, 公益社団法人 日本薬理学会, 2020年
日本語, Endothelin receptors (ETRs) is one of G protein coupled receptors, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). The overexpression of endothelin (ET)-1 or ETRs is related to malignancy of human cancer, although ET-1 was originally identified as an endothelium-derived vasocontractile peptide. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ET_AR and ET_BR. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. Furthermore, we found that annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggested that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation.

カルシウム感受性受容体-G_q/11タンパク質シグナル伝達系はヒト血管内皮細胞からの一酸化窒素遊離に関与する
堀之内 孝広, 真崎 雄一, 三輪 聡一
日本薬理学会年会要旨集, 93, 1-LBS-11, 公益社団法人 日本薬理学会, 2020年
日本語, Ca²⁺-sensing receptor (CaSR) belongs to family C of G protein-coupled receptors and is activated by the endogenous agonists such as Ca²⁺. Stimulation of CaSR expressed in vascular endothelial cells through the increase in extracellular Ca²⁺ concentration ([Ca²⁺]_o) is reported to induce vasorelaxation via the production of nitric oxide (NO). The purpose of the present study is to characterize the CaSR-mediated NO production in human vascular endothelial cells. In human endothelial EA.hy926 cells, the increase in [Ca²⁺]_o from 0.2 to 2 mM induced a concentration-dependent increase in intracellular Ca²⁺ concentration, which was significantly inhibited by NPS 2143 (a CaSR antagonist) and YM-254890 (a G_q/11 protein inhibitor). Stimulation with 2 mM Ca²⁺ for 4 h elicited an increase in the phosphorylation level of eNOS at Ser¹¹⁷⁷, which was significantly depressed by NPS 2143, YM-254890, and removal of Ca²⁺ from the medium. Ca²⁺ (2 mM) induced an increase in NO production, which was inhibited by NPS 2143, YM-254890, removal of Ca²⁺ from the medium, and L-NAME (a competitive eNOS inhibitor). These results provide evidence that activation of CaSR with extracellular Ca²⁺ facilitates NO release from human vascular endothelial cells via a G_q/11 protein-eNOS-dependent pathway.

Ca2+ signal is involved in endothelin-1-induced internalization of endothelin type A receptor expressed in Chinese hamster ovary cells.
Takahiro Horinouchi, Sarita Karki, Koji Terada, Yuichi Mazaki, Soichi Miwa
Journal of pharmacological sciences, 140, 1, 102, 105, 2019年05月, ［査読有り］, ［筆頭著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Endothelin type A receptor (ETAR) is internalized upon agonist stimulation; however, the mechanism thereof remains controversial. In this study, we characterized the endothelin-1 (ET-1)-induced internalization of ETAR expressed in Chinese hamster ovary cells. ET-1 elicited ETAR internalization and increase in intracellular Ca2+ concentration. ET-1-induced ETAR internalization was completely inhibited by a reduction in intracellular and extracellular Ca2+ levels and partially suppressed by inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2), both of which are downstream molecules in ETAR signaling. These results suggest that Ca2+ mobilization, PKC, and ERK1/2 are involved in ET-1-induced ETAR internalization.

Annexin A2 is involved in activation of extracellular signal-regulated kinase upon endothelin-1 stimulation.
Yuichi Mazaki, Tsunehito Higashi, Takahiro Horinouchi, Soichi Miwa
Biochemical and biophysical research communications, 511, 1, 69, 72, Elsevier {BV}, 2019年03月26日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The overexpression of endothelin (ET)-1 or ET receptors (ETRs) is related to initiation and progression of tumor. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase C through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain uncertain. Here, we found that ETRs interacted with annexin A2, which is overexpressed in various cancers. Annexin A2 bound to ET type A receptor and ET type B receptor. Upon ET-1 stimulation, serine phosphorylation of annexin A2 increased, while there is no change in tyrosine phosphorylation of annexin A2. On the other hand, annexin A2 silencing suppressed activation of ERK upon ET-1 stimulation. These results suggest that interaction of ETRs and annexin A2 may play important roles in activation of extracellular signal-regulated kinase upon ET-1 stimulation.

Endothelin type B receptor interacts with the 78-kDa glucose-regulated protein.
Yuichi Mazaki, Tsunehito Higashi, Yasuhito Onodera, Jin-Min Nam, Ari Hashimoto, Shigeru Hashimoto, Takahiro Horinouchi, Soichi Miwa
FEBS letters, 593, 6, 644, 651, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization.

GRP78はエンドセリンB受容体を介したERKの活性化を促す
真崎 雄一, 東 恒仁, 小野寺 康仁, 南 ジンミン, 橋本 あり, 橋本 茂, 堀之内 孝広, 三輪 聡一
日本薬理学会年会要旨集, 92, 1-P-110, 公益社団法人 日本薬理学会, 2019年
日本語, Endothelin (ET)-1 is involved in various diseases, including cancer, hypertension, atherosclerosis, diabetes, and fibrotic diseases, although ET-1 is originally identified as endothelium-derived vasocontractile peptide. ET receptors belong to the class A of G protein-coupled receptor, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). ET_AR and ET_BR generally exhibit the opposite responses, although many exceptions exist. Here, we attempted to identify ET_AR or ET_BR specific binding proteins to understand difference of ET_AR- and ET_BR-mediated responses upon ET-1 stimulation. We found that GRP78 exhibited a stronger binding affinity toward ET_BR than ET_AR. Overexpression of GRP78 promotes ET_BR-mediated ERK activation. In addition, the silencing of GRP78 suppressed ET_BR-mediated ERK activation. On the other hand, ET_BR can localize GRP78 to cell periphery. Our results suggest that interaction of ET_BR with GRP78 affects the ERK activation and GRP78 localization.

ヒト血管内皮細胞の一酸化窒素合成酵素の活性化におけるカルシウム感受性受容体の関与
堀之内 孝広, 真崎 雄一, 三輪 聡一
日本薬理学会年会要旨集, 92, 1-O-01, 公益社団法人 日本薬理学会, 2019年
日本語, Ca²⁺-sensing receptor (CaSR) is a seven-transmembrane G protein-coupled receptor (GPCR), and is activated by an increase in extracellular Ca²⁺ concentration. In vascular endothelial cells, stimulation of CaSR induces nitric oxide (NO) release via activation of endothelial nitric oxide synthase (eNOS) and membrane hyperpolarization via activation of intermediate Ca²⁺-activated K⁺ channels, contributing to vasodilation. In the present study, we have pharmacologically characterized eNOS activation in response to stimulation of CaSR in human endothelial EA.hy926 cells. In 2 mM Ca²⁺-containing Krebs-HEPES solution, the phosphorylation level of eNOS at serine 1177 was markedly reduced by NPS 2143 (a CaSR antagonist) and YM-254890 (a G_q/11 protein inhibitor). In organ bath study with endothelium-removed ring preparations of rat thoracic aorta, addition of EA.hy926 cell suspension produced relaxation of the rings precontracted with phenylephrine. The endothelium-dependent relaxant response was inhibited by pretreatment of EA.hy926 cells with NPS 2143, YM-254890, and L-NAME (an eNOS inhibitor). These results suggest that stimulation of CaSR expressed in endothelial cells with extracellular Ca²⁺ induces NO-mediated vasorelaxation via G_q/11-protein-dependent activation of eNOS.

Chinese herbal medicine Qing-Dai-induced pulmonary arterial hypertension in a patient with ulcerative colitis: A case report and experimental investigation.
Kazuki Sato, Hiroshi Ohira, Takahiro Horinouchi, Toshitaka Nakaya, Yuichi Mazaki, Ayako Sugimoto, Taku Watanabe, Ichizo Tsujino, Masaharu Nishimura
Respiratory medicine case reports, 26, 265, 269, Elsevier {BV}, 2019年, ［査読有り］, ［国際誌］
英語, A recent case report described a case of pulmonary arterial hypertension (PAH) associated with use of the Chinese herbal medicine Qing-Dai; however, the clinical course and possible mechanisms have not been characterized. We present the case of a man with ulcerative colitis who was diagnosed with idiopathic PAH. After initiating oral beraprost therapy, the patient showed significant hemodynamic improvements and an unusual course of clinical recovery. In 2016, the Japanese Ministry of Health, Labour, and Welfare issued a warning regarding the possible side effects of Qing-Dai. We learned that our patient had been taking self-purchased Qing-Dai for 2 years. Therefore, we performed an experimental study and determined that Qing-Dai may cause PAH through a mechanism involving nitric oxide synthase inhibition and pulmonary artery endothelial dysfunction.

Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells
Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
Folia Pharmacologica Japonica, 151, 4, 140, 147, Japanese Pharmacological Society, 2018年, ［査読有り］, ［招待有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr308 and Ser473, and [3H]-labelled 2-deoxy-D-glucose ([3H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [3H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ETAR), inhibition of Gq/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ETAR with ET-1 inhibits insulin-induced Akt phosphorylation and [3H]2-DG uptake in a Gq/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ETAR and GRK2 are potential targets for insulin resistance.

ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis.
Yuichi Mazaki, Yasuhito Onodera, Tsunehito Higashi, Takahiro Horinouchi, Tsukasa Oikawa, Hisataka Sabe
Cell communication and signaling : CCS, 15, 1, 36, 36, BIOMED CENTRAL LTD, 2017年10月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. RESULTS: We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. CONCLUSIONS: Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.

Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity.
Hitoshi Kashiwagi, Koh-Ichi Yuhki, Yoshitaka Imamichi, Fumiaki Kojima, Shima Kumei, Tsunehito Higashi, Takahiro Horinouchi, Soichi Miwa, Shuh Narumiya, Fumitaka Ushikubi
TH open : companion journal to thrombosis and haemostasis, 1, 2, e122-e129, e129, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid-induced TXA 2 production in murine platelets with an IC 50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.

Phos-tag電気泳動法の応用 Phos-tag biotinを用いた受容体作動性TRPC6チャネルのPKAリン酸化部分の同定
堀之内 孝広, 寺田 晃士, 東 恒仁, 真崎 雄一, 三輪 聡一
電気泳動, 60, Suppl., s19, s19, 日本電気泳動学会, 2016年08月
日本語

エンドセリン受容体シグナルを標的にした肺高血圧症治療の新展開　　　　　　　　　　　　　　　
堀之内 孝広, 堀口 美香, 真崎 雄一, 東 恒仁, 原田 拓弥, 三輪 聡一
呼吸と循環, 64, 5, S9, S10, (株)医学書院, 2016年05月
日本語

Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.
Takahiro Horinouchi, Akimasa Hoshi, Takuya Harada, Tsunaki Higa, Sarita Karki, Koji Terada, Tsunehito Higashi, Yosuke Mai, Prabha Nepal, Yuichi Mazaki, Soichi Miwa
British journal of pharmacology, 173, 6, 1018, 32, WILEY, 2016年03月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling. EXPERIMENTAL APPROACH: We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [ (3) H]-2-deoxy-d-glucose ([ (3) H]2-DG). The C-terminus region of GPCR kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of the corresponding short-interfering RNA (siRNA). KEY RESULTS: In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [ (3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [ (3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [ (3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONS: Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [ (3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.

骨格筋細胞においてエンドセリン-1はGタンパク質共役型受容体キナーゼ2を介してインスリン抵抗性を惹起する　　　　　　　　　　　　　　　
堀之内孝広, 星暁壮, 原田拓弥, 比嘉綱己, サリタ・カルキ, 寺田晃士, 東恒仁, 眞井洋輔, ネパル・プラハ, 真崎雄一, 三輪聡一
北海道医学雑誌, 91, 77, 2016年, ［筆頭著者, 責任著者］
日本語, 研究論文（学術雑誌）

The dynamics of mucosal-associated invariant T cells in multiple sclerosis.
Chie Sugimoto, Makoto Hirotani, Kazunori Yoshikiyo, Uichi Koshimizu, Rika Wakao, Takahiro Horinouchi, Yuichi Mazaki, Tsunehiko Higashi, Toshiyuki Fukazawa, Hiroyoshi Fujita, Hidenao Sasaki, Hiroshi Wakao
SpringerPlus, 5, 1, 1259, 1259, SPRINGER INTERNATIONAL PUBLISHING AG, 2016年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination, gliosis and axonal loss in the Central Nervous System. Although the etiology of the disease has remained enigmatic, recent studies have suggested a role of the innate-like T cells, called Mucosal Associated Invariant T cells (MAITs) in the pathophysiology. In the present study, we have analyzed the relative frequency of MAITs and the expression of the cell surface antigens in MAITs to seek a possible link to the disease. RESULTS: There was little difference in the frequency of total MAITs between healthy donors (HDs) and untreated MS patients, whereas the latter harbored more CD8(lo/neg) (DN) MAITs concomitant with a decrease in CD8(high) MAITs and in CD4 MAITs compared with those in HDs. While the expression of CCR5, CCR6, CD95, CD127, and CD150 has increased in untreated subjects compared with that in HDs, CD45RO has declined in untreated subjects in both DN MAITs and CD8(hi) MAITs. FTY720 therapy has increased the relative frequency of total MAITs in a time-dependent fashion up to 2 years. Intriguingly, FTY720 therapy for 3 years reversed the above phenotype, engendering more CD8(high) MAITs accompanied with decreased DN MAITs. FTY720 therapy affected the cytokine production from CD4 T cells and also enhanced the relative frequency of cells producing both TNF-α and IFN-γ from MAITs, CD8 T cells, and CD4 T cells compared with that in untreated subjects. CONCLUSIONS: FTY 720 therapy enhanced the relative frequency of MAITs in MS patients in a time-dependent manner. Although the expression of CD8 in MAITs has been affected early by FTY720, longer treatment has reversed the phenotypic change. These data demonstrated that FTY720 induced dynamic change in the relative frequency and in the phenotype of MAITs in MS.

肺高血圧症治療薬の最近の進歩：エンドセリンシステムからみた作用機序
堀之内孝広, 真崎雄一, 寺田晃士, 東恒仁, 三輪聡一
日本薬理学雑誌, 148, 5, 231, 238, 2016年, ［査読有り］, ［招待有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

A Standardized Method for the Preparation of a Gas Phase Extract of Cigarette Smoke.
Tsunehito Higashi, Yosuke Mai, Yuichi Mazaki, Takahiro Horinouchi, Soichi Miwa
Biological & pharmaceutical bulletin, 39, 6, 898, 902, PHARMACEUTICAL SOC JAPAN, 2016年, ［査読有り］, ［招待有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The gas phase of cigarette smoke is important from the viewpoint of human health, because it can pass through alveolar epithelium and enter the circulation. There is no standard method for the preparation of a gas phase extract of cigarette smoke (CSE), although CSE is widely used for research instead of whole cigarette smoke. We have established a standard method for the preparation of CSE. One cigarette per trial is continuously combusted under a reduced pressure generated by an aspiration pump with a velocity of 1.050 L/min: the main stream of the smoke is passed through a Cambridge filter to remove tar, and subsequently, bubbled through a glass ball filter (pore size, 20-30 µm) into 15 mL of phosphate-buffered saline (PBS). To express the concentration of CSE, a virtual tar concentration is introduced, which is calculated assuming that tar trapped on the Cambridge filter is dissolved in the PBS. CSEs prepared from smaller numbers of cigarettes (original virtual tar concentration≤15 mg/mL) show similar concentration-response curves for cytotoxicity versus virtual tar concentrations. CSEs prepared from various brands of cigarettes and by different smoking regimes (continuous and puff smoking) show similar cytotoxic potency if the virtual tar concentrations are the same. In conclusion, using the standardized method for CSE preparation in combination with the virtual tar concentration, it becomes possible to simply and rapidly prepare standard CSEs with defined concentrations from any brand of cigarettes, which are toxicologically equivalent to CSE prepared by puff smoking.

Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties.
Takahiro Horinouchi, Tsunehito Higashi, Yuichi Mazaki, Soichi Miwa
Biological & pharmaceutical bulletin, 39, 6, 909, 14, PHARMACEUTICAL SOC JAPAN, 2016年, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］, ［国内誌］
英語, 研究論文（学術雑誌）, Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking.

Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.
Takahiro Horinouchi, Koji Terada, Tsunehito Higashi, Soichi Miwa
Methods in molecular biology (Clifton, N.J.), 1397, 267, 277, 2016年, ［査読有り］, ［招待有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

第146回東邦医学会例会 パネルディスカッション:肺血管リモデリングの発症機序に迫る エンドセリン受容体シグナルからの視点
堀之内 孝広, 東 恒仁, 真崎 雄一, 三輪 聡一
東邦医学会雑誌, 62, 3, 197, 199, 東邦大学医学会, 2015年09月
日本語, type:TOHO University Scholarly Publication
総説
要約: 肺血管リモデリングは,肺動脈内皮細胞・平滑筋細胞の異常増殖とアポトーシス耐性獲得によって惹起される器質的な肺血管病変であり,肺動脈性肺高血圧症(pulmonary arterial hypertension:PAH)の発症・進展に深く関与している.エンドセリン-1(endothelin-1:ET-1)は,血管内皮細胞で産生される血管収縮性・炎症性ペプチドであり,PAHの病態形成において,重要な役割を担っている.ET-1によるエンドセリンA型受容体刺激は,transient receptor potential canonical 6(TRPC6)チャネルの活性化に伴う持続的なCa2+流入やRhoキナーゼの活性化を介して,肺血管リモデリングを引き起こす.一方,ET-1によるエンドセリンB型受容体の活性化は,血管内皮細胞における内皮由来弛緩因子の遊離および血中ET-1のクリアランスを介して,肺血管リモデリングの抑制に寄与する.本稿では,エンドセリン受容体シグナルに焦点を当て,肺血管リモデリングの発症機序と治療戦略について概説する.

肺血管リモデリングの発症機序に迫る エンドセリン受容体シグナルからの視点
堀之内 孝広, 東 恒仁, 真崎 雄一, 三輪 聡一
東邦医学会雑誌, 62, 3, 197, 199, 東邦大学医学会, 2015年09月
日本語, 肺血管リモデリングは、肺動脈内皮細胞・平滑筋細胞の異常増殖とアポトーシス耐性獲得によって惹起される器質的な肺血管病変であり、肺動脈性肺高血圧症(pulmonary arterial hypertension:PAH)の発症・進展に深く関与している。エンドセリン-1(endothelin-1:ET-1)は、血管内皮細胞で産生される血管収縮性・炎症性ペプチドであり、PAHの病態形成において、重要な役割を担っている。ET-1によるエンドセリンA型受容体刺激は、transient receptor potential canonical 6(TRPC6)チャネルの活性化に伴う持続的なCa2+流入やRhoキナーゼの活性化を介して、肺血管リモデリングを引き起こす。一方、ET-1によるエンドセリンB型受容体の活性化は、血管内皮細胞における内皮由来弛緩因子の遊離および血中ET-1のクリアランスを介して、肺血管リモデリングの抑制に寄与する。本稿では、エンドセリン受容体シグナルに焦点を当て、肺血管リモデリングの発症機序と治療戦略について概説する。(著者抄録)

『エンドセリン受容体のユビキチン化』 ユビキチン化によるエンドセリン受容体の細胞内トラフィッキング制御
寺田晃士, 堀之内孝広, 東恒仁, Prabha Nepal, 三輪聡一
日本薬理学雑誌, 145, 1, 4, 9, 2015年01月, ［査読有り］, ［招待有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

肺血管リモデリングの発症機序に迫る－エンドセリン受容体シグナルからの視点－
堀之内孝広, 東恒仁, 真崎雄一, 三輪聡一
東邦医学誌, 62, 3, 197, 199, The Medical Society of Toho University, 2015年, ［招待有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.
Koji Terada, Takahiro Horinouchi, Yoichiro Fujioka, Tsunehito Higashi, Prabha Nepal, Mika Horiguchi, Sarita Karki, Chizuru Hatate, Akimasa Hoshi, Takuya Harada, Yosuke Mai, Yusuke Ohba, Soichi Miwa
The Journal of biological chemistry, 289, 51, 35283, 95, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014年12月19日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor in rat L6 myoblasts.
Takuya Harada, Takahiro Horinouchi, Tsunaki Higa, Akimasa Hoshi, Tsunehito Higashi, Koji Terada, Yosuke Mai, Prabha Nepal, Mika Horiguchi, Chizuru Hatate, Soichi Miwa
Life sciences, 104, 1-2, 24, 31, PERGAMON-ELSEVIER SCIENCE LTD, 2014年05月28日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, AIMS: Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitivity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR-mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat skeletal muscle. MAIN METHODS: Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) was overexpressed in L6 myoblasts using adenovirus-mediated gene transfer. KEY FINDINGS: ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was abolished by BQ123 (a selective ET type A receptor (ETAR) antagonist), YM-254890 (a Gαq/11 protein inhibitor), and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholipase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of dominant-negative dynamin (K44A). These results suggest that ETAR stimulation induces ERK1/2 phosphorylation in L6 myoblasts through Gq/11 protein-dependent, PLC-independent PDGFR transactivation which requires dynamin-dependent ETAR internalization. SIGNIFICANCE: Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the development of insulin sensitivity, the ETAR-mediated PDGFR transactivation and subsequent ERK1/2 activation play an important role in ET-1-induced insulin resistance.

A simple and rapid method for standard preparation of gas phase extract of cigarette smoke.
Tsunehito Higashi, Yosuke Mai, Yoichi Noya, Takahiro Horinouchi, Koji Terada, Akimasa Hoshi, Prabha Nepal, Takuya Harada, Mika Horiguchi, Chizuru Hatate, Yuji Kuge, Soichi Miwa
PloS one, 9, 9, e107856, PUBLIC LIBRARY SCIENCE, 2014年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤ 15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥ 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.
Yoichi Noya, Koh-Ichi Seki, Hiroshi Asano, Yosuke Mai, Takahiro Horinouchi, Tsunehito Higashi, Koji Terada, Chizuru Hatate, Akimasa Hoshi, Prabha Nepal, Mika Horiguchi, Yuji Kuge, Soichi Miwa
Toxicology, 314, 1, 1, 10, Elsevier Ireland Ltd, 2013年12月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent cytotoxicity with an EC50 value of 264.0±16.9μM (n=3). The concentrations of acrolein, MVK and CPO in the CSE were 3368±334, 2429±123 and 392.9±31.8μM (n=4), respectively, which were higher than the cytotoxic concentrations. The cytotoxicity of acrolein and MVK consisted of plasma membrane damage and decreased cell viability: the plasma membrane damage was totally prevented by treatment with an inhibitor of PKC or NOX, whereas the decreased cell viability was only partially prevented by these inhibitors. The cytotoxicity of CPO consisted only of decreased cell viability, which was totally resistant to these inhibitors. These results show that acrolein and MVK are responsible for the acute cytotoxicity of the CSE through PKC/NOX-dependent and -independent mechanisms, whereas CPO is responsible for the delayed cytotoxicity of the CSE through a PKC/NOX-independent mechanism.

Endothelin receptor signaling: new insight into its regulatory mechanisms.
Takahiro Horinouchi, Koji Terada, Tsunehito Higashi, Soichi Miwa
Journal of pharmacological sciences, 123, 2, 85, 101, JAPANESE PHARMACOLOGICAL SOC, 2013年, ［査読有り］, ［招待有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The endothelin (ET) system consists of two G protein coupled-receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca(2+) concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca(2+) entry and receptor-operated Ca(2+) entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca(2+) sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1-induced Ca(2+) mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca(2+) signaling and to GIPs in the signal transduction, modification, and degradation of ETRs.

Different binding property of STIM1 and its novel splice variant STIM1L to Orai1, TRPC3, and TRPC6 channels.
Takahiro Horinouchi, Tsunehito Higashi, Tsunaki Higa, Koji Terada, Yosuke Mai, Hiroyuki Aoyagi, Chizuru Hatate, Prabha Nepal, Mika Horiguchi, Takuya Harada, Soichi Miwa
Biochemical and biophysical research communications, 428, 2, 252, 8, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年11月16日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum (ER) Ca(2+) sensor to control ER Ca(2+) levels. A recent study has shown that STIM1L, a new splice variant of STIM1, is expressed in various tissues of rodent and in human skeletal muscle, and that the interaction of STIM1L with actin filament allows rapid activation of store-operated Ca(2+) entry (SOCE) mediated through Orai1 channels. Here, we characterize mRNA expression and function of human STIM1 and STIM1L, and compare their binding property to Orai1 functioning as store-operated Ca(2+) channels (SOCCs), and TRPC3 (transient receptor potential canonical 3) and TRPC6 channels functioning as endothelin type A receptor (ET(A)R)-operated Ca(2+) channels (ROCCs). Although mRNA for STIM1 was ubiquitously expressed in human tissues, STIM1L was detected only in skeletal muscle. STIM1L augmented thapsigargin- and endothelin-1-induced SOCE more strongly than STIM1 in human embryonic kidney 293 cells stably expressing ET(A)R, whereas, it tends to suppress ET(A)R-operated Ca(2+) entry (ROCE) via TRPC3 and TRPC6 more strongly than STIM1. Coimmunoprecipitation experiments have revealed that when compared with STIM1, STIM1L binds more abundantly to Orai1 and also to TRPC3 and TRPC6. These results suggest that the higher binding capacity of STIM1L to SOCCs and ROCCs plays an important role in the regulation of Ca(2+) signaling such as the augmentation of SOCE via Orai1 and the inhibition of ROCE via TRPC3 and TRPC6.

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase.
Hiroshi Asano, Takahiro Horinouchi, Yosuke Mai, Osamu Sawada, Shunsuke Fujii, Tadashi Nishiya, Masabumi Minami, Takahiro Katayama, Toshihiko Iwanaga, Koji Terada, Soichi Miwa
Journal of pharmacological sciences, 118, 2, 275, 87, JAPANESE PHARMACOLOGICAL SOC, 2012年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-L-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of (•)OH) but not by superoxide dismutase, indicating involvement of (•)OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H(2)O(2) or O(2)(-) generated by the xanthine/xanthine oxidase system, indicating involvement of H(2)O(2) or O(2)(-) in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.

Nicotine- and tar-free cigarette smoke extract induces cell injury via intracellular Ca2+-dependent subtype-specific protein kinase C activation.
Yosuke Mai, Tsunehito Higashi, Koji Terada, Chizuru Hatate, Prabha Nepal, Mika Horiguchi, Takuya Harada, Soichi Miwa, Takahiro Horinouchi
Journal of pharmacological sciences, 120, 4, 310, 4, JAPANESE PHARMACOLOGICAL SOC, 2012年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Nicotine- and tar-free cigarette smoke extract (CSE) is reported to induce cell damage via activation of protein kinase C (PKC) and NADPH oxidase (NOX) in rat C6 glioma cells. Here we determined PKC isozyme(s) activated by CSE and their activation mechanism. In C6 glioma cells, mRNAs for PKCα, PKCδ, PKCε, and PKCι were expressed. CSE triggered translocation of PKCα and PKCε to plasma membrane. CSE-induced cell damage and PKC translocation were inhibited by chelating intracellular Ca(2+) but not extracellular Ca(2+). These results suggest that CSE induces cell damage through intracellular Ca(2+)-dependent activation of PKCα and PKCε and subsequent NOX activation.

Adenylate cyclase/cAMP/protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca²⁺ entry mediated via transient receptor potential canonical 6 channels.
Takahiro Horinouchi, Tsunaki Higa, Hiroyuki Aoyagi, Tadashi Nishiya, Koji Terada, Soichi Miwa
The Journal of pharmacology and experimental therapeutics, 340, 1, 143, 51, AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2012年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Receptor-operated Ca²⁺ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca²⁺ concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca²⁺ measurements with thapsigargin-induced Ca²⁺-depletion/Ca²⁺-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase.
Kazuma Matsumoto, Tadashi Nishiya, Satoshi Maekawa, Takahiro Horinouchi, Kouetsu Ogasawara, Takashi Uehara, Soichi Miwa
Biochemical and biophysical research communications, 409, 1, 46, 51, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年05月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime.

Regulation of inducible nitric-oxide synthase by the SPRY domain- and SOCS box-containing proteins.
Tadashi Nishiya, Kazuma Matsumoto, Satoshi Maekawa, Emi Kajita, Takahiro Horinouchi, Masahiro Fujimuro, Kouetsu Ogasawara, Takashi Uehara, Soichi Miwa
The Journal of biological chemistry, 286, 11, 9009, 19, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011年03月18日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.

Juvenile stress attenuates the dorsal hippocampal postsynaptic 5-HT1A receptor function in adult rats.
Hirokazu Matsuzaki, Takeshi Izumi, Takahiro Horinouchi, Shuken Boku, Takeshi Inoue, Taku Yamaguchi, Takayuki Yoshida, Machiko Matsumoto, Hiroko Togashi, Soichi Miwa, Tsukasa Koyama, Mitsuhiro Yoshioka
Psychopharmacology, 214, 1, 329, 37, SPRINGER, 2011年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, RATIONALE: Traumatic events in early life are associated with an increased risk of psychiatric diseases in adulthood. 5-hydroxytryptamine (5-HT)(1A) receptors play a pivotal role in the 5-HTergic mechanisms associated with the etiology of stress-related disorders. OBJECTIVE: The goal of the present study was to investigate whether juvenile stress influences emotional control via postsynaptic 5-HT(1A) receptor in the hippocampus and amygdala using contextual fear conditioning test in adult rats. METHODS: The rats were subjected to aversive footshock (FS) during the third week of the postnatal period (3wFS group). During the postadolescent period (10-14 weeks postnatal), experiments were performed. RESULTS: The systemic administration of the 5-HT(1A) receptor agonist R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.2 mg/kg, i.p.) attenuated the freezing behavior in the non-FS group, but not in the 3wFS group. The bilateral local injection of 8-OH-DPAT (1 μg/side) into the amygdala decreased the freezing behavior in the non-FS group and the 3wFS group. However, the local injection of 8-OH-DPAT (1 μg/side) into the hippocampus decreased the freezing behavior in the non-FS group, but not in the 3wFS group. In a 5-HT(1A) receptor binding study, the Bmax of the 3wFS group decreased in the dorsal hippocampus, but not the amygdala in comparison with the non-FS group. CONCLUSIONS: The juvenile stress attenuated the hippocampal postsynaptic 5-HT(1A) receptor function in context-dependent conditioned fear.

Effect of cigarette smoke components on vascular smooth muscle cell migration toward platelet-derived growth factor BB.
Shinji Yoshiyama, Takahiro Horinouchi, Soichi Miwa, Hong Hui Wang, Kazuhiro Kohama, Akio Nakamura
Journal of pharmacological sciences, 115, 4, 532, 5, JAPANESE PHARMACOLOGICAL SOC, 2011年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Cigarette smoking is one of the factors causing accumulation of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. Changes in cell migration toward platelet-derived growth factor BB were investigated using a Boyden chamber after 48-h preincubation of GBaSM-4 VSMCs with nicotine or nicotine-free cigarette smoke extract (CSE). A nicotine concentration of 0.1 µM maximally promoted cell migration; 0.1% CSE also promoted cell migration, while high CSE concentrations damaged GBaSM-4 cells. Fetal bovine serum (FBS) long-depletion induced decrease in migration of GBaSM-4 cells. Our results suggest that nicotine and some CSE components can induce GBaSM-4 cell migration.

Function and regulation of endothelin type A receptor-operated transient receptor potential canonical channels.
Takahiro Horinouchi, Koji Terada, Tsunaki Higa, Hiroyuki Aoyagi, Tadashi Nishiya, Hiroyuki Suzuki, Soichi Miwa
Journal of pharmacological sciences, 117, 4, 295, 306, JAPANESE PHARMACOLOGICAL SOC, 2011年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation.
Arata Nishimoto, Lingyun Lu, Misato Hayashi, Tadashi Nishiya, Takahiro Horinouchi, Soichi Miwa
Biochemical and biophysical research communications, 391, 4, 1616, 22, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010年01月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Endothelin type A receptor (ET(A)R) plays an important role in some cardiovascular disorders where ET(A)R levels are increased. However, regulatory mechanisms for ET(A)R levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET(A)R-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET(A)R as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET(A)R-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET(A)R and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET(A)R, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET(A)R after protein synthesis inhibition as an index of a degradation rate. ET(A)R was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET(A)R degradation and increased the amount of Jab1 bound to ET(A)R with a maximal level of 500% at 3h. In the absence of ET-1 stimulation, the level of ET(B)R was lower than that of ET(A)R and the degradation rate of ET(B)R was markedly faster than that of ET(A)R. Notably, the amount of Jab1 bound to ET(B)R and ubiquitination level of ET(B)R were markedly higher than those for ET(A)R. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET(A)R and ET(B)R by modulating ubiquitination of these receptors, leading to changes in ET(A)R and ET(B)R levels.

Endothelin type B receptor-induced sustained Ca2+ influx involves G(q/11)/phospholipase C-independent, p38 mitogen-activated protein kinase-dependent activation of Na+/H+ exchanger.
Tsunaki Higa, Takahiro Horinouchi, Hiroyuki Aoyagi, Hiroshi Asano, Tadashi Nishiya, Arata Nishimoto, Ikunobu Muramatsu, Soichi Miwa
Journal of pharmacological sciences, 113, 3, 276, 80, JAPANESE PHARMACOLOGICAL SOC, 2010年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The mechanism for sustained Ca2+ influx activated by G protein-coupled receptors was examined. In Chinese hamster ovary cells expressing recombinant human endothelin type B receptor (ET(B)R) and endogenous P2Y receptor (P2Y-R), endothelin-1 elicited a sustained Ca2+ influx depending on G(q/11 )protein, phospholipase C (PLC), Na+/H+ exchanger (NHE), and p38 mitogen-activated protein kinase (p38MAPK), whereas P2Y-R-induced sustained Ca2+ influx was negligible. Functional studies showed that NHE activation by ET(B)R was mediated via p38MAPK but not G(q/11)/PLC, while that by P2Y-R involves only G(q/11)/PLC/p38MAPK. These results suggest that G(q/11)/PLC-independent NHE activation via p38MAPK plays an important role in ET(B)R- mediated sustained Ca2+ influx.

Differential coupling of human endothelin type A receptor to G(q/11) and G(12) proteins: the functional significance of receptor expression level in generating multiple receptor signaling.
Takahiro Horinouchi, Hiroshi Asano, Tunaki Higa, Arata Nishimoto, Tadashi Nishiya, Ikunobu Muramatsu, Soichi Miwa
Journal of pharmacological sciences, 111, 4, 338, 51, JAPANESE PHARMACOLOGICAL SOC, 2009年12月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, This study examines the influence of receptor expression level on signaling pathways activated via endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary cells at 32,100 (ET(A)R-high-CHO) and 893 (ET(A)R-low-CHO) fmolmg protein(-1). Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was dependent on G(q/11) protein, phospholipase C (PLC), Na(+)/H(+) exchanger (NHE), and p38 mitogen-activated protein kinase (p38MAPK) in ET(A)R-high-CHO, whereas the sustained [Ca(2+)](i) increase was negligible in ET(A)R-low-CHO. Functional study with Cytosensor(TM) microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ET(A)R-high-CHO and ET(A)R-low-CHO. In ET(A)R-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to G(q/11) and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ET(A)R-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1-induced p38MAPK phosphorylation in ET(A)R-low-CHO but not in ET(A)R-high-CHO was mediated via G(q/11) and PLC. The G(q/11)/PLC-independent p38MAPK phosphorylation in ET(A)R-high-CHO was suppressed by expression of the C terminus of G(alpha12) protein to disrupt receptor-G(12) protein coupling. These results provide evidence for multiple signaling pathways of ET(A)R that were activated via at least the G(q/11)/PLC/NHE, G(12)/p38MAPK/NHE, and G(q/11)/PLC/p38MAPK/NHE cascades in an expression level-dependent manner.

Gタンパク質共役型受容体を介した持続性Ca2+流入の活性化機構：エンドセリンA型受容体を中心に．
堀之内孝広, 三輪聡一
日本薬理学雑誌, 134, 1, 8, 12, 2009年07月, ［招待有り］, ［国内誌］
日本語, 研究論文（学術雑誌）

Pharmacological evaluation of ocular beta-adrenoceptors in rabbit by tissue segment binding method.
Takahiro Horinouchi, Shigeru Morishima, Yoshio Tanaka, Katsuo Koike, Soichi Miwa, Ikunobu Muramatsu
Life sciences, 84, 5-6, 181, 7, PERGAMON-ELSEVIER SCIENCE LTD, 2009年01月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, AIMS: This study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) beta-adrenoceptors in rabbit to characterize the plasma membrane beta-adrenoceptors and binding affinities of beta-adrenoceptor antagonists. MAIN METHODS: The tissue segment binding method with a hydrophilic radioligand (-)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-(3)H]benzimidazol-2-one ([ (3)H]-CGP12177) was employed. KEY FINDINGS: Specific and saturable binding of [ (3)H]-CGP12177 to intact tissue segments was detected by using (+/-)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective beta(1)- and beta(2)-adrenoceptor antagonists revealed a single population of beta(2)-adrenoceptors in ocular tissues and of beta(1)-adrenoceptors in atria, but mixed populations of beta(1)- and beta(2)-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for beta(2)-adrenoceptors was approximately 158-fold higher than for beta(1)-adrenoceptors, indicating the beta(2)-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (-)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [ (3)H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol. SIGNIFICANCE: This study with tissue segment binding method suggests that the binding affinity of (-)-enantiomers of beta-adrenoceptor antagonists for plasma membrane beta-adrenoceptors (beta(1)-adrenoceptors of atria, beta(2)-adrenoceptors of ocular tissues, and mixed beta(1)-/beta(2)-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between beta-adrenoceptor antagonists.

Ligand-directed signaling at the beta3-adrenoceptor produced by 3-(2-Ethylphenoxy)-1-[ (1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) relative to receptor agonists.
Masaaki Sato, Takahiro Horinouchi, Dana S Hutchinson, Bronwyn A Evans, Roger J Summers
Molecular pharmacology, 72, 5, 1359, 68, 2007年11月, ［国際誌］
英語, 研究論文（学術雑誌）, This study examines signaling pathways activated by the mouse beta(3)-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHObeta(3)H) or low (CHObeta(3)L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (-)-Isoproterenol and the beta(3)-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHObeta(3)H and CHObeta(3)L cells. For cAMP accumulation, the beta(3)-AR ligand SR59230A was a partial agonist in CHObeta(3)H and an antagonist in CHObeta(3)L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (-)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHObeta(3)H cells, whereas p38 MAPK is a major contributor to ECAR in CHObeta(3)L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHObeta(3)H and CHObeta(3)L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHObeta(3)H and CHObeta(3)L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol.
Takahiro Horinouchi, Shigeru Morishima, Takashi Tanaka, Fumiko Suzuki, Yoshio Tanaka, Katsuo Koike, Soichi Miwa, Ikunobu Muramatsu
Life sciences, 81, 5, 399, 404, 2007年07月12日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Recently, tissue segment binding method with a hydrophilic radioligand [ (3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.

Endothelin-1 decreases [Ca2+]i via Na+/Ca2+ exchanger in CHO cells stably expressing endothelin ETA receptor.
Takahiro Horinouchi, Arata Nishimoto, Tadashi Nishiya, Lingyun Lu, Emi Kajita, Soichi Miwa
European journal of pharmacology, 566, 1-3, 28, 33, 1-3, 2007年07月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Endothelin ET(A) receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent 'decrease' in [Ca2+]i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 microM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+]i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+]i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+]i was markedly suppressed by 3',4'-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+]i. Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+]i.

OCTN2VT, a splice variant of OCTN2, does not transport carnitine because of the retention in the endoplasmic reticulum caused by insertion of 24 amino acids in the first extracellular loop of OCTN2
Satoshi Maekawa, Daisuke Mori, Tadashi Nishiya, Osamu Takikawa, Takahiro Horinouchi, Arata Nishimoto, Emi Kajita, Soichi Miwa
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1773, 6, 1000, 1006, ELSEVIER SCIENCE BV, 2007年06月, ［査読有り］
英語, 研究論文（学術雑誌）, A novel organic cation transporter OCTN2 is indispensable for carnitine transport across plasma membrane and subsequent fatty acid metabolism in the mitochondria. Here, we report a novel splice variant of OCTN2 (OCTN2VT), in which a 72-base-pair sequence located in the first intron of OCTN2 gene was spliced between exons 1 and 2 of OCTN2, causing the insertion of 24 amino acids in the first extracellular loop of OCTN2. Despite the similarity between OCTN2 and OCTN2VT regarding primary structure and tissue distribution, their biochemical characteristics were significantly different. OCTN2 was expressed on the plasma membrane with robust N-glycosylation,whereas OCTN2VT was retained in the endoplasmic reticulum (ER) with poor N-glycosylation. In addition, the retention in the ER caused no carnitine uptake into the cells. These results demonstrate that the biochemical and functional characteristics of OCTN2VT are distinct from OCTN2 due to the insertion of 24 amino acids in the first extracellular loop. (c) 2007 Elsevier B.V. All rights reserved.

OCTN2VT, a splice variant of OCTN2, does not transport carnitine because of the retention in the endoplasmic reticulum caused by insertion of 24 amino acids in the first extracellular loop of OCTN2.
Satoshi Maekawa, Daisuke Mori, Tadashi Nishiya, Osamu Takikawa, Takahiro Horinouchi, Arata Nishimoto, Emi Kajita, Soichi Miwa
Biochimica et biophysica acta, 1773, 6, 1000, 6, 6, 2007年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A novel organic cation transporter OCTN2 is indispensable for carnitine transport across plasma membrane and subsequent fatty acid metabolism in the mitochondria. Here, we report a novel splice variant of OCTN2 (OCTN2VT), in which a 72-base-pair sequence located in the first intron of OCTN2 gene was spliced between exons 1 and 2 of OCTN2, causing the insertion of 24 amino acids in the first extracellular loop of OCTN2. Despite the similarity between OCTN2 and OCTN2VT regarding primary structure and tissue distribution, their biochemical characteristics were significantly different. OCTN2 was expressed on the plasma membrane with robust N-glycosylation, whereas OCTN2VT was retained in the endoplasmic reticulum (ER) with poor N-glycosylation. In addition, the retention in the ER caused no carnitine uptake into the cells. These results demonstrate that the biochemical and functional characteristics of OCTN2VT are distinct from OCTN2 due to the insertion of 24 amino acids in the first extracellular loop.

循環系調節因子としてのエンドセリンにより活性化される細胞内カルシウムシグナルとその調節系
三輪聡一, 堀之内孝広, 西本新, 西屋禎
適応医学, 11, 1, 20, 2007年05月20日
日本語

Pharmacological characterization of the beta-adrenoceptor that mediates the relaxant response to noradrenaline in guinea-pig tracheal smooth muscle.
Yoshio Tanaka, Yoko Yamashita, Hiromi Michikawa, Takahiro Horinouchi, Katsuo Koike
Naunyn-Schmiedeberg's archives of pharmacology, 375, 1, 51, 64, 2007年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Pharmacological characteristics of beta-adrenoceptors (beta-ARs) mediating noradrenaline-induced relaxation were investigated in guinea-pig tracheal smooth muscle. The inhibitory effects of several types of beta-AR antagonists on noradrenaline-induced relaxation against histamine contraction were scrutinized with Schild plot analysis. The concentration-response curve for noradrenaline obtained in the absence of phentolamine and uptake inhibitors was competitively antagonized by all of the beta-AR antagonists used in this study (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551). However, their pA2 values were markedly less than the expected values for beta1-AR and beta2-AR. On the other hand, pA2 values of ICI-118,551 (6.85) determined in the presence of phentolamine suggested a contribution of a beta1 -AR rather than beta2 -AR. In the presence of phentolamine and uptake inhibitors (desipramine and deoxycorticosterone), the Schild plot for atenolol was a better fit, with two distinct straight lines. The pA2 values of atenolol provided by the regression were: approximately 7.0, which corresponds to the expected beta1-AR value, and approximately 6.5, which was 3 times less than the expected value for beta1 -AR, and thus the possible presence of two classes of beta1 -AR (beta1(Low) and beta1(High)) was suggested. This view was also supported by Schild plot analysis for propranolol, which fit two straight lines each with a slope of 1.0. The present findings indicate that beta1 -ARs contributing to noradrenaline-elicited relaxation in guinea-pig tracheal smooth muscle exhibit diverse pharmacological characteristics and may be subdivided into at least two classes with distinct affinities for atenolol. Under physiological conditions, beta1(Low) rather than beta1(High) seems to play a more significant role in noradrenaline-regulated airway smooth muscle tone.

Pharmacological evaluation of plasma membrane beta-adrenoceptors in rat hearts using the tissue segment binding method.
Takahiro Horinouchi, Shigeru Morishima, Takashi Tanaka, Fumiko Suzuki, Yoshio Tanaka, Katsuo Koike, Ikunobu Muramatsu
Life sciences, 79, 10, 941, 8, 2006年08月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [ (3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [ (3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.

Functional domains of the mouse beta3-adrenoceptor associated with differential G protein coupling.
Masaaki Sato, Dana S Hutchinson, Tore Bengtsson, Anders Floren, Ulo Langel, Takahiro Horinouchi, Bronwyn A Evans, Roger J Summers
The Journal of pharmacology and experimental therapeutics, 315, 3, 1354, 61, 2005年12月, ［国際誌］
英語, 研究論文（学術雑誌）, Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [ (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[ (1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.

Quantifying receptor properties: the tissue segment binding method - a powerful tool for the pharmacome analysis of native receptors.
Ikunobu Muramatsu, Takashi Tanaka, Fumiko Suzuki, Zhang Li, Yasuko Hiraizumi-Hiraoka, Abu Syed Md Anisuzzaman, Hatsumi Yamamoto, Takahiro Horinouchi, Shigeru Morishima
Journal of pharmacological sciences, 98, 4, 331, 9, JAPANESE PHARMACOLOGICAL SOC, 2005年08月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The radioligand binding assay technique is an extremely powerful tool for studying receptors. It allows an analysis of the interactions of hormones, neurotransmitters, and related drugs with their receptors. Most of the binding assays have widely been applied to crude membrane fractions prepared from many tissues, but in the conventional method, there are some limitations such as a yield loss of receptor-bearing membranes and a change in receptor environment upon homogenization and fractionation. Recently, in order to overcome these problems, a binding assay has been developed using intact tissue segments. This article presents a brief overview of the tissue segment binding assay that has been developed mainly in our department. Practical guidelines for setting up this new assay are presented, including segment preparation, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. The unique advantages and disadvantages of the tissue segment binding method are discussed in comparison with those of conventional membrane binding methods. We suggest that the tissue segment binding method is a powerful tool for detecting the native properties of receptors occurring in tissues and cells without altering their environment.

New insights into beta-adrenoceptors in smooth muscle: distribution of receptor subtypes and molecular mechanisms triggering muscle relaxation.
Yoshio Tanaka, Takahiro Horinouchi, Katsuo Koike
Clinical and experimental pharmacology & physiology, 32, 7, 503, 14, BLACKWELL PUBLISHING, 2005年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 1. The beta-adrenoceptor is currently classified into beta(1), beta(2) and beta(3) subtypes and all three subtypes are expressed in smooth muscle. Each beta-adrenoceptor subtype exhibits tissue-specific distribution patterns, which may be a determinant controlling the mechanical functions of corresponding smooth muscle. Airway and uterine smooth muscles abundantly express the beta(2)-adrenoceptor, the physiological significance of which is established as a fundamental regulator of the mechanical activities of these muscles. Recent pharmacomechanical and molecular approaches have revealed roles for the beta(3)-adrenoceptor in the gastrointestinal tract and urinary bladder smooth muscle. 2. The beta-adrenoceptor is a G(s)-protein-coupled receptor and its activation elevates smooth muscle cAMP. A substantial role for a cAMP-dependent mechanism(s) is generally believed to be the key trigger for eliciting beta-adrenoceptor-mediated relaxation of smooth muscle. Downstream effectors activated via a cAMP-dependent mechanism(s) include plasma membrane K(+) channels, such as the large-conductance, Ca(2+)-activated K(+) (MaxiK) channel. 3. Beta-Adrenoceptor-mediated relaxant mechanisms also include cAMP-independent signalling pathways. This view is supported by numerous pharmacological and electrophysiological lines of evidence. In airway smooth muscle, direct activation of the MaxiK channel by G(s)alpha is a mechanism by which stimulation of beta(2)-adrenoceptors elicits muscle relaxation independently of the elevation of cAMP. 4. The cAMP-independent mechanism(s) is also substantial in beta(3)-adrenoceptor-mediated relaxation of gastrointestinal tract smooth muscle. However, in the case of the beta(3)-adrenoceptor, a delayed rectified K(+) channel rather than the MaxiK channel seems to mediate, in part, cAMP-independent relaxant mechanisms. 5. In the present article, we review the distribution of beta-adrenoceptor subtypes in smooth muscle tissues and discuss the molecular mechanisms by which each subtype elicits muscle relaxation, focusing on the roles of cAMP and plasma membrane K(+) channels.

Adrenaline produces the relaxation of guinea-pig airway smooth muscle primarily through the mediation of beta(2)-adrenoceptors.
Yoshio Tanaka, Yoko Yamashita, Takahiro Horinouchi, Katsuo Koike
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi, 41, 3, 153, 61, 2005年06月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The beta-adrenoceptor subtype that mediates adrenaline-induced relaxation was pharmacologically identified in smooth muscle cells of the isolated guinea-pig trachea. Adrenaline produced a concentration-dependent relaxation with a pD(2) value of 7.1. The concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by the beta(1)-/beta(2)-nonselective antagonists propranolol and bupranolol, with pA(2) values of 8.85 and 8.97, respectively. Adrenaline-induced relaxation was not affected by the beta(1)-selective antagonists atenolol and CGP-20, 712A within the concentration ranges supposed to antagonize the beta(1)-subtype (atenolol, or=3x10(-6) M with a pA(2) value of 5.77. The concentration-response curve for adrenaline was also competitively antagonized by the beta(2)-selective antagonists butoxamine and ICI-118,551 with pA(2) values of 6.86 and 8.73, respectively. The pA(2) values of beta-adrenoceptor antagonists (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551) tested against adrenaline were consistent with the values when tested against salbutamol, a beta(2)-selective adrenoceptor agonist. The present findings provide evidence that the relaxant response of the smooth muscle of the guinea-pig trachea to the adrenal medulla hormone, adrenaline, is mainly mediated through beta(2)-adrenoceptors.

cAMP-independent mechanism is significantly involved in β₂-adrenoceptor-mediated tracheal relaxation.
Katsuo Koike, Yoko Yamashita, Takahiro Horinouchi, Fumiko Yamaki, Yoshio Tanaka
European Journal of Pharmacology, 492, 1, 65, 70, ELSEVIER SCIENCE BV, 2004年05月10日, ［国際誌］
英語, 研究論文（学術雑誌）, The role of cAMP in the beta2-adrenoceptor-mediated relaxation in response to salbutamol was examined in guinea pig tracheal smooth muscle. The concentration-dependent salbutamol-induced relaxation was antagonized in a competitive fashion by a beta2-selective adrenoceptor antagonist, butoxamine, with a pA2 value of 6.90. Salbutamol (10 microM) elevated the tracheal smooth muscle cAMP content by about fivefold, a response which was significantly inhibited by an adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 100 microM). However, the salbutamol-elicited relaxation was not diminished by SQ 22,536 (100 microM). These results provide evidence for the first time that a cAMP-independent mechanism(s) is involved in beta2-adrenoceptor-mediated tracheal smooth muscle relaxation in the guinea pig.

cAMP-independent mechanism is significantly involved in beta(2)-adrenoceptor-mediated tracheal relaxation
K Koike, Y Yamashita, T Horinouchi, F Yamaki, Y Tanaka
EUROPEAN JOURNAL OF PHARMACOLOGY, 492, 1, 65, 70, ELSEVIER SCIENCE BV, 2004年05月
英語, 研究論文（学術雑誌）, The role of cAMP in the beta(2)-adrenoceptor-mediated relaxation in response to salbutamol was examined in guinea pig tracheal smooth muscle. The concentration-dependent salbutamol-induced relaxation,was antagonized in a competitive fashion by a beta(2)-selective adrenoceptor antagonist, butoxamine, with a pA(2) value of 6.90. Salbutamol (10 muM) elevated the tracheal smooth muscle cAMP content by about fivefold, a response which was significantly inhibited by an adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 100 muM). However, the salbutamol-elicited relaxation was not diminished by SQ 22,536 (100 muM). These results provide evidence for the first time that a cAMP-independent mechanism(s) is involved in beta(2)-adrenoceptor-mediated tracheal smooth muscle relaxation in the guinea pig. (C) 2004 Elsevier B.V. All rights reserved.

Beta1-adrenoceptor-mediated relaxation with isoprenaline and the role of MaxiK channels in guinea-pig esophageal smooth muscle.
Yoshio Tanaka, Keiko Shinoda, Sarasa Sekiya, Fumiko Yamaki, Mari Shibano, Yoko Yamashita, Takahiro Horinouchi, Katsuo Koike
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi, 40, 2, 43, 52, 2004年04月, ［国内誌］
英語, 研究論文（学術雑誌）, The possible functional coupling between beta1-adrenoceptor and MaxiK channels which results in smooth muscle relaxation was examined in the guinea-pig esophageal muscularis mucosae. Isoprenaline-elicited relaxation of esophageal smooth muscle was confirmed to be mediated through beta1-adrenoceptors as the response was competitively antagonized by a beta1-selective antagonist atenolol with a pA2 value of 7.01. Iberiotoxin (IbTx, 10(-7) M), a selective MaxiK channel inhibitor, substantially diminished the relaxant response to isoprenaline. The extent of the MaxiK channel contribution to the relaxant response was 15-40% of the control response when estimated as the E50%-Emax responses to isoprenaline. The relaxation to isoprenaline was also attenuated by high-KCl (80 mM) to the same degree as the relaxant response generated in the presence of IbTx, and thus the estimated extent of the K+ channel contribution was 10-40%. These findings indicate that beta1-adrenoceptors are substantially coupled with MaxiK channels to produce relaxation of esophageal smooth muscle in the guinea-pig. Although MaxiK channels account for the contribution of K+ channels to the beta1-adrenoceptor-mediated relaxation in this smooth muscle preparation, their contribution seems to be less when compared to the beta2-adrenoceptor-mediated relaxation of tracheal smooth muscle.

β₁-Adrenoceptor-mediated relaxation with isoprenaline and the role of MaxiK channels in guinea-pig esophageal smooth muscle.
Yoshio Tanaka, Keiko Shinoda, Sarasa Sekiya, Fumiko Yamaki, Mari Shibano, Yoko Yamashita, Takahiro Horinouchi, Katsuo Koike
Journal of Smooth Muscle Research, 40, 2, 43, 52, 2004年04月
英語, 研究論文（学術雑誌）, The possible functional coupling between β1-adrenoceptor and MaxiK channels which results in smooth muscle relaxation was examined in the guinea-pig esophageal muscularis mucosae. Isoprenaline-elicited relaxation of esophageal smooth muscle was confirmed to be mediated through β1-adrenoceptors as the response was competitively antagonized by a β1-selective antagonist atenolol with a pA2 value of 7.01. Iberiotoxin (IbTx, 10-7 M), a selective MaxiK channel inhibitor, substantially diminished the relaxant response to isoprenaline. The extent of the MaxiK channel contribution to the relaxant response was 15-40% of the control response when estimated as the E50%-Emax responses to isoprenaline. The relaxation to isoprenaline was also attenuated by high-KCl (80 mM) to the same degree as the relaxant response generated in the presence of IbTx, and thus the estimated extent of the K+ channel contribution was 10-40%. These findings indicate that β1- adrenoceptors are substantially coupled with MaxiK channels to produce relaxation of esophageal smooth muscle in the guinea-pig. Although MaxiK channels account for the contribution of K+ channels to the β1-adrenoceptor-mediated relaxation in this smooth muscle preparation, their contribution seems to be less when compared to the β2-adrenoceptor-mediated relaxation of tracheal smooth muscle.

Comparison of the b-adrenoceptor subtypes which mediate relaxant responses of guinea-pig airway smooth muscle to noradrenaline and adrenaline.　　　　　　　　　　　　　　　
Minemura A, Yamaki F, Yamashita Y, Horinouchi T, Tanaka Y, Koike K
Yakugaku Zasshi, 124(Suppl. 2), 155, 156, 2004年, ［査読有り］

Evidence showing that b-adrenoceptor subtype responsible for the relaxation induced by isoprenaline is principally b2 but not b1 in guinea-pig tracheal smooth muscle.
Tanaka Y, Yamashita Y, Horinouchi T, Yamaki F, Koike K
Auton. Autacoid Pharmacol., 24, 2, 37, 43, 2004年, ［査読有り］

Characterization of beta 3-adrenoceptor-mediated relaxation in rat abdominal aorta smooth muscle.
Mayumi Matsushita, Takahiro Horinouchi, Yoshio Tanaka, Hiromichi Tsuru, Katsuo Koike
European journal of pharmacology, 482, 1-3, 235, 44, 2003年12月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The present study was carried out to characterize beta-adrenoceptor subtypes mediating relaxation of rat abdominal aorta smooth muscle. (-)-Isoprenaline and a nonconventional beta(3)-adrenoceptor agonist, (+/-)-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A), induced concentration-dependent relaxation of (-)-phenylephrine (0.3 microM) preconstricted spiral preparations. Pretreatment with a combination of (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A, a selective beta(1)-adrenoceptor antagonist) and (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[ (1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511, a selective beta(2)-adrenoceptor antagonist) (0.1 microM for each) produced a 14-fold rightward shift of the concentration-response curve for (-)-isoprenaline; however, the relaxation in response to (+/-)-CGP12177A was unaffected by the blockade of beta(1)- and beta(2)-adrenoceptors. In the presence of CGP20712A and ICI-118,551 (0.1 microM for each), the concentration-response curves for (-)-isoprenaline and (+/-)-CGP12177A were shifted to the right by a nonselective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist, (+/-)-bupranolol (3 and 10 microM). These results clearly suggest that beta(3)-adrenoceptors are involved in beta-adrenoceptor-mediated relaxation of rat abdominal aorta smooth muscle.

MaxiK Channel Mediates β2-Adrenoceptor-activated Relaxation to Isoprenaline through cAMP-dependent and -independent Mechanisms in Guinea-pig Tracheal Smooth Muscle
Yoshio Tanaka, Yoko Yamashita, Fumiko Yamaki, Takahiro Horinouchi, Koki Shigenobu, Katsuo Koike
Journal of Smooth Muscle Research, 39, 6, 205, 219, 2003年12月
英語, 研究論文（学術雑誌）, We examined the contribution of large-conductance, Ca 2+-sensitive K+ (MaxiK) channel to β 2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between β2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through β2-type of adrenoceptor since the response was antagonized in a competitive fashion by a β2-selective adrenoceptor antagonist butoxamine with a pA 2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 μM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 μM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 μM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX 5 μg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein G s These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates β 2-adrenoceptor-activated relaxation
2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between β 2-adrenoceptor and MaxiK channel to induce muscle relaxation
and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between β2-adrenoceptor and this ion channel.

MaxiK channel mediates β₂-adrenoceptor-activated relaxation to isoprenaline through cAMP-dependent and -independent mechanisms in guinea-pig tracheal smooth muscle.
Yoshio Tanaka, Yoko Yamashita, Fumiko Yamaki, Takahiro Horinouchi, Koki Shigenobu, Katsuo Koike
Journal of Smooth Muscle Research, 39, 6, 205, 19, 2003年12月, ［国内誌］
英語, 研究論文（学術雑誌）, We examined the contribution of large-conductance, Ca(2+)-sensitive K+ (MaxiK) channel to beta2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between beta2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through beta2-type of adrenoceptor since the response was antagonized in a competitive fashion by a beta2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 microM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 microM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 microM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 microg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates beta2-adrenoceptor-activated relaxation; 2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between beta2-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between beta2-adrenoceptor and this ion channel.

Evidence for a significant role of a G(s)-triggered mechanism unrelated to the activation of adenylyl cyclase in the cyclic AMP-independent relaxant response of guinea-pig tracheal smooth muscle
Y Tanaka, Y Yamashita, F Yamaki, T Horinouchi, K Shigenobu, K Koike
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 368, 5, 437, 441, SPRINGER-VERLAG, 2003年11月
英語, 研究論文（学術雑誌）, Cyclic AMP is a key molecule in the regulation of airway smooth muscle tone. Increased cyclic AMP leads to relaxation of this smooth muscle and its inhibition results in the muscle contraction. A constitutive role for cyclic AMP in the contraction and relaxation of airway muscle is supported by the observations that direct activators of adenylyl cyclase, such as forskolin and membrane-permeable cyclic AMP analogues, relax this smooth muscle potently. This traditional view of the role for cyclic AMP is the basis for the idea that relaxation of airway smooth muscle mediated through adenylyl cyclase-linked, G(s)-coupled receptors, including the beta(2)-adrenoceptor, is achieved mainly by the elevation of cyclic AMP content [cyclic AMP-dependent mechanism(s)]. However, recent pharmacological and biochemical evidence raises a fundamental question concerning the role of cyclic AMP; can G(s)-coupled receptor-mediated relaxation of tracheal smooth muscle be attributed exclusively to cyclic AMP-dependent mechanism(s)? In the present study, we show that cholera toxin (CTX, 5 mug/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein G(s), relaxes guinea-pig tracheal smooth muscle. CTX also elevates tissue cyclic AMP content by about 30-fold and this is practically abolished by an adenylyl cyclase inhibitor, SQ 22,536 (100 muM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of G(s) is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. G(s)-triggered, cyclic AMP-unrelated cellular mechanism(s) seem(s) to play a substantial role in smooth muscle relaxation mediated through adenylyl cyclase-linked receptors. This mechanism may account in part for the cyclic AMP-independent relaxant response of tracheal smooth muscle.

Evidence for a significant role of a Gs-triggered mechanism unrelated to the activation of adenylyl cyclase in the cyclic AMP-independent relaxant response of guinea-pig tracheal smooth muscle.
Yoshio Tanaka, Yoko Yamashita, Fumiko Yamaki, Takahiro Horinouchi, Koki Shigenobu, Katsuo Koike
Naunyn-Schmiedeberg's archives of pharmacology, 368, 5, 437, 41, SPRINGER-VERLAG, 2003年11月, ［国際誌］
英語, 研究論文（学術雑誌）, Cyclic AMP is a key molecule in the regulation of airway smooth muscle tone. Increased cyclic AMP leads to relaxation of this smooth muscle and its inhibition results in the muscle contraction. A constitutive role for cyclic AMP in the contraction and relaxation of airway muscle is supported by the observations that direct activators of adenylyl cyclase, such as forskolin and membrane-permeable cyclic AMP analogues, relax this smooth muscle potently. This traditional view of the role for cyclic AMP is the basis for the idea that relaxation of airway smooth muscle mediated through adenylyl cyclase-linked, G(s)-coupled receptors, including the beta(2)-adrenoceptor, is achieved mainly by the elevation of cyclic AMP content [cyclic AMP-dependent mechanism(s)]. However, recent pharmacological and biochemical evidence raises a fundamental question concerning the role of cyclic AMP; can G(s)-coupled receptor-mediated relaxation of tracheal smooth muscle be attributed exclusively to cyclic AMP-dependent mechanism(s)? In the present study, we show that cholera toxin (CTX, 5 microg/ml), an activator of the heterotrimeric guanine-nucleotide-binding protein G(s), relaxes guinea-pig tracheal smooth muscle. CTX also elevates tissue cyclic AMP content by about 30-fold and this is practically abolished by an adenylyl cyclase inhibitor, SQ 22,536 (100 microM). However, unexpectedly, the relaxant response to CTX is not affected by SQ 22,536. These results firstly show that activation of G(s) is able to produce a relaxation in tracheal smooth muscle independently of the elevation of cyclic AMP. G(s)-triggered, cyclic AMP-unrelated cellular mechanism(s) seem(s) to play a substantial role in smooth muscle relaxation mediated through adenylyl cyclase-linked receptors. This mechanism may account in part for the cyclic AMP-independent relaxant response of tracheal smooth muscle.

[Molecular mechanisms for cAMP-independent activation of MaxiK channel: G protein direct channel regulation and contribution to the tuning of vascular smooth muscle tone].
Fumiko Yamaki, Yoshio Tanaka, Takahiro Horinouchi, Koki Shigenobu, Katsuo Koike
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 122 Suppl, 37P-39P, 39P, 2003年11月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, MaxiK channel, the large conductance Ca(2+)-sensitive K+ channel, is expressed abundantly in vascular smooth muscles and plays a key role in the tuning of their excitability and contractility. The present study was carried out to elucidate the contribution of MaxiK channel to prostacyclin receptor (IP receptor)-mediated vascular relaxation with a special reference to the role of cAMP. An IP agonist, beraprost, induced a strong relaxation in de-endothelialized guinea pig thoracic aorta, which was almost abolished by a MaxiK channel selective blocker, iberiotoxin (IbTx). Beraprost produced a 30-fold rise in tissue cAMP contents. In addition, beraprost-induced relaxation potentiated in the presence of Ro-20-1724 (a selective inhibitor of cAMP-specific phosphodiesterase) was completely counteracted by IbTx. However, beraprost-induced relaxation was not affected by SQ22,536, an adenylyl cyclase inhibitor, which abolished this IP agonist-induced elevation of cAMP contents. SQ22,536-insensitive relaxant component was significantly inhibited by IbTx. Cholera toxin, a Gs activator, qualitatively mimicked the effects of beraprost. Furthermore, MaxiK channel currents in aortic myocytes were increased by beraprost in a GTP-dependent manner. These results indicate that both cAMP-dependent and -independent pathways contribute to MaxiK channel-mediated vascular relaxation following IP receptor stimulation. Direct regulation by Gs seems to partly account for MaxiK channel-mediated, cAMP-independent mechanism.

[beta-adrenoceptor subtypes involved in relaxations of guinea-pig gastrointestinal smooth muscles: distribution and signaling pathway of beta 3-adrenoceptors].
Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 122 Suppl, 54P-56P, 56P, 2003年11月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, The distribution and signaling pathway of beta 3-adrenoceptors involved in relaxations were examined in guinea-pig gastrointestinal smooth muscles. In esophagus, beta 1-adrenoceptors participate in isoprenaline-induced relaxations. However, both beta 2- and beta 3-adrenoceptors contribute to the relaxations in taenia caecum. In gastric fundus, duodenum, and ileum, beta 3-adrenoceptors were responsible for the relaxant responses. These results suggest that functional beta 3-adrenoceptors are present in four smooth muscles except esophagus. This was strongly supported by molecular analyses with RT-PCR, showing the expression of beta 3-adrenoceptor mRNA in the four tissues. beta 3-Adrenoceptor-mediated relaxations were unaffected by both an adenylyl cyclase inhibitor, SQ-22,536 (100 microM), and a PKA inhibitor, H-89 (10 microM), in guinea-pig gastric fundus smooth muscle. Furthermore, in the presence of SQ-22,536 (100 microM), the stimulation of beta 3-adrenoceptors elicited relaxations without an elevation of cAMP, indicating the involvement of cAMP-independent mechanism(s). beta 3-Adrenoceptor-mediated, cAMP-independent relaxations were significantly diminished by a Kv channel blocker, 4-aminopyridine (3 mM). These results indicate that beta 3-adrenoceptors play a primary role in relaxations of guinea-pig gastrointestinal smooth muscles and 4-aminopyridine-sensitive Kv channels participate in cAMP-independent smooth muscle relaxations in response to the activation of beta 3-adrenoceptors.

Phospholipase C inhibitors suppress spontaneous mechanical activity of guinea pig urinary bladder smooth muscle
Y Tanaka, T Okamoto, T Imai, T Horinouchi, H Tanaka, K Shigenobu, K Koike
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 26, 8, 1192, 1194, PHARMACEUTICAL SOC JAPAN, 2003年08月
英語, 研究論文（学術雑誌）, Urinary bladder smooth muscle (UBSM) exhibits spontaneous rhythmic contraction. This spontaneous mechanical activity is generated in the presence of neuronal blockade and thus is myogenic in origin. The spontaneous myogenic contraction of UBSM may be the fundamental determinant of the physiological functions of the urinary bladder to store and excrete urine. Although the mechanisms by which UBSM generates spontaneous contraction have not been completely ascertained, its induction has been suggested to be intimately associated with smooth muscle cell action potentials to enhance extracellular Ca2+ influx through voltage-gated L-type Ca2+ channels. However, the alteration of membrane electrical activity does not seem to be the exclusive trigger mechanism for the generation of the spontaneous contraction. In the present study, we show that spontaneous mechanical activity of guinea pig UBSM is substantially diminished by an inhibitor of phospholipase C (PLC), U-73122, but is not affected by its inactive form, U-73343. Significant attenuation of the mechanical activity can be also obtained with another PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. Our present findings suggest a significant role for the activation of PLC and subsequent inositol 1,4,5-trisphosphate-induced Ca2+ release mechanism as an alternative triggering system for inducing spontaneous mechanical activity of UBSM. The present results support the idea that the action potential is not the sole pacemaker mechanism by which spontaneous contraction is induced in UBSM.

Phospholipase C inhibitors suppress spontaneous mechanical activity of guinea pig urinary bladder smooth muscle.
Yoshio Tanaka, Takao Okamoto, Toshiyasu Imai, Takahiro Horinouchi, Hikaru Tanaka, Koki Shigenobu, Katsuo Koike
Biological & pharmaceutical bulletin, 26, 8, 1192, 4, PHARMACEUTICAL SOC JAPAN, 2003年08月, ［国内誌］
英語, 研究論文（学術雑誌）, Urinary bladder smooth muscle (UBSM) exhibits spontaneous rhythmic contraction. This spontaneous mechanical activity is generated in the presence of neuronal blockade and thus is myogenic in origin. The spontaneous myogenic contraction of UBSM may be the fundamental determinant of the physiological functions of the urinary bladder to store and excrete urine. Although the mechanisms by which UBSM generates spontaneous contraction have not been completely ascertained, its induction has been suggested to be intimately associated with smooth muscle cell action potentials to enhance extracellular Ca(2+) influx through voltage-gated L-type Ca(2+) channels. However, the alteration of membrane electrical activity does not seem to be the exclusive trigger mechanism for the generation of the spontaneous contraction. In the present study, we show that spontaneous mechanical activity of guinea pig UBSM is substantially diminished by an inhibitor of phospholipase C (PLC), U-73122, but is not affected by its inactive form, U-73343. Significant attenuation of the mechanical activity can be also obtained with another PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. Our present findings suggest a significant role for the activation of PLC and subsequent inositol 1,4,5-trisphosphate-induced Ca(2+) release mechanism as an alternative triggering system for inducing spontaneous mechanical activity of UBSM. The present results support the idea that the action potential is not the sole pacemaker mechanism by which spontaneous contraction is induced in UBSM.

Function of β₁-adrenoceptors and mRNA expression of β₁- and β₂-adrenoceptors in guinea-pig esophagus.
Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
European Journal of Pharmacology, 473, 1, 79, 82, ELSEVIER SCIENCE BV, 2003年07月18日, ［国際誌］
英語, 研究論文（学術雑誌）, beta-Adrenoceptor subtypes mediating relaxation were examined by using pharmacological and molecular analyses in guinea-pig esophageal muscularis mucosae. (-)-Isoprenaline-induced relaxations were antagonized by (+/-)-propranolol (pA2 = 8.47+/-0.07), a selective beta1-adrenoceptor antagonist, (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A; pA(2)=9.43+/-0.09), and a selective beta(2)-adrenoceptor antagonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[ (1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511; pA2 = 7.11+/-0.04), indicating that beta(1)-adrenoceptors but not beta2- or beta3-adrenoceptors were essentially involved in beta-adrenoceptor-mediated relaxations. However, the expression of messenger RNA (mRNA) for beta1- and beta2-adrenoceptors, but not for beta3-adrenoceptors, was detected by reverse transcription-polymerase chain reaction (RT-PCR). These results clearly suggest that not all beta-adrenoceptor mRNA expressed strictly reflect functional receptors in guinea-pig esophageal muscularis mucosae.

Function of beta(1)-adrenoceptors and mRNA expression of beta(1)- and beta(2)-adrenoceptors in guinea-pig esophagus
T Horinouchi, Y Tanaka, K Koike
EUROPEAN JOURNAL OF PHARMACOLOGY, 473, 1, 79, 82, ELSEVIER SCIENCE BV, 2003年07月
英語, 研究論文（学術雑誌）, beta-Adrenoceptor subtypes mediating relaxation were examined by using pharmacological and molecular analyses in guinea-pig esophageal muscularis mucosae. (-)-Isoprenaline-induced relaxations were antagonized by (+/-)-propranolol (pA(2) = 8.47+/-0.07), a selective beta(1)-adrenoceptor antagonist, (+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP20712A; pA(2) = 9.43+/-0.09), and a selective beta(2)-adrenoceptor antagonist, (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[ (1-methylethyl)amino]-2-butanol hydrochloride (ICI-118,5511; pA(2) = 7.11+/-0.04), indicating that beta(1)-adrenoceptors but not beta(2)- or beta(3)-adrenoceptors were essentially involved in beta-adrenoceptor-mediated relaxations. However, the expression of messenger RNA (mRNA) for beta(1)- and beta(2)-adrenoceptors, but not for beta(3)-adrenoccptors, was detected by reverse transcription-polymerase chain reaction (RT-PCR). These results clearly suggest that not all beta-adrenoceptor mRNA expressed strictly reflect functional receptors in guinea-pig esophageal muscularis mucosae. (C) 2003 Elsevier B.V. All rights reserved.

The β3-adrenoceptor-mediated relaxation induced by dopamine in guinea pig taenia caecum
Yurie Akimoto, Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 39, 3, 39, 45, 2003年06月
英語, 研究論文（学術雑誌）, The mechanisms of the β-adrenoceptor-mediated relaxation induced by dopamine in guinea pig taenia caecum were examined. The relaxant response to dopamine was unaffected by propranolol (10-1-10-5 M) or phentolamine (10-8-10-5 M). Atenolol (3 × 10 -7-3 × 10-4 M), butoxamine (10-7-10 -4 M), prazosin (10-8-10-5 M), yohimbine (10-8-10-5 M), SCH 23390 (10-8-10-5 M) and haloperidol (10-8-10-5 M) had no effect on the potency of dopamine. The response to dopamine was antagonized in a concentration-dependent manner by bupranolol (3 × 10-6-3 × 10-5 M), and Schild plot of the data revealed the pA 2 value of 5.55 and the slope of the regression line was 1.13. These results suggest that the relaxant response to dopamine in the guinea pig taenia caecum is mainly mediated by the β3-adrenoceptors.

The β₃-adrenoceptor-mediated relaxation induced by dopamine in guinea pig taenia caucum.
Yurie Akimoto, Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 39, 3, 39, 45, 2003年06月, ［国内誌］
英語, 研究論文（学術雑誌）, The mechanisms of the beta-adrenoceptor-mediated relaxation induced by dopamine in guinea pig taenia caecum were examined. The relaxant response to dopamine was unaffected by propranolol (10(-8)-10(-5) M) or phentolamine (10(-8)-10(-5) M). Atenolol (3 x 10(-7)-3 x 10(-4) M), butoxamine (10(-7)-10(-4) M), prazosin (10(-8)-10(-5) M), yohimbine (10(-8)-10(-5) M), SCH 23390 (10(-8)-10(-5) M) and haloperidol (10(-8)-10(-5) M) had no effect on the potency of dopamine. The response to dopamine was antagonized in a concentration-dependent manner by bupranolol (3 x 10(-6)-3 x 10(-5) M), and Schild plot of the data revealed the pA2 value of 5.55 and the slope of the regression line was 1.13. These results suggest that the relaxant response to dopamine in the guinea pig taenia caecum is mainly mediated by the beta3-adrenoceptors.

Evidence for the primary role for 4-aminopyridine-sensitive K-v channels in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles
T Horinouchi, Y Tanaka, K Koike
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 367, 2, 193, 203, SPRINGER-VERLAG, 2003年02月
英語, 研究論文（学術雑誌）, Gastrointestinal smooth muscles exhibit relaxation in response to the stimulation of beta-adrenoceptors with catecholamines. Subtypes of beta-adrenoceptors which mediate catecholamine-elicited relaxations in gastrointestinal smooth muscles are predominantly atypical beta-adrenoceptors including beta(3)-adrenoceptors. Gastrointestinal smooth muscle relaxations mediated via beta(3)-adrenoceptors can occur independently of intracellular cyclic adenosine monophosphate (AMP) elevation. One of the mechanisms responsible for cyclic AMP-independent smooth muscle relaxation following activation of G(s) protein-coupled receptors could be activation of voltage-gated K+ channels. In the present study, possible contribution of two types of K+ (large-conductance, Ca2+-sensitive and voltage-gated K+, BKCa; voltage-gated, K-v) channels to beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations was compared in gastric fundus and duodenum smooth muscles isolated from the guinea-pig. In these gastrointestinal smooth muscles, three catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and two beta(3)-adrenoceptor agonists ((R*, R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl) amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) elicited a concentration-dependent relaxation in the presence of beta(1)- and beta(2)-adrenoceptor antagonists. The relaxations were unaffected by an adenylyl cyclase inhibitor, SQ-22,536 (100 muM), which indicates their characteristic of cyclic AMP-independency. On the other hand, the SQ-22,536-resistant, beta(3)-adrenoceptor-mediated relaxant components were potently attenuated when the tone was raised using high-KCl (80 mM) or in the presence of a K-v channel blocker, 4-aminopyridine (4-AP, 1-3 mM). Iberiotoxin (100 nM), a selective blocker of BKCa channels which significantly contribute to cyclic AMP-independent vascular smooth muscle relaxations induced through activation of G(s) protein-coupled receptors, did not apparently show any inhibitory effects on SQ-22,536-resistant, beta(3)-adrenoceptor-mediated relaxations in these gastrointestinal smooth muscles. The present results indicate that 4-AP-sensitive K-v channels play a primary role in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles. In these smooth muscles, BKCa, channels seem to apparently contribute insignificantly to cyclic AMP-independent relaxations following stimulation Of beta(3)-type of adrenoceptors.

Pharmacological evidence that tetraethylammonium-sensitive, iberiotoxin-insensitive K+ channels function as a negative feedback element for sympathetic neurotransmission by suppressing omega-conotoxin-GVIA-insensitive Ca2+ channels in the relaxation of rabbit facial vein (vol 367, pg 218, 2003)
Y Tanaka, A Akutsu, H Tanaka, T Horinouchi, H Tsuru, K Koike, K Shigenobu
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 367, 2, 218, 218, SPRINGER-VERLAG, 2003年02月
英語, Pharmacological evidence that tetraethylammonium-sensitive, Iberiotoxin- insensitive K+ channels function as a negative-feedback element for sympathetic neurotransmission via suppression of ω-conotoxin-GVIA-insensitive Ca2+ channels in the relaxation of rabbit facial vein.

Evidence for the primary role for 4-aminopyridine-sensitive Kv channels in β₃-adrenoceptor-mediated, cyclic AMP- independent relaxations of guinea-pig gastrointestinal smooth muscles.
Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Naunyn-Schmiedeberg’s Archives of Pharmacology, 367, 2, 193, 203, SPRINGER-VERLAG, 2003年02月, ［国際誌］
英語, 研究論文（学術雑誌）, Gastrointestinal smooth muscles exhibit relaxation in response to the stimulation of beta-adrenoceptors with catecholamines. Subtypes of beta-adrenoceptors which mediate catecholamine-elicited relaxations in gastrointestinal smooth muscles are predominantly atypical beta-adrenoceptors including beta(3)-adrenoceptors. Gastrointestinal smooth muscle relaxations mediated via beta(3)-adrenoceptors can occur independently of intracellular cyclic adenosine monophosphate (AMP) elevation. One of the mechanisms responsible for cyclic AMP-independent smooth muscle relaxation following activation of G(s) protein-coupled receptors could be activation of voltage-gated K(+) channels. In the present study, possible contribution of two types of K(+) (large-conductance, Ca(2+)-sensitive and voltage-gated K(+), BK(Ca); voltage-gated, K(v)) channels to beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations was compared in gastric fundus and duodenum smooth muscles isolated from the guinea-pig. In these gastrointestinal smooth muscles, three catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and two beta(3)-adrenoceptor agonists ((R(*), R(*))-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) elicited a concentration-dependent relaxation in the presence of beta(1)- and beta(2)-adrenoceptor antagonists. The relaxations were unaffected by an adenylyl cyclase inhibitor, SQ-22536 (100 microM), which indicates their characteristic of cyclic AMP-independency. On the other hand, the SQ-22536-resistant, beta(3)-adrenoceptor-mediated relaxant components were potently attenuated when the tone was raised using high-KCl (80 mM) or in the presence of a K(v) channel blocker, 4-aminopyridine (4-AP, 1-3 mM). Iberiotoxin (100 nM), a selective blocker of BK(Ca) channels which significantly contribute to cyclic AMP-independent vascular smooth muscle relaxations induced through activation of G(s) protein-coupled receptors, did not apparently show any inhibitory effects on SQ-22536-resistant, beta(3)-adrenoceptor-mediated relaxations in these gastrointestinal smooth muscles. The present results indicate that 4-AP-sensitive K(v) channels play a primary role in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles. In these smooth muscles, BK(Ca) channels seem to apparently contribute insignificantly to cyclic AMP-independent relaxations following stimulation of beta(3)-type of adrenoceptors.

Pharmacological evidence that tetraethylammonium-sensitive, Iberiotoxin- insensitive K⁺ channels function as a negative-feedback element for sympathetic neurotransmission via suppression of ω-conotoxin-GVIA-insensitive Ca²⁺ channe・・・
Y Tanaka, A Akutsu, H Tanaka, T Horinouchi, H Tsuru, K Koike, K Shigenobu
Naunyn-Schmiedeberg’s Archives of Pharmacology, 367, 2, 218, 218, SPRINGER-VERLAG, 2003年02月
英語, Pharmacological evidence that tetraethylammonium-sensitive, Iberiotoxin- insensitive K⁺ channels function as a negative-feedback element for sympathetic neurotransmission via suppression of ω-conotoxin-GVIA-insensitive Ca²⁺ channels in the relaxation of rabbit facial vein.

Cholinesterase阻害薬distigmine bromideの膀胱及び尿道に対する作用．
堀之内 孝広, 青木 智子, 秋山 理恵, 小野 友子, 芝野 真理, 田中 芳夫, 小池 勝夫
応用薬理, Vol. 64, P45-P52, 3, 45, 52, 2003年
日本語

Pharmacological evidence that tetraethylammonium-sensitive, iberiotoxin-insensitive K+ channels function as a negative feedback element for sympathetic neurotransmission by suppressing omega-conotoxin-GVIA-insensitive Ca2+ channels in the relaxation of rabbit facial vein.
Yoshio Tanaka, Aya Akutsu, Hikaru Tanaka, Takahiro Horinouchi, Hiromichi Tsuru, Katsuo Koike, Koki Shigenobu
Naunyn-Schmiedeberg's archives of pharmacology, 367, 1, 35, 42, SPRINGER-VERLAG, 2003年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The facial vein isolated from various species relaxes in response to electrical field stimulation (EFS). EFS-elicited relaxation of the facial vein is mediated through the release of noradrenaline (NA) from sympathetic nerve endings and the subsequent activation of smooth muscle beta-adrenoceptors. The release of NA from sympathetic nerve endings in arterial tissues requires transmembrane Ca2+ influx, mediated predominantly by voltage-gated N-type Ca2+ channels. The present pharmaco-mechanical study was undertaken to determine whether the N-type channel is the exclusive pre-junctional Ca2+ channel mediating NA release from sympathetic nerve endings in the rabbit facial vein. Possible roles of K+ channels in the sympathetic neurotransmission were also examined, especially focusing on the contribution of voltage-dependent, Ca2+-activated K+ (BKCa) channels. An isolated ring preparation of the rabbit facial vein exhibited intrinsic myogenic tone which lasted for several hours when stretched. EFS produced frequency-dependent (0.25-2 Hz) relaxation in this preparation. EFS-elicited relaxation was abolished by tetrodotoxin (TTX, 1 microM), guanethidine (5 microM) or propranolol (1 microM), indicating that NA released from sympathetic nerve endings was mediating the relaxant response. NA-mediated neurogenic relaxation was almost eliminated by omega-conotoxin-GVIA (1 microM), an N-type Ca2+ channel blocker. On the other hand, tetraethylammonium (TEA, 2 mM) strongly potentiated EFS-elicited relaxation without affecting the relaxation induced by exogenously applied NA. This potentiation by TEA was not profoundly diminished by omega-conotoxin-GVIA (1 microM) alone or omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM, P-type channel blocker), but was almost abolished by omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM) plus omega-conotoxin-MVIIC (3 microM, N-, P- and Q-type channel blocker). The potentiating effect of TEA was not mimicked by iberiotoxin (100 nM) or charybdotoxin (3 microM), both of which block BKCa channels. These findings suggest that pre-junctional N-type Ca2+ channels play the predominant role in the sympathetic nerve transmission in the rabbit facial vein, as in peripheral arterial vascular beds. In addition, Ca2+ channels resistant to 1 microM omega-conotoxin-GVIA, most probably Q-type channels, appear to be present at the sympathetic nerve endings in the rabbit facial vein and contribute substantially to the regulation of NA release from the nerve endings. Prejunctional K+ channels, sensitive to TEA but pharmacologically distinct from iberiotoxin-sensitive BKCa channels, seem to be functionally coupled intimately with the omega-conotoxin-GVIA-resistant Ca2+ channels, and thus function as a negative feedback element in sympathetic neurotransmission in the rabbit facial vein.

Bk(Ca) channel activity enhances with muscle stretch in guinea-pig urinary bladder smooth muscle.
Yoshio Tanaka, Takao Okamoto, Toshiyasu Imai, Yoshihisa Yamamoto, Takahiro Horinouchi, Hikaru Tanaka, Katsuo Koike, Koki Shigenobu
Research communications in molecular pathology and pharmacology, 113-114, 247, 52, 2003年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Possible effects of muscle stretch on the spontaneous rhythmic contractile activity and the extent of contribution of voltage-dependent and Ca2+ -activated K+ (BK(Ca)) channels were examined mechanically in guinea-pig urinary bladder smooth muscle (UBSM). The amplitude of the spontaneous mechanical activity showed stretch-dependency. Iberiotoxin, a selective blocker of BK(Ca) channels, potently increased contraction amplitude, and this effect was more prominent when muscle length was more than twice of its initial length. BK(Ca) channels seem activated more strongly to counteract enhanced spontaneous mechanical activity with UBSM stretch.

Pharmacological characterization of α₁-adrenoceptor in mouse iliac artery.
Mari Shibano, Yoshihisa Yamamoto, Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
European Journal of Pharmacology, 456, 1-3, 77, 9, ELSEVIER SCIENCE BV, 2002年12月05日, ［国際誌］
英語, 研究論文（学術雑誌）, Subtypes of alpha(1)-adrenoceptor-mediated contraction to noradrenaline in the mouse iliac artery were determined (pharmaco-mechanically). Prazosin, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) and 5-methylurapidil shifted the concentration-response curve for noradrenaline to the right, giving the pA(2) values of 9.30, 9.55 and 8.71, respectively. 8-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]-ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) shifted the concentration-response curve for noradrenaline to the right and the pA(2) value was 6.62. These results indicate that the contractile response to noradrenaline in the mouse iliac artery is predominantly mediated by the alpha(1A) -adrenoceptor subtype.

Pharmacological characterization of alpha(1)-adrenoceptor in mouse iliac artery
M Shibano, Y Yamamoto, T Horinouchi, Y Tanaka, K Koike
EUROPEAN JOURNAL OF PHARMACOLOGY, 456, 1-3, 77, 79, ELSEVIER SCIENCE BV, 2002年12月
英語, 研究論文（学術雑誌）, Subtypes of alpha(1)-adrenoceptor-mediated contraction to noradrenaline in the mouse iliac artery were determined (pharmaco-mechanically). Prazosin, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) and 5-methylurapidil shifted the concentration-response curve for noradrenaline to the right, giving the pA(2) values of 9.30, 9.55 and 8.71, respectively. 8-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]-ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) shifted the concentration-response curve for noradrenaline to the right and the pA(2) value was 6.62. These results indicate that the contractile response to noradrenaline in the mouse iliac artery is predominantly mediated by the alpha(1A)-adrenoceptor subtype. (C) 2002 Elsevier Science B.V. All rights reserved.

[Beta 3-adrenoceptor-mediated relaxation of guinea-pig gastric funds smooth muscle: cAMP-independent characteristics and a primary role of 4-aminopyridine-sensitive voltage-dependent K+ (Kv) channels].
Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 120, 1, 109P-111P, 111P, 2002年11月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, beta-Adrenoceptor subtypes which mediate relaxation of guinea-pig gastrointestinal smooth muscles in response to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and beta 3-adrenoceptor agonists (BRL37344 and (+/-)-CGP12177A) are predominantly beta 3-adrenoceptors. Although cAMP-PKA system is thought to play a substantial role in the smooth muscle relaxation mediated via beta 1- and beta 2-adrenoceptors, there is little information on the role of cAMP in beta 3-adrenoceptor-meidated relaxation. The present study was carried out to elucidate the role of cAMP in beta 3-adrenoceptor-meidated relaxation of guinea-pig gastric fundus smooth muscle. Furthermore, possible contribution of two types of K+ (voltage-dependent and Ca(2+)-activated K+, BKCa; voltage-dependent, Kv) channels was also examined pharmaco-mechanically. In gastric fundus smooth muscle, catecholamines and beta 3-adrenoceptor agonists elicited potent relaxations in the presence of beta 1- and beta 2-adrenoceptor antagonists. All of these relaxations were not diminished by an adenylate cyclase inhibitor, SQ-22,536 (100 microM), which indicates their characteristic of cAMP-independency. SQ-22,536-resistant, beta 3-adrenoceptor-mediated relaxations were strongly attenuated by a Kv channel blocker, 4-aminopyridine (3 mM), but not by iberiotoxin (100 nM), a selective blocker of BKCa channel. The present results indicate that 4-aminopyridine-sensitive Kv channels play a primary role in cAMP-independent relaxation of guinea-pig gastric fundus smooth muscle in response to the stimulations of beta 3-adrenoceptors.

[BK channels play an important role as a negative feedback mechanism in the regulation of spontaneous rhythmic contraction of urinary bladder smooth muscles].
Yoshio Tanaka, Takahiro Horinouchi, Hikaru Tanaka, Koki Shigenobu, Katsuo Koike
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 120, 1, 106P-108P, 108P, 2002年11月, ［査読有り］, ［国内誌］
日本語, 研究論文（学術雑誌）, Smooth muscles of urinary bladder wall exhibit spontaneous rhythmic contraction which is myogenic in origin. Although the precise mechanism responsible for the generation of this mechanical activity remains to be established, it can be related closely to the action potential (AP) in urinary bladder smooth muscle (UBSM) cell, and may be the fundamental constituent to determine urinary bladder physiological functions to store and micturate urine. In the present study, possible roles of voltage-dependent and Ca(2+)-sensitive K+ (BK) channels, highly expressed in UBSM cells, were examined in the regulation of spontaneous UBSM contraction with reference to the generation of AP. Iberiotoxin (IbTx), a selective BK channel blocker, strongly increased mechanical activity and AP generation in guinea-pig UBSM. In contrast, BK channel openers (NS-1619, niflumic acid; estradiol, tamoxifen: BK channel alpha- and beta-subunit activators, respectively) significantly diminished AP generation and spontaneous mechanical activity. The present study indicates that BK channels play the primary role as a negative feedback element to limit extracellular Ca2+ influx through affecting AP configurations in the generation of UBSM contraction. BK channel openers including beta-subunit activators may be a potentially useful therapeutic remedy for the treatment of urinary bladder dysfunctions such as frequent urination.

The β2- and β3-adrenoceptor-mediated relaxation induced by fenoterol in guinea pig taenia caecum
Yurie Akimoto, Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 38, 4-5, 145, 151, 2002年10月
英語, 研究論文（学術雑誌）, Fenoterol, a β2-adrenoceptor selective agonist, belongs to the arylethanolamine class. To understand the receptor subtypes responsible for β-adrenoceptor-mediated relaxation of guinea pig taenia caecum, we investigated the effect of fenoterol. Fenoterol caused concentration-dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration-response curve for fenoterol. Schild regression analyses carried out for propranolol, butoxamine and bupranolol against fenoterol gave pA2 values of 8.41, 6.33 and 8.44, respectively. However, in the presence of 3 × 10-4 M atenolol, 10-4 M butoxamine and 10-6 M phentolamine to block the β1-, β2- and α-adrenoceptor effects, respectively, Schild regression analysis carried out for bupranolol against fenoterol gave pA2 values of 5.80. These results suggest that the relaxant response to fenoterol in the guinea pig taenia caecum is mediated by both the β2- and the β3-adrenoceptors.

Nitric oxide (NO) primarily accounts for endothelium-dependent component of α-adrenoceptor-activated smooth muscle relaxation of mouse aorta in response to isoprenaline
Yurie Akimoto, Takahiro Horinouchi, Mari Shibano, Mayumi Matsushita, Yoko Yamashita, Takao Okamoto, Fumiko Yamaki, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 38, 4-5, 87, 99, 2002年10月
英語, 研究論文（学術雑誌）, Isoprenaline is known to produce vascular relaxation through activation of β-adrenoceptors. In recent years, β-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to β-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae pre-contracted with phenylephrine (3 x 10-7-10-6 M), isoprenaline elicited relaxation in a concentration-dependent fashion (10-9-10-5 M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40-50% of the response obtained in endothelium-intact preparations. In the preparations treated with NG-nitro-L-arginine methyl ester (L-NAME, 3 x 10-4 M
an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ, 10-5 M
a soluble guanylyl cyclase inhibitor), isoprenaline-elicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 x 10-6 M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 x 10-6 M)-inhibitable β-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited relaxant response of mouse aorta.

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.
Kazuoki Otsuka, Hikaru Tanaka, Takahiro Horinouchi, Katsuo Koike, Koki Shigenobu, Yoshio Tanaka
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi, 38, 4-5, 117, 29, 2002年10月, ［国内誌］
英語, 研究論文（学術雑誌）, We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP > CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

The β₂- and β₃-adrenoceptor-mediated relaxation induced by fenoterol in guinea pig taenia caucum.
Yurie Akimoto, Takahiro Horinouchi, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 38, 4-5, 145, 51, 2002年10月, ［国内誌］
英語, 研究論文（学術雑誌）, Fenoterol, a beta2-adrenoceptor selective agonist, belongs to the arylethanolamine class. To understand the receptor subtypes responsible for beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum, we investigated the effect of fenoterol. Fenoterol caused concentration-dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration-response curve for fenoterol. Schild regression analyses carried out for propranolol, butoxamine and bupranolol against fenoterol gave pA2 values of 8.41, 6.33 and 8.44, respectively. However, in the presence of 3 x 10(-4) M atenolol, 10(-4) M butoxamine and 10(-6) M phentolamine to block the beta1-, beta2- and a-adrenoceptor effects, respectively, Schild regression analysis carried out for bupranolol against fenoterol gave pA2 values of 5.80. These results suggest that the relaxant response to fenoterol in the guinea pig taenia caecum is mediated by both the beta2- and the beta3-adrenoceptors.

Nitric oxide (NO) primarily accounts for endothelium-dependent component of β-adrenoceptor-activated smooth muscle relaxation of mouse aorta in response to isoprenaline.
Yurie Akimoto, Takahiro Horinouchi, Mari Shibano, Mayumi Matsushita, Yoko Yamashita, Takao Okamoto, Fumiko Yamaki, Yoshio Tanaka, Katsuo Koike
Journal of Smooth Muscle Research, 38, 4-5, 87, 99, 2002年10月, ［国内誌］
英語, 研究論文（学術雑誌）, Isoprenaline is known to produce vascular relaxation through activation of beta-adrenoceptors. In recent years, beta-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to beta-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae pre-contracted with phenylephrine (3 x 10(-7)-10(-6) M), isoprenaline elicited relaxation in a concentration-dependent fashion (10(-9)-10(-5) M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40-50% of the response obtained in endothelium-intact preparations. In the preparations treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M; an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10(-5) M; a soluble guanylyl cyclase inhibitor), isoprenaline-elicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 x 10(-6) M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 x 10(-6) M)-inhibitable beta-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited relaxant response of mouse aorta.

Functional contribution of voltage-dependent and Ca2+-activated K+ (BKCa) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides
Kazuoki Otsuka, Hikaru Tanaka, Takahiro Horinouchi, Katsuo Koike, Koki Shigenobu, Yoshio Tanaka
Journal of Smooth Muscle Research, 38, 4-5, 117, 129, 2002年10月
英語, 研究論文（学術雑誌）, We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated K+ (BKCa) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP
brain natriuretic peptide, BNP
C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP ≥ BNP >
>
CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10-7 M), a selective BKCa channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation
when estimated at 10-7 M of each peptide, the apparent extent of BKCa channel contribution to the total relaxant response was ≈ 60% for CNP >
≈ 20% for either ANP or BNP. Supporting the substantial role of BKCa channels in the vascular responses, high-KC1 (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10-7 M) and high-KC1 (80 mM), which indicates the key role of cyclic GMP in the BKCa channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BKCa channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K+ channel may function as a primary relaxant mediator in this conduit artery.

2-aminoethoxydiphenyl borate causes dissociation between membrane electrical and mechanical activity in guinea-pig urinary bladder smooth muscle
T Imai, Y Tanaka, T Okamoto, T Horinouchi, H Tanaka, K Koike, K Shigenobu
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 366, 3, 282, 285, SPRINGER-VERLAG, 2002年09月
英語, 研究論文（学術雑誌）, Physiological functions of urinary bladder profoundly reflect smooth muscle mechanical activity. Urinary bladder smooth muscle itself produces myogenic rhythmic contraction, and this spontaneous mechanical event could be the fundamental determinant of urinary bladder functions. The spontaneous contraction of urinary bladder smooth muscle is thought to be triggered primarily by the action potential generated in this smooth muscle cell. Modulators of ion channels contributing to the configuration of action potential also affect urinary bladder smooth muscle mechanical activity as expected exactly from the effects on the electrical event. In the present study, we show that the frequency of action potential recorded in intact strip of guinea-pig urinary bladder smooth muscle is dramatically increased by 2-aminoethoxydiphenyl borate (2-APB; 30 muM) from 0.2 Hz to 1 Hz (similar to500% increments). In contrast to an increasing effect expected from the membrane electrical alterations, mechanical activity (both contraction amplitude and frequency) of this smooth muscle is unexpectedly reduced by the same concentration of 2-APB to similar to35% of the control. The present results firstly show an apparent dissociation of electrical-mechanical coupling in urinary bladder smooth muscle. The alteration of membrane electrical activity might not be the exclusive trigger mechanism responsible for the generation of spontaneous rhythmic contraction of this smooth muscle.

Evidence that action potential generation is not the exclusive determinant to trigger spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle
T Imai, Y Tanaka, T Okamoto, Y Yamamoto, T Horinouchi, H Tanaka, K Koike, K Shigenobu
ACTA PHYSIOLOGICA SCANDINAVICA, 176, 1, 57, 63, BLACKWELL PUBLISHING LTD, 2002年09月
英語, 研究論文（学術雑誌）, Urinary bladder smooth muscle (UBSM) exhibits spontaneous contraction. This spontaneous mechanical activity is myogenic and can be closely related to the UBSM cell action potential to facilitate Ca2+ influx through voltage-gated Ca2+ channels. In the present study, to know whether this membrane electrical event is the exclusive mechanism to trigger spontaneous smooth muscle contraction, we compared the inhibitory effects of Ca2+ channel blockers on the spontaneous action potential and mechanical activity in the isolated guinea-pig UBSM. Both action potential and rhythmic contraction were generated spontaneously in the presence of atropine (1 mu M), phentolamine (1 mu M), propranolol (1 mu M), suramin (10 mu M) and tetrodotoxin (1 mu M), which suggest that both phenomena were myogenic in origin. Nisoldipine (100 nM) and diltiazem (10 mu M) completely eliminated the generation of action potential whereas its frequency was dramatically increased by a dihydropyridine Ca2+ agonist, BayK 8644 (1 mu M). In contrast to disappearance of action potential in the presence of Ca2+ channel blockers, spontaneous contraction of UBSM was inhibited only partly by nisoldipine or diltiazem and most of the mechanical components persisted in these channel blockers. These results indicate that spontaneous action potential in UBSM cell is generated through the activation of L-type voltage-gated Ca2+ channels. The subsequent elevation of intracellular Ca2+ concentrations during a burst of action potentials can be partly responsible for the induction of UBSM mechanical activity. In addition, the present study provides evidence that UBSM spontaneous mechanical activity is also attributable to the mechanism(s) other than the generation of Ca2+ spike.

2-Aminoethoxydiphenyl borate causes dissociation between membrane electrical and mechanical activity in guinea-pig urinary bladder smooth muscle.
Toshiyasu Imai, Yoshio Tanaka, Takao Okamoto, Takahiro Horinouchi, Hikaru Tanaka, Katsuo Koike, Koki Shigenobu
Naunyn-Schmiedeberg's archives of pharmacology, 366, 3, 282, 5, SPRINGER-VERLAG, 2002年09月, ［国際誌］
英語, 研究論文（学術雑誌）, Physiological functions of urinary bladder profoundly reflect smooth muscle mechanical activity. Urinary bladder smooth muscle itself produces myogenic rhythmic contraction, and this spontaneous mechanical event could be the fundamental determinant of urinary bladder functions. The spontaneous contraction of urinary bladder smooth muscle is thought to be triggered primarily by the action potential generated in this smooth muscle cell. Modulators of ion channels contributing to the configuration of action potential also affect urinary bladder smooth muscle mechanical activity as expected exactly from the effects on the electrical event. In the present study, we show that the frequency of action potential recorded in intact strip of guinea-pig urinary bladder smooth muscle is dramatically increased by 2-aminoethoxydiphenyl borate (2-APB; 30 microM) from 0.2 Hz to 1 Hz (approximately 500% increments). In contrast to an increasing effect expected from the membrane electrical alterations, mechanical activity (both contraction amplitude and frequency) of this smooth muscle is unexpectedly reduced by the same concentration of 2-APB to approximately 35% of the control. The present results firstly show an apparent dissociation of electrical-mechanical coupling in urinary bladder smooth muscle. The alteration of membrane electrical activity might not be the exclusive trigger mechanism responsible for the generation of spontaneous rhythmic contraction of this smooth muscle.

Evidence that action potential generation is not the exclusive determinant to trigger spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle
T Imai, Y Tanaka, T Okamoto, Y Yamamoto, T Horinouchi, H Tanaka, K Koike, K Shigenobu
ACTA PHYSIOLOGICA SCANDINAVICA, 176, 1, 57, 63, BLACKWELL PUBLISHING LTD, 2002年09月
英語, 研究論文（学術雑誌）, Urinary bladder smooth muscle (UBSM) exhibits spontaneous contraction. This spontaneous mechanical activity is myogenic and can be closely related to the UBSM cell action potential to facilitate Ca2+ influx through voltage-gated Ca2+ channels. In the present study, to know whether this membrane electrical event is the exclusive mechanism to trigger spontaneous smooth muscle contraction, we compared the inhibitory effects of Ca2+ channel blockers on the spontaneous action potential and mechanical activity in the isolated guinea-pig UBSM. Both action potential and rhythmic contraction were generated spontaneously in the presence of atropine (1 mu M), phentolamine (1 mu M), propranolol (1 mu M), suramin (10 mu M) and tetrodotoxin (1 mu M), which suggest that both phenomena were myogenic in origin. Nisoldipine (100 nM) and diltiazem (10 mu M) completely eliminated the generation of action potential whereas its frequency was dramatically increased by a dihydropyridine Ca2+ agonist, BayK 8644 (1 mu M). In contrast to disappearance of action potential in the presence of Ca2+ channel blockers, spontaneous contraction of UBSM was inhibited only partly by nisoldipine or diltiazem and most of the mechanical components persisted in these channel blockers. These results indicate that spontaneous action potential in UBSM cell is generated through the activation of L-type voltage-gated Ca2+ channels. The subsequent elevation of intracellular Ca2+ concentrations during a burst of action potentials can be partly responsible for the induction of UBSM mechanical activity. In addition, the present study provides evidence that UBSM spontaneous mechanical activity is also attributable to the mechanism(s) other than the generation of Ca2+ spike.

Cyclic AMP-independent relaxation mediated by beta3-adrenoceptors on guinea pig gastrointestine.
Takahiro Horinouchi, Katsuo Koike
European journal of pharmacology, 442, 1-2, 137, 46, 2002年05月03日, ［国際誌］
英語, 研究論文（学術雑誌）, In this study, we investigated the signal transduction pathway involved in beta(3)-adrenoceptor-mediated relaxations of guinea pig gastric fundus and duodenum. In the presence of beta1- and beta2-adrenoceptor blockade, the potency (pD2 value) of catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and beta(3)-adrenoceptor agonists ((R*, R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) to induce relaxation was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22,536, 100 microM). Catecholamines induced an elevation of cyclic AMP and SQ-22,536 significantly abolished the responses of gastric fundus. However, cyclic AMP levels were unaltered by the beta3-adrenoceptor agonists in gastric fundus and by the five agonists in duodenum. Furthermore, the relaxant responses to catecholamines and to beta3-adrenoceptor agonists were unaffected by the cyclic AMP-dependent protein kinase inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89, 10 microM) in gastric fundus. These results suggest that beta3-adrenoceptor-induced relaxation is mediated through both cyclic AMP-dependent and cyclic AMP-independent pathways in gastric fundus and through a cyclic AMP-independent pathway in duodenum.

Cyclic AMP-independent relaxation mediated by β₃-adrenoceptors on guinea pig gastrointestine.
T Horinouchi, K Koike
European Journal of Pharmacology, 442, 1-2, 137, 146, ELSEVIER SCIENCE BV, 2002年05月
英語, 研究論文（学術雑誌）, In this study we investigated the signal transduction pathway involved in beta(3)-adrenoceptor-mediated relaxations of guinea pig gastric fundus and duodenum. In the presence of beta(1)- and beta(2)-adrenoceptor blockade, the potency (pD(2) value) of catecholamines (( - )-isoprenaline, ( - )-noradrenaline and ( - )-adrenaline) and beta(3)-adrenoceptor agonists ((R*, R*)-( +/- )-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and ( +/- )-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxyl-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride (( +/- )-CGP12177A)) to induce relaxation was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22,536, 100 muM). Catecholamines induced an elevation of cyclic AMP and SQ-22,536 significantly abolished the responses of gastric fundus. However, cyclic AMP levels were unaltered by the,adrenoceptor agonists in gastric fundus and by the five agonists in duodenum. Furthermore, the relaxant responses to catecholamines and to beta(3)-adrenoceptor agonists were unaffected by the cyclic AMP-dependent protein kinase inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89, 10 muM) in gastric fundus. These results suggest that beta(3)-adrenoceptor-induced relaxation is mediated through both cyclic AMP-dependent and cyclic AMP-independent pathways in gastric fundus and through a cyclic AMP-independent pathway in duodenum. (C) 2002 Elsevier Science B.V All rights reserved.

Pharmacological characterization and novel signaling pathways of β3-adrenoceptors in guinea pig gastric fundus.　　　　　　　　　　　　　　　
Current Topics in Pharmacology, Vol. 6, P1-P24, 2002年

Partial agonistic activity of labetalol on beta(3)-adrenoceptors in the guinea pig gastric fundus
T Horinouchi, S Asai, M Fukushima, K Koike
JAPANESE JOURNAL OF PHARMACOLOGY, 88, 252P, 252P, JAPANESE PHARMACOLOGICAL SOC, 2002年
英語

Pharmacological characterization and novel signaling pathways of β₃-adrenoceptors in guinea pig gastric fundus.　　　　　　　　　　　　　　　
Current Topics in Pharmacology, Vol. 6, P1-P24, 2002年

Partial agonistic activity of labetalol, the arylethanolamine, on β₃-adrenoceptors in the guinea pig gastric fundus.
T Horinouchi, S Asai, M Fukushima, K Koike
Autonomic and Autacoid Pharmacology, 88, 252P, 252P, JAPANESE PHARMACOLOGICAL SOC, 2002年
英語

ウサギ瞳孔括約筋におけるβ－アドレナリン受容体の立体選択性に関する研究.　　　　　　　　　　　　　　　
新しい眼科, Vol. 19, P99-P105, 2002年

Pharmacological characterization and novel signaling pathways of beta3-adrenoceptors in guinea pig gastric fundus.　　　　　　　　　　　　　　　
Horinouchi, T, Koike, K
Curr. Top. Pharmacol., 6, 1, 24, 2002年, ［査読有り］, ［招待有り］
英語, 研究論文（学術雑誌）

MaxiK channel-mediated relaxation of guinea-pig aorta following stimulation of IP receptor with beraprost via cyclic AMP-dependent and -independent mechanisms
F Yamaki, M Kaga, T Horinouchi, H Tanaka, K Koike, K Shigenobu, L Toro, Y Tanaka
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 364, 6, 538, 550, SPRINGER-VERLAG, 2001年12月
英語, 研究論文（学術雑誌）, The present study was aimed to elucidate the cellular pathway(s) controlling vascular relaxation triggered by stimulation of prostaglandin I-2 (PGI(2), IP) receptor with a stable PGI(2) analog, beraprost. Beraprost caused a concentration-dependent relaxation in de-endothelialized guinea-pig aorta contracted with prostaglandin F-2 alpha (PGF(2 alpha)). Beraprost-induced relaxation was almost abolished in high-KCI-contracted tissue, indicating a major role of K+ conductances. In contrast to other PGI(2) analogs (e.g. cicaprost and iloprost), beraprost-induced relaxation was practically abolished by a selective voltage and Ca2+-activated K+ (MaxiK, BK) channel blocker Iberiotoxin (10(-7) M) or by tetraethylammonium (2 X 10(-3) M). The relaxation induced by beraprost was not significantly affected by other K+ channel blockers glibenclamide (10(-6) M) or Ba2+ (10(-5) M), but was slightly attenuated by 4-aminopyridine (10(-4) M). Beraprost increased intracellular cyclic AMP levels, suggesting a role for cyclic AMP-dependent pathways. A selective inhibitor of cyclic AMP-specific phosphodiesterase, RO-20-1724 (10(-4) M), significantly potentiated beraprost-induced relaxation. Iberiotoxin (10(-7) M) completely counteracted this potentiation. Moreover, tension decrement due to forskolin (3 x 10(-7) M) or 8-bromo-cyclic AMP (10(-2) M) was thoroughly restored by Iberiotoxin (10(-7) M), confirming a role for a cyclic AMP-dependent mechanism. However, SQ 22,536 (10(-4) M), an adenylyl cyclase inhibitor, did not affect beraprost-induced relaxation though it almost totally inhibited the elevation of cyclic AMP contents induced by beraprost, suggesting the existence of an additional mechanism that is cyclic AMP-independent. Moreover, cholera toxin (CTX, 1 mug/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (G(s)), significantly suppressed PGF(2 alpha)-induced contraction both in the absence and presence of SQ 22,536 (10(-4) M). Iberiotoxin (10(-7) M) was also capable of restoring the relaxation induced by CTX. These findings suggest that MaxiK channel plays a primary role in mediating smooth muscle relaxation following stimulation of IP receptor with beraprost in guinea-pig aorta. Both cyclic AMP-dependent and -independent pathways contribute to the MaxiK channel-mediated relaxation following IP receptor stimulation in this vascular tissue. Direct regulation of MaxiK channels by G(s) may partly account for the cyclic AMP-independent relaxant mechanism.

Further characterization of β₃-adrenoceptors in the guinea pig gastric fundus: Stereoselectivity, β-adrenoceptor alkylating and structure-activity relationship.
T Horinouchi, K Koike
Canadian Journal of Physiology and Pharmacology, 79, 12, 985, 995, NATL RESEARCH COUNCIL CANADA, 2001年12月
英語, 研究論文（学術雑誌）, The stereoselectivity of beta (3)-adrenoceptors, the effect of a beta -adrenoceptor alkylating agent, and the structure-activity relationship at beta (3)-adrenoceptors were investigated on the guinea pig gastric fundus. Isomeric activity ratios ((+)/(-)) for isomers of isoprenaline and noradrenaline were 20.9-fold and 43.7-fold, respectively, and were less than those obtained for activation of beta (1)- and beta (2)-adrenoceptors in the guinea pig atria and trachea, respectively. The concentration-response curves to the catecholamines ((-)-isoprenaline, (-)-noradrenaline, and (-)-adrenaline), the selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium), and the nonconventional partial beta(3)-adrenoceptor agonist (+/-)-CGP12177A ((+/-)-[4-[3-[ (1,1-dimethylethyl) amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) were resistant to blockade by (+/-)-pindobind (10 muM), the beta -adrenoceptor alkylating agent. Furthermore, (+/-)-nadolol, which belongs to the aryloxy propanolamine class and has beta (1)- and beta (2)-adrenoceptor antagonistic characteristics, displays agonistic activity at beta (3)-adrenoceptors. These results indicate that pharmacological characteristics of the beta (3)-adrenoceptors of guinea pig gastric fundus differ from those of beta (1)- and beta (2)-adrenoceptors. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. In addition, dobutamine was 22-fold more potent than dopamine. These results suggest that the 4-hydroxyl group at the catechol ring and the beta -hydroxyl group and the large moiety on the alkylamine chain characterized efficacy at beta (3)-adrenoceptors.

MaxiK channel-mediated relaxation of guinea-pig aorta following stimulation of IP receptor with beraprost via cyclic AMP-dependent and -independent mechanisms
F Yamaki, M Kaga, T Horinouchi, H Tanaka, K Koike, K Shigenobu, L Toro, Y Tanaka
NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 364, 6, 538, 550, SPRINGER-VERLAG, 2001年12月
英語, 研究論文（学術雑誌）, The present study was aimed to elucidate the cellular pathway(s) controlling vascular relaxation triggered by stimulation of prostaglandin I-2 (PGI(2), IP) receptor with a stable PGI(2) analog, beraprost. Beraprost caused a concentration-dependent relaxation in de-endothelialized guinea-pig aorta contracted with prostaglandin F-2 alpha (PGF(2 alpha)). Beraprost-induced relaxation was almost abolished in high-KCI-contracted tissue, indicating a major role of K+ conductances. In contrast to other PGI(2) analogs (e.g. cicaprost and iloprost), beraprost-induced relaxation was practically abolished by a selective voltage and Ca2+-activated K+ (MaxiK, BK) channel blocker Iberiotoxin (10(-7) M) or by tetraethylammonium (2 X 10(-3) M). The relaxation induced by beraprost was not significantly affected by other K+ channel blockers glibenclamide (10(-6) M) or Ba2+ (10(-5) M), but was slightly attenuated by 4-aminopyridine (10(-4) M). Beraprost increased intracellular cyclic AMP levels, suggesting a role for cyclic AMP-dependent pathways. A selective inhibitor of cyclic AMP-specific phosphodiesterase, RO-20-1724 (10(-4) M), significantly potentiated beraprost-induced relaxation. Iberiotoxin (10(-7) M) completely counteracted this potentiation. Moreover, tension decrement due to forskolin (3 x 10(-7) M) or 8-bromo-cyclic AMP (10(-2) M) was thoroughly restored by Iberiotoxin (10(-7) M), confirming a role for a cyclic AMP-dependent mechanism. However, SQ 22,536 (10(-4) M), an adenylyl cyclase inhibitor, did not affect beraprost-induced relaxation though it almost totally inhibited the elevation of cyclic AMP contents induced by beraprost, suggesting the existence of an additional mechanism that is cyclic AMP-independent. Moreover, cholera toxin (CTX, 1 mug/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (G(s)), significantly suppressed PGF(2 alpha)-induced contraction both in the absence and presence of SQ 22,536 (10(-4) M). Iberiotoxin (10(-7) M) was also capable of restoring the relaxation induced by CTX. These findings suggest that MaxiK channel plays a primary role in mediating smooth muscle relaxation following stimulation of IP receptor with beraprost in guinea-pig aorta. Both cyclic AMP-dependent and -independent pathways contribute to the MaxiK channel-mediated relaxation following IP receptor stimulation in this vascular tissue. Direct regulation of MaxiK channels by G(s) may partly account for the cyclic AMP-independent relaxant mechanism.

Functional properties of atypical β-adrenoceptors on the guinea pig duodenum
Takahiro Horinouchi, Katsuo Koike
European Journal of Pharmacology, 416, 1-2, 153, 163, 2001年03月23日
英語, 研究論文（学術雑誌）, In this study, we attempted to further characterize atypical β-adrenoceptors on the guinea pig duodenum. (-)-Enantiomers of isoprenaline and noradrenaline were more potent than its (+)-enantiomers. The isomeric activity ratios ((+)/(-)) were less than those obtained in the guinea pig atria and trachea. The concentration-response curves to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), to the selective β3-adrenoceptor agonist, BRL37344 ((R*, R*)-(±)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino] propyl]phenoxyacetic acid sodium), and to the non-conventional partial β3-adrenoceptor agonist, (±)-CGP12177A ((±)-[4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3- dihydro-2H-benzimidazol-2-one] hydrochloride), were resistant to blockade by (±)-pindobind, the β-adrenoceptor alkylating agent. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. Selective β2-adrenoceptor agonists possess agonistic activities at atypical β-adrenoceptors. (±)-Propranolol and (±)-bupranolol had no agonistic effect, whereas (±)-alprenolol, (±)-pindolol, (±)-nadolol, (±)-CGP12177A and (±)-carteolol exhibited agonistic activities at atypical β-adrenoceptors. These results suggest that pharmacological properties of atypical β-adrenoceptors differ from those of conventional β1- and β2-adrenoceptors on the guinea pig. © 2001 Published by Elsevier Science B.V.

Agonistic activity of SR59230A at atypical beta-adrenoceptors in guinea pig gastric fundus and duodenum
T Horinouchi, K Koike
EUROPEAN JOURNAL OF PHARMACOLOGY, 416, 1-2, 165, 168, ELSEVIER SCIENCE BV, 2001年03月
英語, 研究論文（学術雑誌）, We have recently suggested that atypical P-adrenoceptors are present in guinea pig gastric fundus and duodenum. In the present study, we have shown that SR59230A (3-(2-ethylphenoxy)-1-[ (1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate), a selective beta (3)-adrenoceptor antagonist, possesses agonistic activities at atypical beta -adrenoceptors in these tissues. SR59230A caused concentration-dependent relaxations. However, (+/-)-propranolol(1 muM) did not affect SR59230A-induced relaxations. Pretreatment of with a combination of (+/-)-propranolol (1 muM) and the non-selective beta (1)-, beta (2)-, beta (3)- and beta (4)-adrenoceptor antagonist, (+/-)-bupranolol (30 muM), significantly antagonized the relaxant effects induced by SR59230A. The results clearly indicate that SR59230A acts as an atypical beta -adrenoceptor agonist on guinea pig gastric fundus and duodenum. (C) 2001 Elsevier Science B.V. All rights reserved.

Functional properties of atypical β-adrenoceptors on the guinea pig duodenum.
T Horinouchi, K Koike
European Journal of Pharmacology, 416, 1-2, 153, 163, ELSEVIER SCIENCE BV, 2001年03月
英語, 研究論文（学術雑誌）, In this study, we attempted to further characterize atypical beta -adrenoceptors on the guinea pig duodenum. (-)-Enantiomers of isoprenaline and noradrenaline were more potent than its(+)-enantiomers. The isomeric activity ratios ((+)/(-)) were less than those obtained in the guinea pig atria and trachea. The concentration-response curves to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), to the selective beta (3)-adrenoceptor agonist, BRL37344 ((R*, R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxy-ethyl)amino]propyl]phenoxyacetic acid sodium), and to the non-conventional partial beta (3)-adrenoceptor agonist, (+/-)-CGP12177A ((+/-)-[4-[3-[ (1,1 -dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride), were resistant to blockade by (+/-)-pindobind, the beta -adrenoceptor alkylating agent. (-)-Noradrenaline and(-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. Selective beta (2)-ladrenoceptor agonists possess agonistic activities at atypical beta -adrenoceptors. (+/-)-Propranolol and (+/-)-bupranolol had no agonistic effect, whereas (+/-)-alprenolol, (+/-)-pindolol, (+/-)-nadolol, (+/-)-CGP12177A and (+/-)-carteolol exhibited agonistic activities at atypical beta -adrenoceptors. These results suggest that pharmacological properties of atypical beta -adrenoceptors differ from those of conventional beta (1)- and beta (2)-adrenoceptors on the guinea pig. (C) 2001 Published by Elsevier Science B.V.

Agonistic activity of SR59230A at atypical β-adrenoceptors in guinea pig gastric fundus and duodenum.
T Horinouchi, K Koike
European Journal of Pharmacology, 416, 1-2, 165, 168, ELSEVIER SCIENCE BV, 2001年03月
英語, 研究論文（学術雑誌）, We have recently suggested that atypical P-adrenoceptors are present in guinea pig gastric fundus and duodenum. In the present study, we have shown that SR59230A (3-(2-ethylphenoxy)-1-[ (1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate), a selective beta (3)-adrenoceptor antagonist, possesses agonistic activities at atypical beta -adrenoceptors in these tissues. SR59230A caused concentration-dependent relaxations. However, (+/-)-propranolol(1 muM) did not affect SR59230A-induced relaxations. Pretreatment of with a combination of (+/-)-propranolol (1 muM) and the non-selective beta (1)-, beta (2)-, beta (3)- and beta (4)-adrenoceptor antagonist, (+/-)-bupranolol (30 muM), significantly antagonized the relaxant effects induced by SR59230A. The results clearly indicate that SR59230A acts as an atypical beta -adrenoceptor agonist on guinea pig gastric fundus and duodenum. (C) 2001 Elsevier Science B.V. All rights reserved.

Further characterization of β3-adrenoceptors in the guinea pig gastric fundus: Stereoselectivity, β-adrenoceptor alkylation, and structure-activity relationship
Takahiro Horinouchi, Katsuo Koike
Canadian Journal of Physiology and Pharmacology, 79, 12, 985, 995, 2001年
英語, 研究論文（学術雑誌）, The stereoselectivity of β3-adrenoceptors, the effect of a β-adrenoceptor alkylating agent, and the structure- activity relationship at β3-adrenoceptors were investigated on the guinea pig gastric fundus. Isomeric activity ratios ((+)/(-)) for isomers of isoprenaline and noradrenaline were 20.9-fold and 43.7-fold, respectively, and were less than those obtained for activation of β1- and β2-adrenoceptors in the guinea pig atria and trachea, respectively. The concentration-response curves to the catecholamines ((-)-isoprenaline, (-)-noradrenaline, and (-)-adrenaline), the selective β3-adrenoceptor agonist BRL37344 ((R*,R*)-(±)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl) amino]propyl]phenoxyacetic acid sodium), and the nonconventional partial β3-adrenoceptor agonist (±)-CGP12177A ((±)-[4-[3-[ (1,1-dimethylethyl) amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) were resistant to blockade by (±)-pindobind (10 μM), the β-adrenoceptor alkylating agent. Furthermore, (±)-nadolol, which belongs to the aryloxy- propanolamine class and has β1- and β2-adrenoceptor antagonistic characteristics, displays agonistic activity at β3-adrenoceptors. These results indicate that pharmacological characteristics of the β3-adrenoceptors of guinea pig gastric fundus differ from those of β1- and β2-adrenoceptors. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. In addition, dobutamine was 22-fold more potent than dopamine. These results suggest that the 4-hydroxyl group at the catechol ring and the β-hydroxyl group and the large moiety on the alkylamine chain characterized efficacy at β3-adrenoceptors.

Effect of SR59230A on atypical beta-adrenoceptor mediating relaxation in the guinea pig gastric fundus
T Horinouchi, Y Yamamoto, K Koike
PHARMACOLOGY, 62, 2, 98, 102, KARGER, 2001年
英語, 研究論文（学術雑誌）, The purpose of the present study was to clarify whether atypical beta -adrenoceptors which presented in the guinea pig gastric fundus are beta (3)-adrenoceptors or putative beta (4)-adrenoceptors, In the presence of both the selective beta (1)-adrenoceptor antagonist atenolol (10(-4) mol/l) and the selective beta (2)-adrenoceptor antagonist butoxamine (10(-4) mol/l), the selective beta (3)-adrenoceptor antagonist SR59230A caused a concentration-dependent rightward shift of the concentration-response curve to catecholamines (isoprenaline, noradrenaline and adrenaline) and beta (3)-adrenoceptor agonists (BRL37344 and CGP12177A) in the guinea pig gastric fundus, Schild plot analyses of SR59230A against these agonists gave pA(2) values of 7.35 +/- 0.03 (isoprenaline), 7.26 +/- 0.04 (noradrenaline), 7.26 +/- 0.05 (adrenaline), 7.79 +/- 0.03 (BRL37344) and 6.74 +/- 0.03 (CGP12177A), respectively, and all Schild slopes were not significantly different from unity, These results suggest that atypical beta -adrenoceptors mediating relaxant responses of these agonists in the guinea pig gastric fundus are beta (3)-adrenoceptors rather than putative beta (4)-adrenoceptors.

Partial agonistic properties of (+/-)-pindolol at atypical beta-adrenoceptors in the guinea pig gastric fundus
T Horinouchi, K Koike
PHARMACOLOGY, 63, 4, 197, 202, KARGER, 2001年
英語, 研究論文（学術雑誌）, (+/-)-Pindolol ([1-(1H-indol-4-yloxy)-3-[ (1-methylethyl)amino]-2-propanol)]) is a partial agonist at atypical beta -adrenoceptors in the guinea pig gastric fundus. (+/-)-Pindolol induced concentration-dependent relaxation in this tissue. However, the relaxant responses of (+/-)-pindolol were not antagonized by a combination of the selective beta (1)-adrenoceptor antagonist atenolol (10(-4) mol/l) and the selective beta (2)-adrenoceptor antagonist butoxamine (10(-4) mol/l). In the presence of both atenolol and butoxamine, the nonselective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist (+/-)-bupranolol (10(-5)-10(-4) Mol/l) caused a concentration-dependent rightward shift of the concentration-response curves for (+/-)-pindolol. Schild plot analyses of (+/-)-bupranolol against (+/-)-pindolol gave the pA(2) value of 5.46 +/- 0.03 and Schild slope was not significantly different from unity. Furthermore, (+/-)-pindolol (10(-5) mol/l weakly but significantly antagonized the relaxant responses to catecholamines ((-)isoprenaline, (-)-nor-adrenaline and H-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium salt) and a nonconventional partial beta (3)-adrenoceptor agonist (+/-)-CGP12177A ([4-[3-[ (1,1 -dimethyl-ethyl) amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one] hydrochloride). These results suggest that (+/-)-pindolol acts as a partial agonist at atypical beta -adrenoceptors in the guinea pig gastric fundus. Copyright (C) 2001 S. Karger AG, Basel.

(+/-)-pindolol acts as a partial agonist at atypical beta-adrenoceptors in the guinea pig duodenum
T Horinouchi, K Koike
JAPANESE JOURNAL OF PHARMACOLOGY, 85, 1, 35, 40, JAPANESE PHARMACOLOGICAL SOC, 2001年01月
英語, 研究論文（学術雑誌）, The agonistic and antagonistic effects of(+/-)-pindolol(1-( 1H-indol-4-yloxy)-3- [ (1-methylethyl)amino]-2-propanol) were estimated to clarify whether (+/-)-pindolol acts as a partial agonist on atypical beta -adrenoceptors in the guinea pig duodenum. (+/-)-Pindolol induced concentration-dependent relaxation with a pD(2) value of 5.10 +/- 0.03 and an intrinsic activity of 0.83 +/- 0.03. However, the relaxations to (+/-)-pindolol were not antagonized by the non-selective beta (1)- and beta (2)-adrenoceptor antagonist (+/-)-propranolol (1 muM). In the presence of (+/-)-propranolol (1 muM), the non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist (+/-)-bupranolol (30 muM) induced a rightward shift of the concentration-response curves for (+/-)-pindolol (apparent pA(2) = 5.41 +/- 0.06). In the presence of (+/-)-propranolol, (+/-)-pindolol (10 muM) weakly but significantly antagonized the relaxant effects to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2 amino] propyl]phenoxyacetic acid sodium salt) and a non-conventional partial pg-adrenoceptor agonist (+/-)-CGP12177A([4-[3-[ (1, 1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride). These results demonstrate that (+/-)-pindolol possesses both agonistic and antagonistic effects on atypical beta -adrenoceptors in the guinea pig duodenum.

Structure-activity relationship studies of (±)-terbutaline and (±)-fenoterol on β3-adrenoceptors in the Guinea pig gastric fundus
Takahiro Horinouchi, Yoshiko Nakagawa, Makiko Wakabayashi, Katsuo Koike
Journal of Smooth Muscle Research, 37, 3-4, 105, 112, 2001年
英語, 研究論文（学術雑誌）, (±)-Terbutaline and (±)-fenoterol are both arylethanolamine analogs that have tertbutyl and aryliso-propyl substituents respectively at the α position on the nitrogen of the ethanolamine side chain. In the present study, we have investigated the structure-activity relationships of (±)-terbutaline and (±)-fenoterol as β3-adrenoceptor agonists in the guinea pig gastric fundus. (±)-Terbutaline and (±)-fenoterol induced concentration-dependent relaxation of the precontracted gastric fundus with pD2 values of 4.45 ± 0.10 and 5.90 ± 0.09, and intrinsic activities of 1.00 ± 0.03 and 0.99 ± 0.01 respectively. The combination of the selective β1-adrenoceptor antagonist (±)-atenolol (100 μM), and the selective β2-adrenoceptor antagonist (±)-butoxamine (100 μM), produced a 2 and 6 fold rightward shift of the concentration-response curves for (±)-terbutaline and (±)-fenoterol respectively, without depressing the maximal responses. The order of potency of these agonists was (pD2 value): (±)-fenoterol (5.09 ± 0.10) >
(±)-terbutaline (4.13 ± 0.08). In the presence of (±)-atenolol and (±)-butoxamine, however, the non-selective β1,β2- and β3-adrenoceptor antagonist (±)-bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for (±)-terbutaline and (±)-fenoterol. Schild plot analyses of the effects of (±)-bupranolol against these agonists gave pA2 values of 6.21 ± 0.07 ((±)-terbutaline) and 6.37 ± 0.06 ((±)-fenoterol) respectively, and the slopes of the Schild plot were not significantly different from unity (p>
0.05). These results suggest that the relaxant responses to (±)-terbutaline and (±)-fenoterol: are mainly mediated through β3-adrenoceptors in the guinea pig gastric fundus. The β3-adrenoceptor agonist potencies of arylethanolamine analogs depend on the size of the end of the alkylamine side chain.

Effect of SR59230A on atypical β-adrenoceptor mediating relaxation in the guinea pig gastric fundus.
T Horinouchi, Y Yamamoto, K Koike
Pharmacology, 62, 2, 98, 102, KARGER, 2001年
英語, 研究論文（学術雑誌）, The purpose of the present study was to clarify whether atypical beta -adrenoceptors which presented in the guinea pig gastric fundus are beta (3)-adrenoceptors or putative beta (4)-adrenoceptors, In the presence of both the selective beta (1)-adrenoceptor antagonist atenolol (10(-4) mol/l) and the selective beta (2)-adrenoceptor antagonist butoxamine (10(-4) mol/l), the selective beta (3)-adrenoceptor antagonist SR59230A caused a concentration-dependent rightward shift of the concentration-response curve to catecholamines (isoprenaline, noradrenaline and adrenaline) and beta (3)-adrenoceptor agonists (BRL37344 and CGP12177A) in the guinea pig gastric fundus, Schild plot analyses of SR59230A against these agonists gave pA(2) values of 7.35 +/- 0.03 (isoprenaline), 7.26 +/- 0.04 (noradrenaline), 7.26 +/- 0.05 (adrenaline), 7.79 +/- 0.03 (BRL37344) and 6.74 +/- 0.03 (CGP12177A), respectively, and all Schild slopes were not significantly different from unity, These results suggest that atypical beta -adrenoceptors mediating relaxant responses of these agonists in the guinea pig gastric fundus are beta (3)-adrenoceptors rather than putative beta (4)-adrenoceptors.

Partial agonistic properties of (±)-pindolol at atypical β-adrenoceptors in the guinea pig gastric fundus.
Takahiro Horinouchi, Katsuo Koike
Pharmacology, 63, 4, 197, 202, 2001年
英語, 研究論文（学術雑誌）, (±)-Pindolol ([1-(1H-indol-4-yloxy)-3-[ (1-methylethyl)-amino]-2-propanol)]) is a partial agonist at atypical β-adrenoceptors in the guinea pig gastric fundus. (±)-Pindolol induced concentration-dependent relaxation in this tissue. However, the relaxant responses of (±)-pindolol were not antagonized by a combination of the selective β1-adrenoceptor antagonist atenolol (10-4 mol/l) and the selective β2-adrenoceptor antagonist butoxamine (10-4 mol/l). In the presence of both atenolol and butoxamine, the nonselective β1-, β2- and β3-adrenoceptor antagonist (±)-bupranolol (10-5-10-4 mol/l) caused a concentration-dependent rightward shift of the concentration-response curves for (±)-pindolol. Schild plot analyses of (±)-bupranolol against (±)-pindolol gave the pA2 value of 5.46 ± 0.03 and Schild slope was not significantly different from unity. Furthermore, (±)-pindolol (10-5 mol/l) weakly but significantly antagonized the relaxant responses to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective β3-adrenoceptor agonist BRL37344 ((R*,R*)-(±)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl) amino]propyl]phenoxyacetic acid sodium salt) and a nonconventional partial β3-adrenoceptor agonist (±)-CGP12177A ([4-[3-[ (1,1-dimethyl-ethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H- benzimidazol-2-one] hydrochloride). These results suggest that (±)-pindolol acts as a partial agonist at atypical β-adrenoceptors in the guinea pig gastric fundus. Copyright © 2001 S. Karger AG, Basel.

(±)-Pindolol acts as a partial agonist at atypical β-adrenoceptors in the guinea pig duodenum.
T Horinouchi, K Koike
The Japanese Journal of Pharmacology, 85, 1, 35, 40, JAPANESE PHARMACOLOGICAL SOC, 2001年01月
英語, 研究論文（学術雑誌）, The agonistic and antagonistic effects of(+/-)-pindolol(1-( 1H-indol-4-yloxy)-3- [ (1-methylethyl)amino]-2-propanol) were estimated to clarify whether (+/-)-pindolol acts as a partial agonist on atypical beta -adrenoceptors in the guinea pig duodenum. (+/-)-Pindolol induced concentration-dependent relaxation with a pD(2) value of 5.10 +/- 0.03 and an intrinsic activity of 0.83 +/- 0.03. However, the relaxations to (+/-)-pindolol were not antagonized by the non-selective beta (1)- and beta (2)-adrenoceptor antagonist (+/-)-propranolol (1 muM). In the presence of (+/-)-propranolol (1 muM), the non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist (+/-)-bupranolol (30 muM) induced a rightward shift of the concentration-response curves for (+/-)-pindolol (apparent pA(2) = 5.41 +/- 0.06). In the presence of (+/-)-propranolol, (+/-)-pindolol (10 muM) weakly but significantly antagonized the relaxant effects to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2 amino] propyl]phenoxyacetic acid sodium salt) and a non-conventional partial pg-adrenoceptor agonist (+/-)-CGP12177A([4-[3-[ (1, 1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride). These results demonstrate that (+/-)-pindolol possesses both agonistic and antagonistic effects on atypical beta -adrenoceptors in the guinea pig duodenum.

Structure-activity relationship studies of (±)-terbutaline and (±)-fenoterol on β₃-adrenoceptors in the guinea pig gastric fundus.
Takahiro Horinouchi, Yoshiko Nakagawa, Makiko Wakabayashi, Katsuo Koike
Journal of Smooth Muscle Research, 37, 3-4, 105, 112, 2001年
英語, 研究論文（学術雑誌）, (±)-Terbutaline and (±)-fenoterol are both arylethanolamine analogs that have tertbutyl and aryliso-propyl substituents respectively at the α position on the nitrogen of the ethanolamine side chain. In the present study, we have investigated the structure-activity relationships of (±)-terbutaline and (±)-fenoterol as β3-adrenoceptor agonists in the guinea pig gastric fundus. (±)-Terbutaline and (±)-fenoterol induced concentration-dependent relaxation of the precontracted gastric fundus with pD2 values of 4.45 ± 0.10 and 5.90 ± 0.09, and intrinsic activities of 1.00 ± 0.03 and 0.99 ± 0.01 respectively. The combination of the selective β1-adrenoceptor antagonist (±)-atenolol (100 μM), and the selective β2-adrenoceptor antagonist (±)-butoxamine (100 μM), produced a 2 and 6 fold rightward shift of the concentration-response curves for (±)-terbutaline and (±)-fenoterol respectively, without depressing the maximal responses. The order of potency of these agonists was (pD2 value): (±)-fenoterol (5.09 ± 0.10) >
(±)-terbutaline (4.13 ± 0.08). In the presence of (±)-atenolol and (±)-butoxamine, however, the non-selective β1,β2- and β3-adrenoceptor antagonist (±)-bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for (±)-terbutaline and (±)-fenoterol. Schild plot analyses of the effects of (±)-bupranolol against these agonists gave pA2 values of 6.21 ± 0.07 ((±)-terbutaline) and 6.37 ± 0.06 ((±)-fenoterol) respectively, and the slopes of the Schild plot were not significantly different from unity (p>
0.05). These results suggest that the relaxant responses to (±)-terbutaline and (±)-fenoterol: are mainly mediated through β3-adrenoceptors in the guinea pig gastric fundus. The β3-adrenoceptor agonist potencies of arylethanolamine analogs depend on the size of the end of the alkylamine side chain.

Partial agonistic effects of carteolol on atypical beta-adrenoceptors in the guinea pig gastric fundus
T Horinouchi, K Koike
JAPANESE JOURNAL OF PHARMACOLOGY, 84, 3, 287, 292, JAPANESE PHARMACOLOGICAL SOC, 2000年11月
英語, 研究論文（学術雑誌）, The properties of the beta (1)-/beta (2)-adrenoceptor partial agonist carteolol were investigated in atypical beta -adrenoceptors on the guinea pig gastric fundus. Carteolol induced concentration-dependent relaxation in this tissue (pD(2) = 5.55, intrinsic activity = 0.94). However, a combination of the selective beta (1)-adrenoceptor antagonist atenolol (100 muM) and the selective beta (2)-adrenoceptor antagonist butoxamine (100 muM) produced only small rightward shifts in the concentration-response curves of carteolol in the gastric fundus (pD(2) = 4.91, intrinsic activity = 0.94). In the presence of both atenolol (100 muM) and butoxamine (100 muM), the non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist (+/-)-bupranolol (10 - 100 muM) caused a concentration-dependent right ward shift of the concentration-response curves for carteolol in the guinea pig gastric fundus. Schild plot analyses of the effects of (+/-)-bupranolol against carteolol gave the pA(2) value of 5.29 and the Schild slope was not significantly different from unity. Furthermore, carteolol (10 muM) weakly but significantly antagonized the relaxant responses to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy-acetic acid sodium salt) and a non-conventional partial beta (3)-adrenoceptor agonist (+/-)-CGP12177A ([4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) in the guinea pig gastric fundus. These results suggest that the partial agonistic effects of carteolol are mediated by atypical P-adrenoceptors in the guinea pig gastric fundus.

Partial agonistic effects of carteolol on atypical β-adrenoceptors in the guinea pig gastric fundus.
T Horinouchi, K Koike
The Japanese Journal of Pharmacology, 84, 3, 287, 292, JAPANESE PHARMACOLOGICAL SOC, 2000年11月
英語, 研究論文（学術雑誌）, The properties of the beta (1)-/beta (2)-adrenoceptor partial agonist carteolol were investigated in atypical beta -adrenoceptors on the guinea pig gastric fundus. Carteolol induced concentration-dependent relaxation in this tissue (pD(2) = 5.55, intrinsic activity = 0.94). However, a combination of the selective beta (1)-adrenoceptor antagonist atenolol (100 muM) and the selective beta (2)-adrenoceptor antagonist butoxamine (100 muM) produced only small rightward shifts in the concentration-response curves of carteolol in the gastric fundus (pD(2) = 4.91, intrinsic activity = 0.94). In the presence of both atenolol (100 muM) and butoxamine (100 muM), the non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist (+/-)-bupranolol (10 - 100 muM) caused a concentration-dependent right ward shift of the concentration-response curves for carteolol in the guinea pig gastric fundus. Schild plot analyses of the effects of (+/-)-bupranolol against carteolol gave the pA(2) value of 5.29 and the Schild slope was not significantly different from unity. Furthermore, carteolol (10 muM) weakly but significantly antagonized the relaxant responses to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy-acetic acid sodium salt) and a non-conventional partial beta (3)-adrenoceptor agonist (+/-)-CGP12177A ([4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) in the guinea pig gastric fundus. These results suggest that the partial agonistic effects of carteolol are mediated by atypical P-adrenoceptors in the guinea pig gastric fundus.

Partial agonist activity of carteolol on atypical beta-adrenoceptors in the guinea pig duodenum
T Horinouchi, K Koike
EUROPEAN JOURNAL OF PHARMACOLOGY, 403, 1-2, 133, 138, ELSEVIER SCIENCE BV, 2000年09月
英語, 研究論文（学術雑誌）, The partial agonist activities of carteolol were investigated on atypical beta-adrenoceptors of duodenum on the guinea pig. Carteolol produced a concentration-dependent relaxation of the guinea pig duodenum (pD(2) = 4.85), which was not significantly affected by propranolol (1 mu M). In the presence of propranolol (1 mu M), however, the non-selective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist, bupranolol (30 mu M), caused a rightward shift of the concentration-response curves for carteolol (apparent pA(2) = 5.31). Moreover, carteolol (10 mu M) weakly, but significantly, antagonized the relaxations in response to catecholamines (isoprenaline, noradrenaline and adrenaline), to a selective beta(3)-adrenoceptor agonist, (R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium salt (BRL37344), and to a non-conventional partial beta(3)-adrenoceptor agonist, [4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride (CGP12177A), also in the guinea pig duodenum (apparent pA(2) = 5.77, 5.92, 6.05, 6.56 and 5.58, respectively). These results suggest that the partial agonist effects of carteolol are mediated by atypical beta-adrenoceptors in the guinea pig duodenum. (C) 2000 Elsevier Science B.V. All rights reserved.

Partial agonist activity of carteolol on atypical β-adrenoceptors in the guinea pig duodenum.
T Horinouchi, K Koike
European Journal of Pharmacology, 403, 1-2, 133, 138, ELSEVIER SCIENCE BV, 2000年09月
英語, 研究論文（学術雑誌）, The partial agonist activities of carteolol were investigated on atypical beta-adrenoceptors of duodenum on the guinea pig. Carteolol produced a concentration-dependent relaxation of the guinea pig duodenum (pD(2) = 4.85), which was not significantly affected by propranolol (1 mu M). In the presence of propranolol (1 mu M), however, the non-selective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist, bupranolol (30 mu M), caused a rightward shift of the concentration-response curves for carteolol (apparent pA(2) = 5.31). Moreover, carteolol (10 mu M) weakly, but significantly, antagonized the relaxations in response to catecholamines (isoprenaline, noradrenaline and adrenaline), to a selective beta(3)-adrenoceptor agonist, (R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium salt (BRL37344), and to a non-conventional partial beta(3)-adrenoceptor agonist, [4-[3-[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride (CGP12177A), also in the guinea pig duodenum (apparent pA(2) = 5.77, 5.92, 6.05, 6.56 and 5.58, respectively). These results suggest that the partial agonist effects of carteolol are mediated by atypical beta-adrenoceptors in the guinea pig duodenum. (C) 2000 Elsevier Science B.V. All rights reserved.

Functional identification of beta(3)-adrenoceptors in the guinea-pig ileum using the non-selective beta-adrenoceptor antagonist (+/-)-bupranolol
T Horinouchi, K Koike
JOURNAL OF AUTONOMIC PHARMACOLOGY, 20, 4, 253, 258, BLACKWELL SCIENCE LTD, 2000年08月
英語, 研究論文（学術雑誌）, 1 To clarify whether there is a species difference or a tissue difference in beta (3)-adrenoceptors, the beta (3)-adrenoceptors mediating relaxations to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 and a non-conventional partial beta (3)-adrenoceptor agonist (+/-)-CGP12177A (a potent beta (1)- and beta (2)-adrenoceptor antagonist with a partial beta (3)-adrenoceptor agonist property) were investigated in the guinea-pig ileum.
2 Catecholamines and beta (3)-adrenoceptor agonists induced concentration-dependent relaxations of pre-contracted strips of the guinea-pig ileum. The rank order for their relaxing potency was (-)-isoprenaline (pD(2): 7.60) > BRL37344 (7.05) > (-)-noradrenaline (6.38) > (+/-)-CGP12177A (6.25) > (-)-adrenaline (6.07).
3 In the presence of the non-selective beta (1)- and beta (2)-adrenoceptor antagonist (+/-)-propranolol (1 muM), only small rightward shifts of the concentration-response curves (CRCs) to these agonists were observed and the rank order of potency of agonists was BRL37344 (pD(2): 7.00) > (+/-)-CGP12177A (6.17) > (-)-isoprenaline (6.01) > (-)-noradrenaline (5.69) > (-)-adrenaline (5.41).
4 In the presence of (+/-)-propranolol (1 muM), the additional presence of (+/-)-bupranolol (3-30 muM), a non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist, caused a concentration-dependent rightward shift of the CRCs to catecholamines and beta (3)-adrenoceptor agonists. Schild plot analyses of (+/-)-bupranolol against these agonists gave pA(2) values of 6.02 ((-)-isoprenaline), 6.03 ((-)-noradrenaline), 6.01 ((-)-adrenaline), 6.56 (BRL37344) and 5.74 ((+/-)-CGP12177A), respectively. All Schild plot slopes were not significantly different from unity. The PA(2) values of (+/-)-bupranolol obtained for the guinea-pig beta (3)-adrenoceptors were about one log unit less than the values obtained for the rat beta (3)-adrenoceptors and about two log units less than the values obtained for dog beta (3)-adrenoceptors.
5 These results confirm that functional beta (3)-adrenoceptors are present in the guinea-pig ileum and that the relaxations of these agonists are mainly mediated via beta (3)-adrenoceptors in this tissue. The differential antagonistic potency of (+/-)-bupranolol may suggest that there is a species difference between the three species (guinea-pig, dog and rat) in their beta (3)-adrenoceptors.

Functional identification of β₃-adrenoceptors in the guinea pig ileum using the non-selective β-adrenoceptor antagonist (±)-bupranolol.
T Horinouchi, K Koike
Journal of Autonomic Pharmacology, 20, 4, 253, 258, BLACKWELL SCIENCE LTD, 2000年08月
英語, 研究論文（学術雑誌）, 1 To clarify whether there is a species difference or a tissue difference in beta (3)-adrenoceptors, the beta (3)-adrenoceptors mediating relaxations to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta (3)-adrenoceptor agonist BRL37344 and a non-conventional partial beta (3)-adrenoceptor agonist (+/-)-CGP12177A (a potent beta (1)- and beta (2)-adrenoceptor antagonist with a partial beta (3)-adrenoceptor agonist property) were investigated in the guinea-pig ileum.
2 Catecholamines and beta (3)-adrenoceptor agonists induced concentration-dependent relaxations of pre-contracted strips of the guinea-pig ileum. The rank order for their relaxing potency was (-)-isoprenaline (pD(2): 7.60) > BRL37344 (7.05) > (-)-noradrenaline (6.38) > (+/-)-CGP12177A (6.25) > (-)-adrenaline (6.07).
3 In the presence of the non-selective beta (1)- and beta (2)-adrenoceptor antagonist (+/-)-propranolol (1 muM), only small rightward shifts of the concentration-response curves (CRCs) to these agonists were observed and the rank order of potency of agonists was BRL37344 (pD(2): 7.00) > (+/-)-CGP12177A (6.17) > (-)-isoprenaline (6.01) > (-)-noradrenaline (5.69) > (-)-adrenaline (5.41).
4 In the presence of (+/-)-propranolol (1 muM), the additional presence of (+/-)-bupranolol (3-30 muM), a non-selective beta (1)-, beta (2)- and beta (3)-adrenoceptor antagonist, caused a concentration-dependent rightward shift of the CRCs to catecholamines and beta (3)-adrenoceptor agonists. Schild plot analyses of (+/-)-bupranolol against these agonists gave pA(2) values of 6.02 ((-)-isoprenaline), 6.03 ((-)-noradrenaline), 6.01 ((-)-adrenaline), 6.56 (BRL37344) and 5.74 ((+/-)-CGP12177A), respectively. All Schild plot slopes were not significantly different from unity. The PA(2) values of (+/-)-bupranolol obtained for the guinea-pig beta (3)-adrenoceptors were about one log unit less than the values obtained for the rat beta (3)-adrenoceptors and about two log units less than the values obtained for dog beta (3)-adrenoceptors.
5 These results confirm that functional beta (3)-adrenoceptors are present in the guinea-pig ileum and that the relaxations of these agonists are mainly mediated via beta (3)-adrenoceptors in this tissue. The differential antagonistic potency of (+/-)-bupranolol may suggest that there is a species difference between the three species (guinea-pig, dog and rat) in their beta (3)-adrenoceptors.

Further evidence that (±)-carteolol-induced relaxation is mediated by β 2-adrenoceptors but not by β 3-adrenoceptors in the guinea pig taenia caecum
Takahiro Horinouchi, Katsuo Koike
Journal of Smooth Muscle Research, 36, 5, 145, 153, 2000年
英語, 研究論文（学術雑誌）, The properties of the β 1- and β 2-adrenoceptor partial agonist (±)-carteolol were investigated against the β 2- and β 3-adrenoceptors of the taenia caecum of the guinea pig. (-)-Isoprenaline and (±)-carteolol induced concentration-dependent relaxation in this tissue. The non-selective β 1- and β 2-adrenoceptor antagonist (±)-propranolol (10-100 nM), the selective β 2-adrenoceptor antagonist ICI 118,551 (10-100 nM) and the non-selective β 1-, β 2- and β 3-adrenoceptor antagonist (±)-bupranolol (10-100 nM), caused a concentration-dependent rightward shift of the concentration-response curves for (-)-isoprenaline and (±)-carteolol. Schild regression plot analyses carried out for (±)-propranolol against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.35 and 8.24, respectively. Schild plot analyses of ICI 118,551 against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.47 and 8.41, respectively. Schild plot analyses of (±)-bupranolol against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.47 and 8.53, respectively. Slopes of the Schild plots were not significantly different from unity. These results suggest that the relaxant effects of (±)-carteolol in the guinea pig taenia caecum are mediated by β 2-adrenoceptors but not by β 3-adrenoceptors.

The β3-adrenoceptor-mediated relaxation induced by epinephrine in guinea pig taenia caecum
Katsuo Koike, Takahiro Horinouchi, Yoshihisa Yamamoto
Journal of Smooth Muscle Research, 36, 3, 93, 99, Japan Society of Smooth Muscle Research, 2000年
英語, 研究論文（学術雑誌）, The mechanisms of the β-adrenoceptor-mediated relaxation induced by epinephrine in guinea pig taenia caecum were examined. The relaxant response to epinephrine was unaffected by propranolol (~10-5 M) or phentolamine (~10-5 M). The response to epinephrine was antagonized in a concentration-dependent manner by bupranolol, and Schild plot of the data revealed the pA2 value of 5.87. Epinephrine significantly increased cyclic AMP level in this preparation. Bupranolol (10-4 M) significantly decreased the cyclic AMP level that was elicited by epinephrine, whereas propranolol (10-5 M) produced no effect. These results suggest that the relaxant response to epinephrine in the guinea pig taenai caecum is mainly mediated by β3-adrenoceptors.

Further evidence that (±)-carteolol-induced relaxation is mediated by β₂-adrenoceptors but not by β₃-adrenoceptors in the guinea pig taenia caecum.
Takahiro Horinouchi, Katsuo Koike
Journal of Smooth Muscle Research, 36, 5, 145, 153, 2000年
英語, 研究論文（学術雑誌）, The properties of the β 1- and β 2-adrenoceptor partial agonist (±)-carteolol were investigated against the β 2- and β 3-adrenoceptors of the taenia caecum of the guinea pig. (-)-Isoprenaline and (±)-carteolol induced concentration-dependent relaxation in this tissue. The non-selective β 1- and β 2-adrenoceptor antagonist (±)-propranolol (10-100 nM), the selective β 2-adrenoceptor antagonist ICI 118,551 (10-100 nM) and the non-selective β 1-, β 2- and β 3-adrenoceptor antagonist (±)-bupranolol (10-100 nM), caused a concentration-dependent rightward shift of the concentration-response curves for (-)-isoprenaline and (±)-carteolol. Schild regression plot analyses carried out for (±)-propranolol against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.35 and 8.24, respectively. Schild plot analyses of ICI 118,551 against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.47 and 8.41, respectively. Schild plot analyses of (±)-bupranolol against (-)-isoprenaline and (±)-carteolol gave pA 2 values of 8.47 and 8.53, respectively. Slopes of the Schild plots were not significantly different from unity. These results suggest that the relaxant effects of (±)-carteolol in the guinea pig taenia caecum are mediated by β 2-adrenoceptors but not by β 3-adrenoceptors.

The β₃-adrenoceptor-mediated relaxation induced by epinephrine in guinea pig taenia caecum.
Katsuo Koike, Takahiro Horinouchi, Yoshihisa Yamamoto
Journal of Smooth Muscle Research, 36, 3, 93, 99, Japan Society of Smooth Muscle Research, 2000年
英語, 研究論文（学術雑誌）, The mechanisms of the β-adrenoceptor-mediated relaxation induced by epinephrine in guinea pig taenia caecum were examined. The relaxant response to epinephrine was unaffected by propranolol (~10-5 M) or phentolamine (~10-5 M). The response to epinephrine was antagonized in a concentration-dependent manner by bupranolol, and Schild plot of the data revealed the pA2 value of 5.87. Epinephrine significantly increased cyclic AMP level in this preparation. Bupranolol (10-4 M) significantly decreased the cyclic AMP level that was elicited by epinephrine, whereas propranolol (10-5 M) produced no effect. These results suggest that the relaxant response to epinephrine in the guinea pig taenai caecum is mainly mediated by β3-adrenoceptors.

Chemical structures of drugs and drug receptor.
Takahiro Horinouchi, Katsuo Koike
Folia Pharmacologica Japonica, 116, 43, 47, 2000年, ［査読有り］
英語, 研究論文（学術雑誌）, The objective of this study was to further characterize the drug specificity for activation of atypical β-adrenoceptors. The relaxant effects of (-)-isoprenaline and (-)-noradrenaline were about 40- and 20-fold as potent as the (+)-isomers, respectively. However, the isomeric activity ratio ((+)/(-)) of isoprenaline and noradrenaline in atypical β-adrenoceptors of guinea pig gastric fundus was less than that obtained for activation of and β1β2-adrenoceptors in the guinea pig atria and trachea, respectively. The β-adrenoceptor alkylating agent, (±)-pindobind (10µM), shifted the concentration-response curves of catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-(-adrenaline) and β3-adrenoceptor agonists (BRL37344 and (±)-CGP12177A) to the right by about 3-fold, respectively. (±)-Pindobind (10µM) produced greater rightward shifts of the concentration-response curves of (-)-isoprenaline in the guinea pig atria and trachea (atria, 600-fold shift, trachea, 300-fold shift). These results suggest that pharmacological properties of atypical β-adrenoceptor differ from that obtained forβ1 and β2-adrenoceptors. © 2000, The Japanese Pharmacological Society. All rights reserved.

Pharmacological analysis of atypical beta-adrenoceptors in the guinea pig gastric fundus using the beta(3)-adrenoceptor antagonist bupranolol
T Horinouchi, K Koike
PHARMACOLOGY, 59, 6, 290, 297, KARGER, 1999年12月
英語, 研究論文（学術雑誌）, Atypical beta-adrenoceptor-mediated relaxations to catecholamines (isoprenaline, noradrenaline and adrenaline) and beta(3)-adrenoceptor agonists, BRL37344 [ (R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]- propyl]phenoxyacetic acid sodium salt] and CGP12177A [ (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one] in guinea pig gastric fundus were investigated. The five agonists induced concentration-dependent relaxation of the gastric fundus. In the presence of both atenolol and butoxamine only small rightward shifts of the concentration-response curves to these agonists were observed. Under this condition, however, bupranolol caused a concentration-dependent rightward shift of the concentration-response curve to catecholamines and beta(3)-adrenoceptor agonists. Schild plot analyses of bupranolol against these agonists gave pA(2) values of 6.08 (isoprenaline), 6.04 (noradrenaline), 5.90 (adrenaline), 6.50 (BRL37344) and 5.80 (CGP12177A), respectively. These results clearly suggest that the existence of functional atypical beta-adrenoceptors in the guinea pig gastric fundus and the relaxation of these agonists in this tissue are mediated via atypical beta-adrenoceptors, Copyright (C) 1999 S.Karger AG, Basel.

Pharmacological analysis of atypical β-adrenoceptors in the guinea pig gastric fundus using the β₃-adrenoceptor antagonist bupranolol.
T Horinouchi, K Koike
Pharmacology, 59, 6, 290, 297, KARGER, 1999年12月
英語, 研究論文（学術雑誌）, Atypical beta-adrenoceptor-mediated relaxations to catecholamines (isoprenaline, noradrenaline and adrenaline) and beta(3)-adrenoceptor agonists, BRL37344 [ (R*,R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]- propyl]phenoxyacetic acid sodium salt] and CGP12177A [ (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one] in guinea pig gastric fundus were investigated. The five agonists induced concentration-dependent relaxation of the gastric fundus. In the presence of both atenolol and butoxamine only small rightward shifts of the concentration-response curves to these agonists were observed. Under this condition, however, bupranolol caused a concentration-dependent rightward shift of the concentration-response curve to catecholamines and beta(3)-adrenoceptor agonists. Schild plot analyses of bupranolol against these agonists gave pA(2) values of 6.08 (isoprenaline), 6.04 (noradrenaline), 5.90 (adrenaline), 6.50 (BRL37344) and 5.80 (CGP12177A), respectively. These results clearly suggest that the existence of functional atypical beta-adrenoceptors in the guinea pig gastric fundus and the relaxation of these agonists in this tissue are mediated via atypical beta-adrenoceptors, Copyright (C) 1999 S.Karger AG, Basel.

Effect of bupranolol for BRL37344 and noradrenaline-induced relaxations mediating atypical beta/beta(3)-adrenoceptor in rat oesophageal muscularis mucosae
T Horinouchi, H Koshikawa, K Koike
GENERAL PHARMACOLOGY, 33, 2, 173, 178, PERGAMON-ELSEVIER SCIENCE LTD, 1999年08月
英語, 研究論文（学術雑誌）, We previously suggested that the existence of atypical beta/beta(3)-adrenoceptor with pA(2)-values for bupranolol, a non-selective beta-adrenoceptor antagonist, against BRL37344 and noradrenaline were 5.79 and 5.53 in guinea pig taenia caecum, respectively. We furthermore determined the affinity of bupranolol to subclassify atypical beta/beta(3)-adrenoceptor in rat oesophageal muscularis mucosae, because it is rich in atypical beta/beta(3)-adrenoceptor. BRL37344 and noradrenaline produced a concentration-dependent relaxation of rat oesophageal muscularis mucosae. The responses to BRL37344 and noradrenaline were resistant to 3 X 10(-6) M propranolol, 10(-4) M atenolol, and 10(-4) M butoxamine. However, bupranolol antagonized the responses to BRL37344 and noradrenaline in a concentration-dependent manner. Schild plot analyses of bupranolol against BRL37344 and noradrenaline gave pA(2)-values of 7.06 and 6.96, respectively. These results suggest that bupranolol can distinguish the difference in affinity between atypical beta/beta(3)-adrenoceptors in rat oesophageal muscularis mucosae and guinea pig taenia caecum. The difference in behavior of bupranolol confirms the existence of some atypical beta/beta(3)-adrenoceptors subtypes. (C) 1999 Elsevier Science Inc. All rights reserved.

Effect of bupranolol for BRL37344 and noradrenaline-induced relaxations mediating atypical β/β₃-adrenoceptor in rat oesophageal muscularis mucosae.
T Horinouchi, H Koshikawa, K Koike
General Pharmacology, 33, 2, 173, 178, PERGAMON-ELSEVIER SCIENCE LTD, 1999年08月
英語, 研究論文（学術雑誌）, We previously suggested that the existence of atypical beta/beta(3)-adrenoceptor with pA(2)-values for bupranolol, a non-selective beta-adrenoceptor antagonist, against BRL37344 and noradrenaline were 5.79 and 5.53 in guinea pig taenia caecum, respectively. We furthermore determined the affinity of bupranolol to subclassify atypical beta/beta(3)-adrenoceptor in rat oesophageal muscularis mucosae, because it is rich in atypical beta/beta(3)-adrenoceptor. BRL37344 and noradrenaline produced a concentration-dependent relaxation of rat oesophageal muscularis mucosae. The responses to BRL37344 and noradrenaline were resistant to 3 X 10(-6) M propranolol, 10(-4) M atenolol, and 10(-4) M butoxamine. However, bupranolol antagonized the responses to BRL37344 and noradrenaline in a concentration-dependent manner. Schild plot analyses of bupranolol against BRL37344 and noradrenaline gave pA(2)-values of 7.06 and 6.96, respectively. These results suggest that bupranolol can distinguish the difference in affinity between atypical beta/beta(3)-adrenoceptors in rat oesophageal muscularis mucosae and guinea pig taenia caecum. The difference in behavior of bupranolol confirms the existence of some atypical beta/beta(3)-adrenoceptors subtypes. (C) 1999 Elsevier Science Inc. All rights reserved.

Characterization of atypical beta-adrenoceptors in the guinea pig duodenum
T Horinouchi, K Koike
EUROPEAN JOURNAL OF PHARMACOLOGY, 376, 1-2, 61, 66, ELSEVIER SCIENCE BV, 1999年07月
英語, 研究論文（学術雑誌）, The atypical beta-adrenoceptors mediating relaxation in the guinea pig duodenum were studied using catecholamines (isoprenaline, noradrenaline and adrenaline), a selective beta(3)-adrenoceptor agonist BRL37344 ((R*, R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium salt) and a non-conventional partial beta(3)-adrenoceptor agonist CGP12177A ((-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one)). Catecholamines and beta(3)-adrenoceptor agonists induced concentration-dependent relaxation in this preparation. Propranolol (1 mu M) produced only small rightward shifts in the concentration-response curves of these agonists. In the presence of propranolol (1 mu M), however, a non-selective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for catecholamines and beta(3)-adrenoceptor agonists. Schild plot analyses of the effects of bupranolol against these agonists gave pA(2) values of 6.02 (isoprenaline), 5.98 (noradrenaline), 5.93 (adrenaline), 6.51 (BRL37344) and 5.70 (CGP12177A), respectively, and all Schild slopes were not significantly different from unity. These results suggest that atypical beta-adrenoceptors are present in the guinea pig duodenum and involved in mediating the functional relaxant response. (C) 1999 Elsevier Science B.V. All rights reserved.

Characterization of atypical β-adrenoceptors in the guinea pig duodenum.
T Horinouchi, K Koike
European Journal of Pharmacology, 376, 1-2, 61, 66, ELSEVIER SCIENCE BV, 1999年07月
英語, 研究論文（学術雑誌）, The atypical beta-adrenoceptors mediating relaxation in the guinea pig duodenum were studied using catecholamines (isoprenaline, noradrenaline and adrenaline), a selective beta(3)-adrenoceptor agonist BRL37344 ((R*, R*)-(+/-)-4-[2-[ (2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium salt) and a non-conventional partial beta(3)-adrenoceptor agonist CGP12177A ((-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one)). Catecholamines and beta(3)-adrenoceptor agonists induced concentration-dependent relaxation in this preparation. Propranolol (1 mu M) produced only small rightward shifts in the concentration-response curves of these agonists. In the presence of propranolol (1 mu M), however, a non-selective beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for catecholamines and beta(3)-adrenoceptor agonists. Schild plot analyses of the effects of bupranolol against these agonists gave pA(2) values of 6.02 (isoprenaline), 5.98 (noradrenaline), 5.93 (adrenaline), 6.51 (BRL37344) and 5.70 (CGP12177A), respectively, and all Schild slopes were not significantly different from unity. These results suggest that atypical beta-adrenoceptors are present in the guinea pig duodenum and involved in mediating the functional relaxant response. (C) 1999 Elsevier Science B.V. All rights reserved.

Pharmacological analysis of atypical β-adrenoceptors in the guinea pig gastrointestinal tissue systems
Takahiro Horinouchi, Michihiko Tsujitani, Katsuo Koike
Folia Pharmacologica Japonica, 114, 1, 1999年, ［査読有り］
日本語, 研究論文（学術雑誌）, The purpose of the present study was to characterize the atypical β- adrenoceptors involved in relaxant responses in guinea pig gastric fundus, duodenum and ileum in functional experiments with catecholamines (isoprenaline, noradrenaline and adrenaline), β3-adrenoceptor agonists (BRL37344 and CGP12177A) and a non-selective β1-, β2- and β3- adrenoceptor antagonist bupranolol, and to obtain further evidence to clarify whether there is a tissue difference in atypical β-adrenoceptors in the guinea pig gastrointestinal tissue systems. The atypical β-adrenoceptors are present in gastric fundus, duodenum and ileum of guinea pig. In the presence of propranolol (1 μM) or atenolol (100 μM) plus butoxamine (100 μM), bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for catecholamines and β3-adrenoceptor agonists. There was not a significant difference of pA2 values for bupranolel against these agonists between gastric fundus, duodenum and ileum of guinea pig. These results suggest that guinea pig gastric fundus, duodenum and ileum relaxation are mediated predominantly by an atypical β- adrenoceptor population whereas the classical β1- or/and β2-adrenoceptors play a subordinate function role and that the receptors of three tissues are pharmacological identified by functional approaches. There is not a tissue difference in atypical β-adrenoceptors in the guinea pig gastrointestinal tissue systems between stomach and ileum.

The β2- and β3-adrenoceptor-mediated relaxation induced by isoprenaline and salbutamol in guinea pig taenia caecum
K. Koike, T. Ichino, T. Horinouchi, I. Takayanagi
Journal of Smooth Muscle Research, 33, 3, 99, 106, Japan Society of Smooth Muscle Research, 1997年
英語, 研究論文（学術雑誌）, To understand the receptor subtypes responsible for β-adrenoceptor- mediated relaxation of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol. Isoprenaline and salbutamol caused dose- dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration-response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respectively. Schild regression analyses carried out for butoxamine against isoprenaline and salbutamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3x10-4 M atenolol, 10-4 M butoxamine and 10- 6 M phentolamine to block the β1-, β2- and α-adrenoceptor effects, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the β2- and the β3-adrenoceptors.

The β₂- and β₃-adrenoceptor-mediated relaxation induced by isoprenaline and salbutamol in guinea pig taenia caecum.
K. Koike, T. Ichino, T. Horinouchi, I. Takayanagi
Journal of Smooth Muscle Research, 33, 3, 99, 106, Japan Society of Smooth Muscle Research, 1997年
英語, 研究論文（学術雑誌）, To understand the receptor subtypes responsible for β-adrenoceptor- mediated relaxation of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol. Isoprenaline and salbutamol caused dose- dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration-response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respectively. Schild regression analyses carried out for butoxamine against isoprenaline and salbutamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3x10-4 M atenolol, 10-4 M butoxamine and 10- 6 M phentolamine to block the β1-, β2- and α-adrenoceptor effects, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the β2- and the β3-adrenoceptors.

MaxiK channel mediates β2-adrenoceptor-activated relaxation to isoprenaline through cAMP-dependent and -independent mechanisms in guinea-pig tracheal smooth muscle　　　　　　　　　　　　　　　
Yoshio Tanaka, Yoko Yamashita, Fumiko Yamaki, Takahiro Horinouchi, Koki Shigenobu, Katsuo Koike
Journal of Smooth Muscle Research, 32, 3, 205, 219, 1996年
英語, 研究論文（学術雑誌）, We examined the contribution of large-conductance, Ca2+-sensitive K+ (MaxiK) channel to β2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between β2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through β2-type of adrenoceptor since the response was antagonized in a competitive fashion by a β2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 μM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 μM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 μM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 μg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates β2-adrenoceptor-activated relaxation
2) both cAMP-dependent and - independent mechanisms underlie the functional coupling between β2-adrenoceptor and MaxiK channel to induce muscle relaxation
and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between β2-adrenoceptor and this ion channel.

Effect of bupranolol on CGP 12177-induced relaxation and cAMP accumulation in the guinea pig Taenia caecum
K Koike, T Horinouchi, Takayanagi, I
GENERAL PHARMACOLOGY, 26, 8, 1791, 1794, PERGAMON-ELSEVIER SCIENCE LTD, 1995年12月
英語, 研究論文（学術雑誌）, 1. The effect of bupranolol on CGP 12177-induced relaxation and cAMP accumulation in the guinea pig taenia caecum was examined.
2. The relaxant response to CGP 12177 was unaffected by propranolol (similar to 10(-6) M), whereas that to CGP 12177 was antagonized in a concentration-dependent manner by bupranolol; Schild plot of the data revealed the pA(2) value of 5.61.
3. CGP 12177 significantly increased cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP level that was elicited by CGP 12177, whereas propranolol(10(-5) M) produced no effect.
4. These results suggest that bupranolol appears to be an efficient beta(3)-antagonist in the guinea pig taenia caecum and confirm that the response to CGP 12177 is mediated by beta(3)-adrenoceptors.

Effect of bupranolol on CGP 12177-induced relaxation and cAMP accumulation in the guinea pig Taenia caecum
K Koike, T Horinouchi, Takayanagi, I
GENERAL PHARMACOLOGY, 26, 8, 1791, 1794, PERGAMON-ELSEVIER SCIENCE LTD, 1995年12月
英語, 研究論文（学術雑誌）, 1. The effect of bupranolol on CGP 12177-induced relaxation and cAMP accumulation in the guinea pig taenia caecum was examined.
2. The relaxant response to CGP 12177 was unaffected by propranolol (similar to 10(-6) M), whereas that to CGP 12177 was antagonized in a concentration-dependent manner by bupranolol; Schild plot of the data revealed the pA(2) value of 5.61.
3. CGP 12177 significantly increased cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP level that was elicited by CGP 12177, whereas propranolol(10(-5) M) produced no effect.
4. These results suggest that bupranolol appears to be an efficient beta(3)-antagonist in the guinea pig taenia caecum and confirm that the response to CGP 12177 is mediated by beta(3)-adrenoceptors.

POSSIBLE MECHANISMS OF BETA-ADRENOCEPTOR-MEDIATED RELAXATION INDUCED BY NORADRENALINE IN GUINEA-PIG TAENIA CECUM
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
EUROPEAN JOURNAL OF PHARMACOLOGY, 279, 2-3, 159, 163, ELSEVIER SCIENCE BV, 1995年06月
英語, 研究論文（学術雑誌）, The mechanisms of the beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum were investigated. Noradrenaline caused graded relaxation of this preparation. However, the concentration-response curves for noradrenaline were unaffected by propranolol (similar to 10(-5) M) or phentolamine (similar to 10(-5) M). The responses to noradrenaline were antagonized in a concentration-dependent manner by bupranolol, and Schild plots of the data revealed a pA(2) value of 5.53. Also, bupranolol antagonized responses to isoprenaline, and Schild plots of the data revealed the pA(2) value to be 8.53. Noradrenaline significantly increased the cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP response elicited by noradrenaline, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to noradrenaline in guinea pig taenia caecum is mainly mediated by beta(3)-adrenoceptors (or atypical beta-adrenoceptors) and that in guinea pig taenia caecum noradrenaline behaves as a beta(3)-selective adrenoceptor agonist.

Possible mechanisms of β-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum.
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
European Journal of Pharmacology, 279, 2-3, 159, 163, ELSEVIER SCIENCE BV, 1995年06月
英語, 研究論文（学術雑誌）, The mechanisms of the beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum were investigated. Noradrenaline caused graded relaxation of this preparation. However, the concentration-response curves for noradrenaline were unaffected by propranolol (similar to 10(-5) M) or phentolamine (similar to 10(-5) M). The responses to noradrenaline were antagonized in a concentration-dependent manner by bupranolol, and Schild plots of the data revealed a pA(2) value of 5.53. Also, bupranolol antagonized responses to isoprenaline, and Schild plots of the data revealed the pA(2) value to be 8.53. Noradrenaline significantly increased the cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP response elicited by noradrenaline, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to noradrenaline in guinea pig taenia caecum is mainly mediated by beta(3)-adrenoceptors (or atypical beta-adrenoceptors) and that in guinea pig taenia caecum noradrenaline behaves as a beta(3)-selective adrenoceptor agonist.

SIGNAL-TRANSDUCTION PATHWAY INVOLVED IN BETA(3)-ADRENOCEPTOR-MEDIATED RELAXATION IN GUINEA-PIG TAENIA CECUM
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
JAPANESE JOURNAL OF PHARMACOLOGY, 68, 1, 41, 46, JAPANESE PHARMACOLOGICAL SOC, 1995年05月
英語, 研究論文（学術雑誌）, Experiments were carried out to examine the components of the intracellular second messenger system that is involved in beta(3)-adrenoceptor (atypical beta-adrenoceptors)-mediated relaxation in the guinea pig taenia caecum. Propranolol and butoxamine caused competitive antagonism of the relaxant response to isoprenaline. However, propranolol or butoxamine did not significantly affect the relaxant responses to CGP 12177 (4-[3-1[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one), a beta(3)-adrenoceptor agonist. The concentration-response curves of the isoprenaline-induced increase in adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels were shifted to the right in a parallel manner by propranolol and butoxamine. However, propranolol or butoxamine did not significantly affect the concentration-response curve for the CGP 12177-induced increase in cyclic AMP levels. MDL 12330 (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine) inhibited the isoprenaline- or CGP 12177-induced increase in cyclic AMP levels. These results suggest that the production of cyclic AMP contributes to the beta(3)-adrenoceptor (or atypical beta-adrenoceptor)-mediated relaxation of the guinea pig taenia caecum.

Signal transduction pathway involved in β₃-adrenoceptor-mediated relaxation in guinea pig taenia caecum.
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
The Japanese Journal of Pharmacology, 68, 1, 41, 46, JAPANESE PHARMACOLOGICAL SOC, 1995年05月
英語, 研究論文（学術雑誌）, Experiments were carried out to examine the components of the intracellular second messenger system that is involved in beta(3)-adrenoceptor (atypical beta-adrenoceptors)-mediated relaxation in the guinea pig taenia caecum. Propranolol and butoxamine caused competitive antagonism of the relaxant response to isoprenaline. However, propranolol or butoxamine did not significantly affect the relaxant responses to CGP 12177 (4-[3-1[ (1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one), a beta(3)-adrenoceptor agonist. The concentration-response curves of the isoprenaline-induced increase in adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels were shifted to the right in a parallel manner by propranolol and butoxamine. However, propranolol or butoxamine did not significantly affect the concentration-response curve for the CGP 12177-induced increase in cyclic AMP levels. MDL 12330 (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine) inhibited the isoprenaline- or CGP 12177-induced increase in cyclic AMP levels. These results suggest that the production of cyclic AMP contributes to the beta(3)-adrenoceptor (or atypical beta-adrenoceptor)-mediated relaxation of the guinea pig taenia caecum.

COMPARISON OF INTERACTIONS OF R(+)-ISOMER AND S(-)-ISOMER OF BETA-ADRENERGIC PARTIAL AGONISTS, BEFUNOLOL AND CARTEOLOL, WITH HIGH-AFFINITY SITE OF BETA-ADRENOCEPTORS IN THE MICROSOMAL FRACTIONS FROM GUINEA-PIG CILIARY BODY, RIGHT ATRIA AND TRACHEA
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
GENERAL PHARMACOLOGY, 25, 7, 1477, 1481, PERGAMON-ELSEVIER SCIENCE LTD, 1994年11月
英語, 研究論文（学術雑誌）, 1. The stereoselectivities of beta-adrenergic partial agonists for the high affinity binding site of beta-adrenoceptors in the guinea-pig ciliary body, right atria and trachea were studied.
2. The inhibition curves by the S(-)-isomers of befunolol and carteolol were not significantly different from that by the R(+)-isomers in the guinea-pig ciliary body.
3. The inhibition curves by the S(-)-isomers of befunolol and carteolol were about 10 times as potent as the R(+)-isomers in the guinea-pig atria and trachea.
4. The pK(i) Values of the S(-)-isomers of befunolol and carteolol were significantly larger than those of R(+)-isomers in the guinea-pig atria and trachea but not larger than those of the R(+)-isomers in the guinea-pig ciliary body.
5. These results suggest that the high affinity binding site of P-adrenoceptors in ciliary body cannot discriminate stereoselectively between the R(+)- and S(-)-isomers, while in other tissues there is stereoselectivity between the two enantiomers.

Comparison of interactions of R(+)- and S(-)-isomers of β-adrenergic partial agonists, befunolol and carteolol, with high affinity site of β-adrenoceptors in the microsomal fractions from guinea-pig ciliary body, right atria and trachea.
K KOIKE, T HORINOUCHI, TAKAYANAGI, I
General Pharmacology, 25, 7, 1477, 1481, PERGAMON-ELSEVIER SCIENCE LTD, 1994年11月
英語, 研究論文（学術雑誌）, 1. The stereoselectivities of beta-adrenergic partial agonists for the high affinity binding site of beta-adrenoceptors in the guinea-pig ciliary body, right atria and trachea were studied.
2. The inhibition curves by the S(-)-isomers of befunolol and carteolol were not significantly different from that by the R(+)-isomers in the guinea-pig ciliary body.
3. The inhibition curves by the S(-)-isomers of befunolol and carteolol were about 10 times as potent as the R(+)-isomers in the guinea-pig atria and trachea.
4. The pK(i) Values of the S(-)-isomers of befunolol and carteolol were significantly larger than those of R(+)-isomers in the guinea-pig atria and trachea but not larger than those of the R(+)-isomers in the guinea-pig ciliary body.
5. These results suggest that the high affinity binding site of P-adrenoceptors in ciliary body cannot discriminate stereoselectively between the R(+)- and S(-)-isomers, while in other tissues there is stereoselectivity between the two enantiomers.

INVOLVEMENT OF BETA-(3)-ADRENOCEPTOR IN THE RELAXATION RESPONSE IN GUINEA-PIG TAENIA CECUM
K KOIKE, TAKAYANAGI, I, M MURAMATSU, S OHKI, T HORINOUCHI
JAPANESE JOURNAL OF PHARMACOLOGY, 66, 2, 213, 220, JAPANESE PHARMACOLOGICAL SOC, 1994年10月
英語, 研究論文（学術雑誌）, beta-Adrenoceptors in the guinea pig taenia caecum were investigated by measuring relaxation responses to agonists and by a radioligand binding assay using [H-3]CGP 12177. The rightward shift of the isoprenaline concentration-response curve was observed by butoxamine, a beta(2)-selective antagonist, and the pA(2) value for butoxamine was 6.46. In control preparations, catecholamines caused relaxation with the following rank order of potency: isoprenaline > adrenaline > noradrenaline. However, in the presence of 10(-6) M phentolamine, 3 x 10(-4) M atenolol and 10(-4) M butoxamine, the rank order of potency of the agonists was: isoprenaline > noradrenaline > adrenaline. CGP 12177 caused graded relaxation of the guinea pig taenia caecum, and this response was not influenced by 10(-6) M phentolamine, 3 x 10(-4) M atenolol, 10(-4) M butoxamine or 10(-6) M propranolol. The Scatchard plot of the specific [3H]CGP 12177 binding to microsomal fractions from the guinea pig taenia caecum showed two affinity sites of the receptor: high affinity (K-D=0.64 nM) and low affinity (K-D=142.21 nM) sites. The pK(D) value of the high affinity site of [H-3]CGP 12177 was in agreement with its pA(2) value, and that of the low affinity site was in agreement with its pD(2) value. These results suggest that isoprenaline-, noradrenaline- and adrenaline-induced relaxations of the guinea pig taenia caecum predominantly involve beta(2)- and beta(2)-adrenoceptors, whereas CGP 12177-induced relaxation is mediated solely through beta(3)-adrenoceptors.

Involvement of β₃-adrenoceptor in the relaxation response in guinea pig taenia caecum.
K KOIKE, TAKAYANAGI, I, M MURAMATSU, S OHKI, T HORINOUCHI
The Japanese Journal of Pharmacology, 66, 2, 213, 220, JAPANESE PHARMACOLOGICAL SOC, 1994年10月
英語, 研究論文（学術雑誌）, beta-Adrenoceptors in the guinea pig taenia caecum were investigated by measuring relaxation responses to agonists and by a radioligand binding assay using [H-3]CGP 12177. The rightward shift of the isoprenaline concentration-response curve was observed by butoxamine, a beta(2)-selective antagonist, and the pA(2) value for butoxamine was 6.46. In control preparations, catecholamines caused relaxation with the following rank order of potency: isoprenaline > adrenaline > noradrenaline. However, in the presence of 10(-6) M phentolamine, 3 x 10(-4) M atenolol and 10(-4) M butoxamine, the rank order of potency of the agonists was: isoprenaline > noradrenaline > adrenaline. CGP 12177 caused graded relaxation of the guinea pig taenia caecum, and this response was not influenced by 10(-6) M phentolamine, 3 x 10(-4) M atenolol, 10(-4) M butoxamine or 10(-6) M propranolol. The Scatchard plot of the specific [3H]CGP 12177 binding to microsomal fractions from the guinea pig taenia caecum showed two affinity sites of the receptor: high affinity (K-D=0.64 nM) and low affinity (K-D=142.21 nM) sites. The pK(D) value of the high affinity site of [H-3]CGP 12177 was in agreement with its pA(2) value, and that of the low affinity site was in agreement with its pD(2) value. These results suggest that isoprenaline-, noradrenaline- and adrenaline-induced relaxations of the guinea pig taenia caecum predominantly involve beta(2)- and beta(2)-adrenoceptors, whereas CGP 12177-induced relaxation is mediated solely through beta(3)-adrenoceptors.

血管内皮細胞における加熱式たばこ主流煙水抽出物の作用解析
堀之内孝広, 真崎雄一, 三輪聡一, 日本薬理学雑誌, 156, Supplement, 2021年

エンドセリン-1によるERKの活性化におけるAnnexin A2の役割
真崎雄一, 東恒仁, 堀之内孝広, 三輪聡一, 日本分子生物学会年会プログラム・要旨集(Web), 44th, 2021年

血管内皮細胞におけるカルシウム感受性受容体を介した一酸化窒素産生機序
堀之内孝広, 真崎雄一, 寺田晃士, 三輪聡一, 日本薬理学雑誌, 155, Supplement, 2020年

血管内皮細胞におけるカルシウム感受性受容体を介したNO産生に対するタバコ煙ガス相水抽出物及びアクロレインの抑制作用
堀之内孝広, 真崎雄一, 三輪聡一, 日本薬理学雑誌, 153, Supplement, 2019年

エンドセリンB受容体はGRP78と相互作用する
真崎雄一, 東恒仁, 堀之内孝広, 橋本あり, 橋本茂, 南ジンミン, 小野寺康仁, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018年

血管内皮細胞におけるアクロレインの作用解析
堀之内孝広, 三輪聡一, 三輪聡一, 真崎雄一, 東恒仁, 日本薬理学会北部会プログラム・抄録集, 68th, 2017年

好中球のケモタキシスにおいて,ARF1の活性化は,ARF1‐RAC1の相互制御回路を開始する
真崎雄一, 小野寺康仁, 東恒仁, 堀之内孝広, 及川司, 佐邊壽孝, 日本細胞生物学会大会(Web), 69th, ROMBUNNO.T8‐11(P1‐077) (WEB ONLY), 63, 2017年
(一社)日本細胞生物学会, 日本語

Phos-tag biotinを用いた受容体作動性TRPC6チャネルのPKAリン酸化部位の同定
堀之内孝広, 寺田晃士, 東恒仁, 真崎雄一, 三輪聡一, 電気泳動(Web), 60, Suppl, 2016年

Unsaturated carbonyl compounds in the gas phase of cigarette smoke induces cell death through intracellular Ca2+-dependent PKC activation.
T. Higashi, Y. Mai, Y. Mazaki, T. Horinouchi, S. Miwa, MOLECULAR BIOLOGY OF THE CELL, 27, 2016年
AMER SOC CELL BIOLOGY, 英語, 研究発表ペーパー・要旨（国際会議）

Identification of cytotoxic compounds in cigarette smoke gas phase and molecular mechanisms for their cytotoxicity
Tsunehito Higashi, Yosuke Mai, Takahiro Horinouchi, Koji Terada, Akimasa Hoshi, Mika Horiguchi, Prabha Nepal, Takuya Harada, Chizuru Hatate, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 131P, 131P, 2014年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

A critical role of ubiquitination in endothelin receptor trafficking
Soichi Miwa, Koji Terada, Tsunehito Higashi, Nepal Prabha, Takahiro Horinouchi, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 34P, 34P, 2014年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Cell signaling mechanism activated by endothelin-1 in rat skeletal muscle cells
Akimasa Hoshi, Takahiro Horinouchi, Takuya Harada, Tsunaki Higa, Tsunehito Higashi, Koji Terada, Yosuke Mai, Prabha Nepal, Mika Horiguchi, Chizuru Hatate, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 93P, 93P, 2014年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Endothelin receptor signalosome as a target for a novel therapeutic strategy against cardiovascular diseases
Takahiro Horinouchi, Koji Terada, Tsunehito Higashi, Akimasa Hoshi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 22P, 22P, 2014年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

The role of ubiquitination in ET-1-dependent endothelin type B receptor trafficking
Koji Terada, Takahiro Horinouchi, Tsunehito Higashi, Nepal Prabha, Mika Horiguchi, Chizuru Hatate, JOURNAL OF PHARMACOLOGICAL SCIENCES, 124, 196P, 196P, 2014年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

アデニル酸シクラーゼ/cAMP/プロテインキナーゼA系によるTRPC6チャネルを介したエンドセリンA型受容体作動性Ca^<2+>流入の抑制性制御
堀之内 孝広, 比嘉 綱己, 青柳 裕之, 西屋 禎, 寺田 晃士, 三輪 聡一, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 88, 2, 91, 91, 2013年04月01日, ［筆頭著者, 責任著者］
日本語

エンドセリン受容体シグナルソームの形成を介した新たな2型糖尿病発症メカニズムの解明
堀之内 孝広, 寺田 晃士, 東 恒仁, 医科学応用研究財団研究報告, 32, 320, 324, 2013年
鈴木謙三記念医科学応用研究財団, 日本語

Identification and characterization of STIM1L, a novel splice variant of STIM1
Takahiro Horinouchi, Tsunehito Higashi, Koji Terada, Tsunaki Higa, Yosuke Mai, Chizuru Hatate, Prabha Nepal, Mika Horiguchi, Takuya Harada, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 121, 91P, 91P, 2013年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Nicotine- and tar-free cigarette smoke extract induces cell injury via intracellular Ca2+ dependent PKC activation
Tsunehito Higashi, Yosuke Mai, Takahiro Horinouchi, Koji Terada, Chizuru Hatate, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 121, 153P, 153P, 2013年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Negative regulation of endothelin type A receptor-operated TRPC6 by PKA phosphorylation.
Takahiro Horinouchi, Tsunaki Higa, Hiroyuki Aoyagi, Koji Terada, Tadashi Nishiya, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 211P, 211P, 2012年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE): Involvement of reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase.
Yosuke Mai, Takahiro Horinouchi, Hiroshi Asano, Osamu Sawada, Tadashi Nishiya, Masabumi Minami, Takahiro Katayama, Koji Terada, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 256P, 256P, 2012年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Role of ubiquitination in endothelin receptor trafficking
Koji Terada, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 197P, 197P, 2012年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Regulatory mechanism of endothelin type A receptor-operated Ca2+ entry via TRPC3 and TRPC6 channels.
Tsunaki Higa, Takahiro Horinouchi, Hiroyuki Aoyagi, Koji Terada, Tadashi Nishiya, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 118, 211P, 211P, 2012年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Negative regulation of endothelin type A receptor-operated TRPC6 by adenylate cyclase/cAMP/PKA pathway
Takahiro Horinouchi, Tsunaki Higa, Tadashi Nishiya, Hiroyuki Aoyagi, Hiroyuki Suzuki, Arata Nishimoto, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 115, 112P, 112P, 2011年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

iNOS regulation by the SPRY domain- and SOCS box-containing proteins
Tadashi Nishiya, Kazuma Matsumoto, Satoshi Maekawa, Takahiro Horinouchi, Masahiro Fujimuro, Kouetsu Ogasawara, Takashi Uehara, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 115, 102P, 102P, 2011年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Molecular mechanisms for endothelin type A receptor-operated Ca2+ entry via TRPC3 and TRPC6
Takahiro Horinouchi, Tsunaki Higa, Hiroshi Asano, Arata Nishimoto, Tadashi Nishiya, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 95P, 95P, 2010年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Regulation of endothelin receptor levels by receptor binding protein, Jab1
Arata Nishimoto, Tadashi Nishiya, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 75P, 75P, 2010年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Early postnatal stress affects the hippocampal postsynaptic 5-HT1A receptor function in context-dependent conditioned fear in adult rats
Hirokazu Matsuzaki, Takeshi Izumi, Machiko Matsumoto, Taku Yamaguchi, Takayuki Yoshida, Takahiro Horinouchi, Shuken Boku, JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 178P, 178P, 2010年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Visualization of homo- and heteromeric TRPC3/6 interaction in living cells using bimolecular fluorescence complementation analysis
Takahiro Horinouchi, Tsunaki Higa, Hiroshi Asano, Tadashi Nishiya, Arata Nishimoto, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 112, 251P, 251P, 2010年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

REGULATION OF CELL SURFACE ENDOTHELIN TYPE A RECEPTOR LEVEL BY A NOVEL RECEPTOR-INTERACTING PROTEIN, JAB1
Arata Nishimoto, Lingyun Lu, Misato Hayashi, Tadashi Nishiya, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHYSIOLOGICAL SCIENCES, 59, 347, 347, 2009年
SPRINGER TOKYO, 英語, 研究発表ペーパー・要旨（国際会議）

Effects of chronic administration of beta-adrenoceptor antagonists on plasma membrane beta-adrenoceptors of rat heart
Takahiro Horinouchi, Shigeru Morishima, Takashi Tanaka, Fumiko Suzuki, Yoshio Tanaka, Katsuo Koike, Soichi Miwa, Ikunobu Muramatsu, JOURNAL OF PHARMACOLOGICAL SCIENCES, 109, 262P, 262P, 2009年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Jab1, a novel human ETAR-interacting protein, regulates cell surface ETAR level and intracellular signaling by promoting ubiquitination and lysosomal degradation of ETAR
Arata Nishimoto, Lingyun Lu, Tadashi Nishiya, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 109, 165P, 165P, 2009年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

multiplicity-of ligand-directed signaling at the beta(2)-adrenoceptor
Takahiro Horinouchi, Masaaki Sato, Dana. S. Hutchinson, Bronwyn. A. Evans, Yoshio Tanaka, Katsuo Koike, Soichi Miwa, Roger. J. Summers, JOURNAL OF PHARMACOLOGICAL SCIENCES, 109, 97P, 97P, 2009年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Regulation of endothelin receptor levels by receptor binding proteins
Arata Nishimoto, Tadashi Nishiya, Takahiro Horinouchi, JOURNAL OF PHARMACOLOGICAL SCIENCES, 109, 36P, 36P, 2009年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Functional role of Na+/H+ exchanger in Ca2+ influx mediated via human endothelin type A receptor stably expressed in Chinese hamster ovary cells.
Takahiro Horinouchi, Yumie Miyake, Tadashi Nishiya, Arata Nishimoto, Shigeru Morishima, Ikunobu Muramatsu, Soichi Miwa, Journal of pharmacological sciences, 107, 4, 456, 9, 2008年08月, ［国内誌］
This study examines the functional role of Na+/H+ exchanger (NHE) in Ca2+ influx mediated by human endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary (CHO) cells. Endothelin-1 (ET-1) increased extracellular acidification rate (ECAR), which was abolished by 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an NHE inhibitor. EIPA and KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, inhibited ET-1-induced sustained increases in intracellular Ca2+ concentration ([Ca2+]i), and EIPA had no effect on [Ca2+]i after KB-R7943 treatment. ET-1-elicited sustained [Ca2+]i increase was suppressed by reducing extracellular Na+ concentration. These results suggest that possible coupling of NHE with NCX via Na+ transport is involved in ET(A)R-mediated sustained [Ca2+]i increase., 英語

Molecular mechanism underlying the sustained increase in [Ca2+](i) mediated via G protein-coupled receptors
Takahiro Horinouchi, Yumie Miyake, Tadashi Nishiya, Arata Nishimoto, Shigeru Morishima, Ikunobu Muramatsu, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 116P, 116P, 2008年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Molecular mechanism for Ca2+ signaling activated by alpha(1A)-AR
Yumie Miyake, Takahiro Horinouchi, Tadashi Nishiya, Arata Nishimoto, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 210P, 210P, 2008年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

The elucidation of the regulatory mechanism for protein degradation of endothelin type A receptor by ETAR-interacting proteins
Arata Nishimoto, Lingyun Lu, Tadashi Nishiya, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 106, 56P, 56P, 2008年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Molecular mechanisms for Ca2+ signaling activated by endothelin-1
Soichi Miwa, Takahiro Horinouchi, Arata Nishimoto, Tadashi Nishiya, CLINICAL SCIENCE, 113, 7-8, 5P, 5P, 2007年10月
PORTLAND PRESS LTD, 英語, 研究発表ペーパー・要旨（国際会議）

Characterization of noradrenaline-induced increases in intracellular Ca2+ levels in Chinese hamster ovary cells stably expressing human alpha1A-adrenoceptor.
Takahiro Horinouchi, Yumie Miyake, Tadashi Nishiya, Arata Nishimoto, Shoko Yorozu, Atsushi Jinno, Emi Kajita, Soichi Miwa, Journal of pharmacological sciences, 105, 1, 103, 11, 2007年09月, ［国内誌］
The mechanism for noradrenaline (NA)-induced increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and physiological significance of Na(+) influx through receptor-operated channels (ROCs) and store-operated channels (SOCs) were studied in Chinese hamster ovary (CHO) cells stably expressing human alpha(1A)-adrenoceptor (alpha(1A)-AR). [Ca(2+)](i) was measured using the Ca(2+) indicator fura-2. NA (1 microM) elicited transient and subsequent sustained [Ca(2+)](i) increases, which were inhibited by YM-254890 (G(alphaq/11) inhibitor), U-73122 (phospholipase C (PLC) inhibitor), and bisindolylmaleimide I (protein kinase C (PKC) inhibitor), suggesting their dependence on G(alphaq/11)/PLC/PKC. Both phases were suppressed by extracellular Ca(2+) removal, SK&F 96365 (inhibitor of SOC and nonselective cation channel type-2 (NSCC-2)), LOE 908 (inhibitor of NSCC-1 and NSCC-2), and La(3+) (inhibitor of transient receptor potential canonical (TRPC) channel). Reduction of extracellular Na(+) and pretreatment with KB-R7943, a Na(+)/Ca(2+) exchanger (NCX) inhibitor, inhibited both phases of [Ca(2+)](i) increases. These results suggest that 1) stimulation of alpha(1A)-AR with NA elicits the transient and sustained increases in [Ca(2+)](i) mediated through NSCC-2 that belongs to a TRPC family; 2) Na(+) influx through these channels drives NCX in the reverse mode, causing Ca(2+) influx in exchange for Na(+) efflux; and 3) the G(alphaq/11)/PLC/PKC-dependent pathway plays an important role in the increases in [Ca(2+)](i)., 英語

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.
Tadashi Nishiya, Emi Kajita, Takahiro Horinouchi, Arata Nishimoto, Soichi Miwa, FEBS letters, 581, 17, 3223, 9, 2007年07月10日, ［国際誌］
MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88., 英語

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88
Emi Kajita, Tadashi Nishiya, Arata Nishimoto, Takahiro Horinouchi, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 103, 263P, 263P, 2007年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Jab1, a novel endothelin type A receptor interacting protein, regulates ETAR level and signaling
Arata Nishimoto, Lingyun Lu, Tadashi Nishiya, Takahiro Horinouchi, Emi Kajita, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 103, 88P, 88P, 2007年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Role of Na+/Ca2+ exchanger in Ca2+ signaling activated by endothelin-1 in CHO cells stably expressing endothelin type A receptor.
Takahiro Horinouchi, Lingyun Lu, Tadashi Nishiya, Arata Nishimoto, Emi Kajita, Soichi Miwa, JOURNAL OF PHARMACOLOGICAL SCIENCES, 103, 86P, 86P, 2007年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

組織片を用いた新規結合実験法による眼内β-アドレナリン受容体の薬理学的解析　　　　　　　　　　　　　　　
堀之内 孝広, 村松 郁延, 田中 芳夫, 小池 勝夫, 三輪 聡一, 日本薬理学雑誌, 129, 1, 15P, 15P, 2007年01月
(公社)日本薬理学会, 日本語

Tissue segment binding assay, a novel technique, for studying plasma membrane beta-adrenoceptors using intact cardiac tissues
Takahiro Horinouchi, Soichi Miwa, Ikunobu Muramatsu, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 41, 6, 1070, 1070, 2006年12月
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 英語, 研究発表ペーパー・要旨（国際会議）

Multiple signalling pathways of the mouse beta(3)-adrenoceptor stably expressed in CHO-K1 cells.
Masaaki Sato, Takahiro Horinouchi, Dana S. Hutchinson, Bronwyn A. Evans, Roger J. ummers, ACTA PHARMACOLOGICA SINICA, 27, 410, 410, 2006年07月
BLACKWELL PUBLISHING, 英語, 研究発表ペーパー・要旨（国際会議）

モルモットの膀胱運動に対する臭化ジスチグミンの作用とその機序に関する検討　　　　　　　　　　　　　　　
関谷 更沙, 大河 夏美, 青木 智子, 秋山 理恵, 小野 友子, 芝野 真理, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬学会年会要旨集, 126年会, 3, 146, 146, 2006年03月
(公社)日本薬学会, 日本語

ジスチグミンの膀胱運動に対する作用の検討 : シストメトリー法による評価
関谷 更沙, 大河 夏美, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 応用薬理, 70, 1, 29, 34, 2006年02月28日
日本語

Effects of distigmine on the motilities of guinea-pig detrusor and urethral smooth muscles
S Sekiya, N Ookawa, T Aoki, R Akiyama, T Ono, M Shibano, T Horinouchi, Y Tanaka, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 100, 265P, 265P, 2006年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

The role of the C-terminal domain in mouse beta(3)-adrenoceptor signalling
T Horinouchi, M Sato, DS Hutchinson, BA Evans, Y Tanaka, K Koike, RJ Summers, JOURNAL OF PHARMACOLOGICAL SCIENCES, 100, 126P, 126P, 2006年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

内因性カテコラミンによるモルモット気管平滑筋の弛緩反応に関与するβ-アドレナリン受容体(β-AR)サブタイプに関する検討　　　　　　　　　　　　　　　
田中 芳夫, 堀之内 孝広, 小池 勝夫, 日本薬理学雑誌, 126, 4, 6P, 6P, 2005年10月
(公社)日本薬理学会, 日本語

眼のβ-アドレナリン受容体について : 組織片を用いた新しい結合実験法による薬物受容体の解析
堀之内 孝広, 村松 郁延, 田中 芳夫, 小池 勝夫, 眼薬理 = Japanese journal of ocular pharmacology, 19, 1, 46, 48, 2005年06月24日, ［筆頭著者］
日本語

受容体・チャンネルを介する平滑筋の機能調節と臨床応用 膀胱平滑筋の機能調節と臨床応用　　　　　　　　　　　　　　　
田中 芳夫, 堀之内 孝広, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 9, 1, J10, J10, 2005年04月
日本平滑筋学会, 日本語

ノルアドレナリンの気管平滑筋弛緩作用に2種類のβ1-アドレナリン受容体が関与する可能性について　　　　　　　　　　　　　　　
田中 芳夫, 堀之内 孝広, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 9, 1, J29, J29, 2005年04月
日本平滑筋学会, 日本語

Pharmacological evidence that noradrenaline (NA)-elicited relaxation of tracheal smooth muscle is mediated through beta-adrenoceptor (beta-AR) subtypes distinct from established beta(1-3)
A Minemura, F Yamaki, T Horinouchi, Y Tanaka, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 97, 247P, 247P, 2005年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

モルモット気管平滑筋の弛緩反応に関与するβ-アドレナリン受容体(β-AR)サブタイプについて ノルアドレナリンとサルブタモールによる弛緩反応の比較　　　　　　　　　　　　　　　
八巻 史子, 山下 庸子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬理学雑誌, 125, 1, 16P, 16P, 2005年01月
(公社)日本薬理学会, 日本語

New insights into the approaches towards the development of therapeutic agents for urologic diseases (生体機能と創薬シンポジウム2004--イオンチャネル機能と細胞障害--創薬を目指して) -- (シンポジウム2 生体機能解析の新展開)
田中 芳夫, 堀之内 孝広, 小池 勝夫, 薬学雑誌, 124, 61, 63, 2004年09月10日
日本薬学会, 英語

IP受容体ならびにβ-アドレナリン受容体を介した組織cAMP量の上昇を伴わない平滑筋の弛緩反応機構について MaxiKチャネルの役割とその活性化機序　　　　　　　　　　　　　　　
八巻 史子, 山下 庸子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬理学雑誌, 124, 2, 13P, 13P, 2004年08月
(公社)日本薬理学会, 日本語

IP受容体ならびにβ-アドレナリン受容体を介した平滑筋の弛緩反応におけるcAMP非依存性機構について MaxiKチャネルの役割とその活性化機序
八巻 史子, 山下 庸子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 8, 1, J34, J34, 2004年04月
日本平滑筋学会, 日本語

糖尿病モデルマウスを用いた消化管運動障害の検討 ニコチン受容体結合能の変化　　　　　　　　　　　　　　　
千倉 知子, 小池 勝夫, 田中 芳夫, 堀之内 孝広, 小野 景義, 中村 智徳, 矢野 眞吾, 日本薬学会年会要旨集, 124年会, 4, 17, 17, 2004年03月
(公社)日本薬学会, 日本語

β-アドレナリン受容体(β-AR)を介したGs蛋白の活性化による細胞内cAMP量の上昇を伴わない気管平滑筋の弛緩反応について　　　　　　　　　　　　　　　
田中 芳夫, 山下 庸子, 八巻 史子, 堀之内 孝広, 小池 勝夫, 日本薬学会年会要旨集, 124年会, 4, 46, 46, 2004年03月
(公社)日本薬学会, 日本語

G(s)-triggered, cAMP-independent mechanism(s) is involved in beta-adrenoceptor-mediated relaxation of guinea-pig tracheal smooth muscle
Y Tanaka, Y Yamashita, F Yamaki, T Horinouchi, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 94, 142P, 142P, 2004年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

New insights into the cellular mechanisms to control the mechanical activity of urinary bladder smooth muscle and possible approaches towards the development of novel types of therapeutic agents for urologic diseases
Y Tanaka, T Horinouchi, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 94, 31P, 31P, 2004年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Pharmacological and molecular analyses of beta-adrenoceptors in guinea-pig esophagus
T Horinouchi, Y Tanaka, M Tsujitani, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 94, 148P, 148P, 2004年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

β2-アドレナリン受容体を介した気管平滑筋の弛緩反応機構におけるcAMPの役割について　　　　　　　　　　　　　　　
山下 庸子, 八巻 史子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬理学雑誌, 123, 1, 29P, 29P, 2004年01月
(公社)日本薬理学会, 日本語

消化管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプの分布及びβ3-アドレナリン受容体のユニークな抑制性緊張制御機構の解明　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬理学雑誌, 122, 補冊1, 54, 56, 2003年11月
各種β-アドレナリン受容体拮抗薬を用いて,モルモット消化管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプを解析した.その結果,食道ではβ1-サブタイプ,盲腸紐ではβ2,β3-サブタイプ,胃底・十二指腸・回腸ではβ3-サブタイプが主として関与していた.又,モルモット胃底平滑筋においてカテコラミン及びβ3アドレナリン受容体活性薬により引き起こされる弛緩反応は,アデニル酸シクラーゼ阻害薬及びcyclic AMP依存性プロテインキナーゼ阻害薬により影響を受けなかった.アデニル酸シクラーゼ阻害薬存在下では,cyclic AMP量の上昇を伴わない弛緩反応を引き起こし,電位依存性Kチャネル阻害薬の4-アミノピリジンにより有意に抑制された.消化管平滑筋の弛緩反応には,β3-アドレナリン受容体が主として関与しており,その弛緩反応機構の可能性として4-アミノピリジン感受性電位依存性Kチャネルの活性化が考えられた, (公社)日本薬理学会, 日本語

cAMP-非依存性のMaxiK channel活性化機構について:G蛋白を介する活性化調節と血管平滑筋の緊張性制御における役割　　　　　　　　　　　　　　　
八巻 史子, 田中 芳夫, 堀之内 孝広, 重信 弘毅, 小池 勝夫, 日本薬理学雑誌, 122, 補冊1, 37, 39, 2003年11月
モルモット胸部大動脈標本において,プロスタサイクリン(IP)アゴニストのベラプロストによる血管弛緩反応は,イベリオトキシン(IbTx)により強力に抑制された.ベラプロストは組織cAMP量を30倍にまで増加させた.cAMP特異的ホスホジエステラーゼを選択的に阻害するRo-20-1724存在下では,ベラプロストによる弛緩反応は有意に増強されたが,IbTxはこれを完全に抑制した.膜透過性cAMP誘導体である8-Br-cAMPによる弛緩反応もIbTxにより消失した.アデニル酸シクラーゼ阻害作用を有するSQ22536は,ベラプロストの組織cAMP量の増加作用を有意に抑制したが,弛緩作用には全く影響しなかった.SQ22536存在下でのベラプロストによる弛緩反応は,IbTxにより有意に抑制された.又,ベラプロストは,単離大動脈平滑筋細胞で記録されたMaxiK channelをGTP依存性に活性化した.IP受容体刺激に伴うモルモット大動脈の弛緩反応において,MaxiK channelが中心的役割を果たしていることが示唆された, (公社)日本薬理学会, 日本語

モルモット消化管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプの分布とmRNA発現の比較　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 辻谷 典彦, 小池 勝夫, 日本薬理学雑誌, 122, 4, 30P, 30P, 2003年10月
(公社)日本薬理学会, 日本語

カテコラミン刺激により誘発される消化管平滑筋弛緩反応に関与するβ-アドレナリン受容体サブタイプとその部位差　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 7, 1, J27, J27, 2003年04月
日本平滑筋学会, 日本語

β2-アドレナリン受容体刺激に伴う気管平滑筋の弛緩反応機構 イソプレナリンによるcAMPを介さないモルモット気管筋の弛緩機序とマキシKチャネル活性化の関連性について　　　　　　　　　　　　　　　
山下 庸子, 八巻 史子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 7, 1, J28, J28, 2003年04月
日本平滑筋学会, 日本語

イソプレナリンの血管弛緩作用について マウス大動脈における解析　　　　　　　　　　　　　　　
芝野 真理, 秋元 ゆりえ, 松下 真由美, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬学会年会要旨集, 123年会, 4, 33, 33, 2003年03月
(公社)日本薬学会, 日本語

カテコラミンによる気管平滑筋弛緩作用に関与するβ-アドレナリン受容体(β-AR)について ノルアドレナリンとイソプレナリンの作用の相違　　　　　　　　　　　　　　　
山下 庸子, 堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬学会年会要旨集, 123年会, 4, 33, 33, 2003年03月
(公社)日本薬学会, 日本語

イソプレナリンによる弛緩反応におけるβ3-アドレナリン受容体の寄与について ラット胸部大動脈及び腹部大動脈での比較　　　　　　　　　　　　　　　
松下 真由美, 堀之内 孝広, 田中 芳夫, 水流 弘通, 小池 勝夫, 日本薬学会年会要旨集, 123年会, 4, 33, 33, 2003年03月
(公社)日本薬学会, 日本語

交感神経電気刺激により誘発されるウサギ顔面静脈弛緩反応とTEAによる増強効果の薬理学的解析　　　　　　　　　　　　　　　
田中 芳夫, 阿久津 あや, 堀之内 孝広, 田中 光, 水流 弘通, 重信 弘毅, 小池 勝夫, 日本薬理学雑誌, 121, 2, 27P, 27P, 2003年02月
(公社)日本薬理学会, 日本語

β3-アドレナリン受容体刺激により誘発されるcyclic AMPの上昇を介さない胃底平滑筋の弛緩反応機構の解析 電位依存性K+チャネルの関与の可能性について　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 辻谷 典彦, 小池 勝夫, 日本薬理学雑誌, 121, 2, 27P, 27P, 2003年02月
(公社)日本薬理学会, 日本語

Effects of MaxiK channel activators on the rhythmic contraction and action potential generated spontaneously in guinea-pig urinary bladder smooth muscle.
T Okamoto, T Imai, T Horinouchi, H Tanaka, A Mitani, T Nakahara, Y Tanaka, K Ishii, K Shigenobu, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 271P, 271P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Different contribution of classical beta- and atypical beta(3)-adrenoceptors in vasodilation to isoprenaline of rat thoracic and abdominal aortae: analysis using by propranolol and bupranolol in vitro.
M Matsushita, T Horinouchi, Y Tanaka, H Tsuru, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 269P, 269P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Pharmacological characterization of beta-adrenoceptor which mediates noradrenaline (NA)-elicited tracheal smooth muscle relaxation.
Y Yamashita, T Horinouchi, Y Tanaka, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 271P, 271P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Functional coupling between Q-type Ca2+ channels and tetraethylammonium (TEA)-sensitive K+ channels in the sympathetic neurotransmission of rabbit facial vein.
Y Tanaka, A Akutsu, H Tanaka, T Horinouchi, H Tsuru, K Shigenobu, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 269P, 269P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Pharmacological characterization of alpha 1-adrenoceptor in mouse iliac artery.
M Shibano, T Horinouchi, Y Tanaka, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 272P, 272P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

IP receptor-activated, cAMP-independent vasorelaxant mechanisms: a substantial role of Gs protein-mediated, MaxiK channel activation.
F Yamaki, T Horinouchi, Y Tanaka, K Shigenobu, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 272P, 272P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

Gastrointestinal smooth muscle beta(3)-adrenoceptor: its distribution and coupling with a unique transduction system.
T Horinouchi, Y Tanaka, M Tsujitani, K Koike, JOURNAL OF PHARMACOLOGICAL SCIENCES, 91, 109P, 109P, 2003年
JAPANESE PHARMACOLOGICAL SOC, 英語, 研究発表ペーパー・要旨（国際会議）

膀胱平滑筋の自発性収縮調節におけるBKチャネルの役割　　　　　　　　　　　　　　　
田中 芳夫, 堀之内 孝広, 田中 光, 重信 弘毅, 小池 勝夫, 日本薬理学雑誌, 120, 補冊1, 106P, 108P, 2002年11月
膀胱平滑筋に高密度に発現している電位依存性Ca2+感受性K+チャネル(BKチャネル)の役割を,自発性収縮及び活動電位の制御の観点から薬理学的に検討した.モルモット摘出膀胱標本で記録した自発性収縮と活動電位はいずれも筋原性の性質を示し,活動電位はCa2+チャネルブロッカーで完全に消失したが,自発性収縮は抵抗性を示した.K+チャネルブロッカーのうちで,BKチャネルに選択性を有するIberiotoxin(IbTx)に強い収縮増強効果を認めた.IbTxは活動電位の頻度を顕著に増加させ,活動電位持続時間も有意に延長させた.BKチャネルのα-サブユニットに作用するNS-1619,ninumic acid及びβ-サブユニットに作用するestradiol,tamoxifenは,BKチャネルの開口確率を増大させ,活動電位の頻度を有意に減少さ,自発性収縮も顕著に抑制した.IbTxはこの膀胱運動の発生頻度を増大させた, (公社)日本薬理学会, 日本語

モルモット胃底におけるβ3-アドレナリン受容体を介した弛緩反応に関与する細胞内情報伝達系の解析と電位依存性K+ channelの役割　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 小池 勝夫, 日本薬理学雑誌, 120, 補冊1, 109P, 111P, 2002年11月
モルモット胃底平滑筋を用いて,β3-アドレナリン受容体(β3-AR)を介した弛緩反応におけるcAMpの役割と,PKAの標的蛋白の一つであるK+チャネルの関与を検討した.β3-ARを介する消化管平滑筋の弛緩反応では,cAMP非依存性に活性化される4-AP感受性K+チャネルが関与し,血管及び気管平滑筋においてBKCaチャネルの活性化が関与するβ1-,β2-ARを介した弛緩反応との相違が明らかとなった, (公社)日本薬理学会, 日本語

モルモット胃底及び十二指腸におけるβ3-アドレナリン受容体を介したcyclic AMP非依存性弛緩反応について　　　　　　　　　　　　　　　
堀之内 孝広, 田中 芳夫, 辻谷 典彦, 小池 勝夫, 日本薬理学雑誌, 120, 4, 9P, 9P, 2002年10月
(公社)日本薬理学会, 日本語

膀胱平滑筋の自発性収縮の発生と活動電位の関連性　　　　　　　　　　　　　　　
田中 芳夫, 岡本 貴男, 今井 利安, 堀之内 孝広, 田中 光, 重信 弘毅, 小池 勝夫, 日本薬理学雑誌, 120, 4, 9P, 9P, 2002年10月
(公社)日本薬理学会, 日本語

ラット腹部大動脈におけるAtypical β-アドレナリン受容体を介した弛緩反応の特徴
松下 真由美, 堀之内 孝広, 田中 芳夫, 小池 勝夫, Journal of Smooth Muscle Research Japanese Section, 6, 1, J26, J26, 2002年04月
日本平滑筋学会, 日本語

モルモット膀胱平滑筋の自発性収縮の性質とBKチャネルの役割の検討
田中 芳夫, 今井 利安, 岡本 貴男, 堀之内 孝広, 田中 光, 小池 勝夫, 重信 弘毅, Journal of Smooth Muscle Research Japanese Section, 6, 1, J29, J29, 2002年04月
日本平滑筋学会, 日本語

IP受容体刺激に伴うMaxiK channel活性化を介する血管弛緩反応機構の解析　　　　　　　　　　　　　　　
田中 芳夫, 八巻 史子, 加賀 桃子, 堀之内 孝広, 田中 光, 小池 勝夫, Toro Ligia, 重信 弘毅, 日本薬学会年会要旨集, 122年会, 4, 35, 35, 2002年03月
(公社)日本薬学会, 日本語

選択的β_3-アドレナリン受容体拮抗薬SR59230Aのβ_3-アドレナリン受容体活性作用について
堀之内 孝広, 小池 勝夫, 日本薬理学雑誌 : FOLIA PHARMACOLOGICA JAPONICA, 118, 1, 125P, 126P, 2001年10月20日
日本語

IP受容体刺激を介する血管弛緩反応におけるMaxiK channelの役割とその活性化機序について　　　　　　　　　　　　　　　
八巻 史子, 田中 芳夫, 堀之内 孝広, 田中 光, 小池 勝夫, Ligia Toro, 重信 弘毅, 日本薬理学雑誌, 118, 4, 3P, 3P, 2001年10月
(公社)日本薬理学会, 日本語

47. β_3-アドレナリン受容体に対するArylethanolamine類の構造活性相関に関する研究
堀之内 孝広, 小池 勝夫, Journal of smooth muscle research. Japanese section, 5, 1, J, 42, 2001年04月27日
日本平滑筋学会, 日本語

9. モルモット胃底におけるβ_3-アドレナリン受容体に対するカルテオロールの部分活性作用について
堀之内 孝広, 辻谷 典彦, 小池 勝夫, Journal of smooth muscle research. Japanese section, 4, 1, J, 21, 2000年04月30日
日本平滑筋学会, 日本語

モルモット盲腸紐におけるイソプレナリン及びサルブタモールによる弛緩反応に関与するβ-受容体サブタイプについて
堀之内 孝広, 小池 勝夫, Journal of smooth muscle research. Japanese section, 2, 1, J, 53, 1998年04月30日
日本平滑筋学会, 日本語

beta(2)- and beta(3)-adrenoceptors mediated relaxation induced by various beta-adrenergic drugs in guinea pig taenia caecum
K Koike, T Horinouchi, NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 358, 1, R252, R252, 1998年
SPRINGER VERLAG, 英語, 研究発表ペーパー・要旨（国際会議）

モルモット毛様体におけるベフノロールおよびカルテオロールの立体特異性について
小池 勝夫, 堀之内 孝広, 高柳 一成, 眼薬理 = Japanese journal of ocular pharmacology, 9, 1, 31, 33, 1995年08月20日
日本語

モルモット毛様体におけるベフノロールおよびカルテオロールの立体特異性について
小池 勝夫, 堀之内 孝広, 高柳 一成, あたらしい眼科 = Journal of the eye, 12, 4, 629, 631, 1995年04月30日
日本語

Using Phos-tag in Western blotting analysis to evaluate protein phosphorylation　　　　　　　　　　　　　　　
Horinouchi T, Terada K, Higashi T, Miwa S.
Springer, 2016年, ［分担執筆］

LRRK2のリン酸化活性はfMLP刺激による好中球のケモタキシスに関与する　　　　　　　　　　　　　　　
真崎 雄一, 半田 悠, 麓 佳月, 堀之内 孝広, 小野寺 康
第47回日本分子生物学会年会, 2024年11月29日, 日本語, ポスター発表
2024年11月26日 - 2024年11月29日

慢性閉塞性肺疾患 (COPD) の疾患進行に及ぼす動脈硬化の影響．　　　　　　　　　　　　　　　
安井秀樹, 大石享平, 井上裕介, 鈴木勇三, 穗積宏尚, 柄山正人, 古橋一樹, 藤澤朋幸, 榎本紀之, 堀之内孝広, 岩城孝行, 鈴木優子, 乾直輝, 須田隆文
第46回日本血栓止血学会学術集会, 2024年06月13日, 日本語, 口頭発表（一般）
2024年06月13日 - 2024年06月15日

高温加熱式たばこ主流煙抽出物暴露による脳血管内皮細胞および神経細胞の遺伝子発現の変動　　　　　　　　　　　　　　　
道具伸也, 田中泰圭, 田村理美, 有留尚孝, 高田芙友子, 岩尾卓朗, 安永美保, 横谷みき, 溝口絢子, 堀之内孝広
日本薬学会第144年会, 日本語
2024年03月28日 - 2024年03月31日

加熱式たばこ主流煙抽出物の脳血管内皮細胞傷害機序　　　　　　　　　　　　　　　
有留尚孝, 高田芙友子, 岩尾卓郎, 田中泰佳, 横谷みき, 安永美保, 溝口絢子, 堀之内孝広, 道具伸也
日本薬学会第144年会, 日本語
2024年03月28日 - 2024年03月31日

加熱式たばこ研究について．　　　　　　　　　　　　　　　
堀之内孝広
第4回加熱式たばこ研究討論会, 2024年03月11日, 日本語, 口頭発表（招待・特別）
2024年03月11日 - 2024年03月11日, 日本国, ［招待講演］, ［国内会議］

血管平滑筋細胞に対する加熱式たばこ主流煙ガス相水抽出物の細胞傷害作用
堀之内 孝広, 三輪 聡一
第97回日本薬理学会年会, 2023年12月15日, 英語, ポスター発表
2023年12月14日 - 2023年12月16日, 36604331

ヒトiPS細胞の内胚葉分化に対する加熱式タバコ由来抽出液の影響　　　　　　　　　　　　　　　
平田 尚也, 堀之内 孝広, 諫田 泰成
第97回日本薬理学会年会, 2023年12月15日, 英語, ポスター発表
2023年12月14日 - 2023年12月16日

ヒトiPS細胞から誘導した前脳オルガノイドの神経発生における高温加熱式たばこ主流煙抽出物の影響．　　　　　　　　　　　　　　　
田中泰圭, 藤本伊依那, 西田朱里, 堀之内孝広, 道具伸也
第76回日本薬理学会西南部会, 2023年10月07日, 日本語, 口頭発表（一般）
2023年10月07日 - 2023年10月07日

たばこ煙ガス相水抽出物による血管内皮機能障害機序の薬理学的解析：紙巻きたばこと加熱式たばこの比較研究．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第37回喫煙科学研究財団助成研究発表会, 2023年07月20日, 日本語, 口頭発表（一般）
2023年07月20日 - 2023年07月20日

高温加熱式たばこ主流煙抽出物の脳血管構成細胞に対する作用．　　　　　　　　　　　　　　　
道具伸也, 神戸翔太, 高田芙友子, 岩尾卓朗, 松本純一, 安永美保, 横谷みき, 堀之内孝広
日本薬学会第143年会, 日本語, ポスター発表
2023年03月25日 - 2023年03月28日

加熱式たばこについて．　　　　　　　　　　　　　　　
堀之内 孝広
第3回加熱式たばこ研究討論会, 2023年03月17日, 日本語, 口頭発表（招待・特別）
2023年03月17日 - 2023年03月17日, ［招待講演］

加熱式タバコが，ヒト口腔がん細胞の細胞増殖を来す可能性と，その機序について．　　　　　　　　　　　　　　　
長尾景充, 梅村将就, 髙安笙太, 鈴木文菜, 水野雄斗, 石川聡一郎, 中鍛冶里奈, 永迫茜, 堀之内孝広, 石川義弘, 山本哲哉
日本生理学会第100回記念大会, 日本語, 口頭発表（一般）
2023年03月14日 - 2023年03月16日

加熱式タバコの口腔がん細胞への影響．　　　　　　　　　　　　　　　
高安笙太, 梅村将就, 長尾景充, 中鍛冶里奈, 永迫茜, 石川聡一郎, 水野雄斗, 鈴木文菜, 堀之内孝広, 石川義弘
日本生理学会第100回記念大会, 日本語, 口頭発表（一般）
2023年03月14日 - 2023年03月16日

Effects of cigarette smoke extracts (combustible and heated tobacco) on rat and human iPS cell-derived cardiomyocytes.　　　　　　　　　　　　　　　
Yasuda J, Matsumura S, Notomi T, Chen I.S, Horinouchi T, Nakamura T.Y
67th Annual Biophysical Society Meeting, 英語, 口頭発表（一般）
2023年02月18日 - 2023年02月22日

ラット心筋細胞の自動拍動数と収縮機能および細胞内カルシウム動態に及ぼす従来式および加熱式タバコのタバコ煙抽出液の影響の比較検討．　　　　　　　　　　　　　　　
安田純平, 松村早季子, 堀之内孝広, 西谷(中村)友重
第32回日本循環薬理学会, 2023年01月27日, 日本語, 口頭発表（一般）

血管内皮細胞において，加熱式たばこ主流煙ガス相水抽出物は，鉄依存性，フェロトーシス非依存性の細胞傷害を引き起こす．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第96回日本薬理学会年会, 日本語, 口頭発表（一般）
2022年11月30日 - 2022年12月03日

紙巻きタバコおよび加熱式タバコのタバコ煙抽出液がラット心室筋細胞の収縮機能，自動拍動数および細胞内Ca2+動態に及ぼす影響の比較検討．　　　　　　　　　　　　　　　
安田純平, 松村早希子, 陳以珊, 納富拓也, 堀之内孝広, 西谷(中村)友重
第96回日本薬理学会年会, 日本語, 口頭発表（一般）
2022年11月30日 - 2022年12月03日

加熱式タバコ由来抽出液は肺癌細胞株の癌幹細胞性を促進する．　　　　　　　　　　　　　　　
平田尚也, 堀之内孝広, 諫田泰成
第96回日本薬理学会年会, 日本語, 口頭発表（一般）
2022年11月30日 - 2022年12月03日

Astrocyte及び悪性神経膠腫における加熱式タバコと燃焼タバコのガス相抽出物の細胞毒性について．　　　　　　　　　　　　　　　
長尾景充, 梅村将就, 髙安笙太, 堀之内孝広, 石川義弘, 山本哲哉
日本脳神経外科学会第81回学術総会, 日本語, 口頭発表（一般）
2022年09月28日 - 2022年10月01日

種々のタバコ煙抽出液がラット心室筋細胞の収縮機能，自動拍動数および細胞内Ca2+動態に及ぼす影響の検討：紙巻きタバコおよび加熱式タバコ煙の比較．　　　　　　　　　　　　　　　
安田純平, 松村早季子, 陳以珊, 納冨拓也, 堀之内孝広, 西谷(中村)友重
第165回日本獣医学会学術集会, 日本語, 口頭発表（一般）
2022年09月06日 - 2022年09月08日

たばこ煙ガス相水抽出物による血管内皮機能障害機序の薬理学的解析：紙巻きたばこと加熱式たばこの比較研究．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第36回喫煙科学研究財団助成研究発表会, 2022年07月21日, 日本語, 口頭発表（一般）

メラノーマ細胞においてAnnexin A2のリン酸化はエンドセリン-1刺激によるAKTの活性化に関与する．　　　　　　　　　　　　　　　
真崎雄一, 東恒仁, 堀之内孝広, 三輪聡一
第95回日本薬理学会年会, 英語, ポスター発表
2022年03月07日 - 2022年03月09日

加熱式たばこ及び紙巻きたばこの主流煙ガス相水抽出物に含まれるカルボニル化合物は血管内皮機能障害を引き起こす．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第95回日本薬理学会年会, 英語, ポスター発表
2022年03月07日 - 2022年03月09日

加熱式たばこについて．　　　　　　　　　　　　　　　
堀之内孝広
第2回加熱式たばこ研究討論会, 2022年01月26日, 日本語, 口頭発表（招待・特別）
［招待講演］

たばこ煙ガス相水抽出物による血管内皮機能障害機序の薬理学的解析：紙巻きたばこと加熱式たばこの比較研究．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第35回喫煙科学研究財団助成研究発表会, 2021年09月02日, 日本語, 口頭発表（一般）

加熱式たばこ及び紙巻きたばこの主流煙ガス相水抽出物がヒト血管内皮細胞に及ぼす影響．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 三輪聡一
第94回日本薬理学会年会, 英語, 口頭発表（一般）
2021年03月08日 - 2021年04月10日

メラノーマ細胞において，Annexin A2は、エンドセリン-1の刺激によるAKTの活性化に関与する．　　　　　　　　　　　　　　　
真崎雄一, 東恒仁, 堀之内孝広, 三輪聡一
第94回日本薬理学会年会, 英語, 口頭発表（一般）
2021年03月08日 - 2021年03月11日

加熱式たばこ主流煙水抽出物の調製方法．　　　　　　　　　　　　　　　
堀之内孝広
第1回加熱式たばこ研究討論会, 2021年02月09日, 日本語, 口頭発表（招待・特別）
［招待講演］

たばこ煙ガス相水抽出物による血管内皮機能障害機序の薬理学的解析：紙巻きたばこと加熱式たばこの比較研究．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一
第34回喫煙科学研究財団助成研究発表会, 2020年09月08日, 日本語, その他

血管内皮細胞における加熱式たばこ主流煙水抽出物の作用解析．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 三輪聡一
第71回日本薬理学会北部会, 2020年09月04日, 日本語, 口頭発表（一般）

カルシウム感受性受容体-Gq/11タンパク質シグナル伝達系はヒト血管内皮細胞からの一酸化窒素遊離に関与する．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 三輪聡一
第93回日本薬理学会年会, 英語, その他
2020年03月16日 - 2020年03月18日

Involvement of Ca2+-sensing receptor-Gq/11 protein signaling pathway in nitric oxide release from human vascular endothelial cells.　　　　　　　　　　　　　　　
Horinouchi T, Mazaki Y, Miwa S
Pharmacology 2019., 2019年12月, British Pharmacological Society., 英語, ポスター発表
2019年12月15日 - 2019年12月17日, Edinburgh, Scotland, グレートブリテン・北アイルランド連合王国(英国)

血管内皮細胞におけるカルシウム感受性受容体を介した一酸化窒素産生機序．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 寺田晃士, 三輪聡一
第70回日本薬理学会北部会, 2019年09月20日, 日本語, 口頭発表（一般）

GRP78はエンドセリンB受容体を介したERKの活性化を促す．　　　　　　　　　　　　　　　
真崎雄一, 東恒仁, 小野寺康仁, 南ジンミン, 橋本あり, 橋本茂, 堀之内孝広, 三輪聡一
第92回日本薬理学会年会, 英語, 口頭発表（一般）
2019年03月14日 - 2019年03月16日

ヒト血管内皮細胞の一酸化窒素合成酵素の活性化におけるカルシウム感受性受容体の関与．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 三輪聡一
第92回日本薬理学会年会, 英語, 口頭発表（一般）
2019年03月14日 - 2019年03月16日

血管内皮細胞におけるカルシウム感受性受容体を介したNO産生に対するタバコ煙ガス相水抽出物及びアクロレインの抑制作用．　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 三輪聡一
第69回日本薬理学会北部会, 2018年09月21日, 日本語, 口頭発表（一般）

青黛の摂取に伴う肺動脈性肺高血圧症の発症機序に関する薬理学的考察．　　　　　　　　　　　　　　　
堀之内孝広, 大平洋, 佐藤一紀, 崎山信哉, 中谷資隆, 中村順一, 鈴木奈緒子, 杉本絢子, 林下晶子, 渡部拓, 真崎雄一, 三輪聡一, 西村正治, 辻野一三
第60回日本平滑筋学会総会, 日本語, ポスター発表
2018年08月16日 - 2018年08月18日

たばこ煙ガス相中の不飽和カルボニル化合物の標的タンパク質の同定とカルボニル化によるタンパク質・細胞の機能変化の解析．　　　　　　　　　　　　　　　
三輪聡一, 堀之内孝広, 東恒仁, 真崎雄一
第33回喫煙科学研究財団助成研究発表会, 2018年07月24日, 日本語, 口頭発表（一般）

Molecular mechanism for negative regulation of insulin-stimulated Akt phosphorylation and glucose uptake by endothelin-1 in skeletal muscle cells.　　　　　　　　　　　　　　　
Horinouchi T, Mazaki Y, Hoshi A, Terada T, Miwa S
18th World Congress of Basic and Clinical Pharmacology, 英語, ポスター発表
2018年07月01日 - 2018年07月06日

青黛起因性肺動脈性肺高血圧症の発症メカニズムについて．　　　　　　　　　　　　　　　
大平洋, 堀之内孝広, 佐藤一紀, 中谷資隆, 杉本絢子, 渡部拓, 西村正治, 辻野一三
第3回日本肺高血圧・肺循環学会学術集会, 日本語, 口頭発表（一般）
2018年06月22日 - 2018年06月23日

たばこ煙ガス相中に含まれる不飽和カルボニル化合物の血管壁構成細胞に対する作用．　　　　　　　　　　　　　　　
堀之内孝広, 東恒仁, 真崎雄一, 三輪聡一
第27回日本循環薬理学会, 2017年12月01日, 日本語, 口頭発表（一般）

血管内皮細胞におけるアクロレインの作用解析．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 真崎雄一, 東恒仁
第68回日本薬理学会北部会, 日本語, 口頭発表（一般）
2017年09月15日 - 2017年09月16日

たばこ煙ガス相中の不飽和カルボニル化合物の標的タンパク質の同定とカルボニル化によるタンパク質・細胞の機能変化の解析．　　　　　　　　　　　　　　　
三輪聡一, 堀之内孝広, 東 恒仁, 真崎 雄一
第32回喫煙科学研究財団助成研究発表会, 2017年07月27日, 日本語, 口頭発表（一般）

ISAの考え方と安全性について．　　　　　　　　　　　　　　　
堀之内孝広
Keep the visual field in 札幌, 2017年04月02日, 日本語, 口頭発表（招待・特別）
［招待講演］

エンドセリン受容体トラフィック研究の新展開　　　　　　　　　　　　　　　
堀之内孝広, 真崎雄一, 東恒仁, 寺田晃士, 三輪聡一
第90回日本薬理学会年会, シンポジウム・ワークショップパネル（指名）
2017年03月15日 - 2017年03月17日, ［招待講演］

ISAのフシギ　　　　　　　　　　　　　　　
堀之内孝広
ミケルナ配合点眼液 発売記念講演会, 2017年02月04日, 日本語, 口頭発表（招待・特別）
［招待講演］

ISAの考え方と安全性について．　　　　　　　　　　　　　　　
堀之内孝広
ミケルナ配合点眼液 発売記念講演会, 2017年01月14日, 日本語, 口頭発表（招待・特別）
［招待講演］

Phos-tag biotinを用いた受容体作動性TRPC6チャネルのPKAリン酸化部位の同定　　　　　　　　　　　　　　　
堀之内孝広, 寺田晃士, 東恒仁, 真崎雄一, 三輪聡一
第67回日本電気泳動学会総会, シンポジウム・ワークショップパネル（指名）
2016年08月25日 - 2016年09月26日, ［招待講演］

β受容体の最新の考え方．　　　　　　　　　　　　　　　
堀之内孝広
第27回日本緑内障学会 モーニングセミナー3, 2016年09月18日, 日本語, 公開講演，セミナー，チュートリアル，講習，講義等
［招待講演］

たばこ煙ガス相中の不飽和カルボニル化合物の標的タンパク質の同定とカルボニル化によるタンパク質・細胞の機能変化の解析．　　　　　　　　　　　　　　　
三輪聡一, 東恒仁, 堀之内孝広, 真崎雄一
第31回喫煙科学研究財団助成研究発表会, 2016年07月20日, 日本語, 口頭発表（一般）

Pharmacological and functional implication of heterodimer formation of endothelin type A and type B receptors.　　　　　　　　　　　　　　　
Horinouchi T
Actelion Academia Prize 2016, 2016年07月02日, 英語, 口頭発表（招待・特別）
［招待講演］

タバコ煙ガス相水抽出液の簡便な標準的作製法の開発．　　　　　　　　　　　　　　　
真崎雄一, 東恒仁, 眞井洋輔, 堀之内孝広, 三輪聡一
第89回日本薬理学会年会, 英語, ポスター発表
2016年03月09日 - 2016年03月11日

タバコ煙ガス相による細胞傷害の抑制因子の探索と抑制メカニズムの解明．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 真崎雄一, 三輪聡一
第89回日本薬理学会年会, 英語, 口頭発表（一般）
2016年03月09日 - 2016年03月11日

エンドセリンA型受容体のインターナリゼーションにおけるCa2+動員機構の関与．　　　　　　　　　　　　　　　
堀之内孝広, サリタ・カルキ, 真崎雄一, 東恒仁, 三輪聡一
89回日本薬理学会年会, 英語, 口頭発表（一般）
2016年03月09日 - 2016年03月11日

タバコ煙に起因する細胞傷害の抑制物質の探索と抑制メカニズムの解明．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 真崎雄一, 堀口美香, 三輪聡一
第25回日本循環薬理学会, 2015年12月04日, 日本語, 口頭発表（一般）

エンドセリンA型受容体のインターナリゼーションに関与する細胞内領域の同定．　　　　　　　　　　　　　　　
堀之内孝広, カルキ・サリタ, 真崎雄一, 東恒仁, 堀口美香, 三輪聡一
第25回日本循環薬理学会, 2015年12月04日, 日本語, 口頭発表（一般）

エンドセリン受容体シグナルを標的にした肺高血圧症治療の新展開　　　　　　　　　　　　　　　
堀之内孝広, 堀口美香, 真崎雄一, 東恒仁, 原田拓弥, 三輪聡一
第4回日本肺循環学会・第3回日本肺高血圧学会 合同学術集会, シンポジウム・ワークショップパネル（指名）
2015年10月 - 2015年10月04日, ［招待講演］

タバコ煙に起因する細胞傷害の抑制方法の開発及びその分子機構の解明．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 真崎雄一, 堀口美香, 三輪聡一
第66回日本薬理学会北部会, 2015年09月18日, 日本語, 口頭発表（一般）

エンドセリン受容体を介して惹起されるインスリン抵抗性におけるGタンパク質共役型受容体キナーゼ2の関与．　　　　　　　　　　　　　　　
堀之内孝広, 原田拓弥, 東恒仁, 真崎雄一, カルキ・サリタ, 堀口美香, 三輪聡一
第66回日本薬理学会北部会, 2015年09月18日, 日本語, 口頭発表（一般）

タバコ煙抽出液で同定された細胞傷害活性因子群の心血管系に対する影響の解析と傷害活性除去法開発の試み．　　　　　　　　　　　　　　　
三輪聡一, 東恒仁, 堀之内孝広, 真崎雄一
第30回喫煙科学研究財団助成研究発表会, 2015年07月23日, 日本語, 口頭発表（一般）

「肺血管リモデリングの発症機序に迫る」エンドセリン受容体シグナルからの視点　　　　　　　　　　　　　　　
堀之内孝広
第146回東邦医学会例会, 2015年06月08日, シンポジウム・ワークショップパネル（指名）
［招待講演］

エンドセリンA型およびB型受容体の細胞内動態制御におけるユビキチン化の役割　　　　　　　　　　　　　　　
三輪聡一, 寺田晃士, 堀之内孝広, 堀口美香, 真崎雄一, 東恒仁
第12回GPCR研究会, 口頭発表（基調）
2015年05月15日 - 2015年05月16日, ［招待講演］

グルタチオン・N-アセチルシステイン・システインはタバコ煙ガス相抽出物による細胞傷害に対する保護作用を有する．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, エルメリジ・イナス, 堀之内孝広, 寺田晃士, 星暁壮, 堀口美香, ネパル・プラハ, サリタ・カルキ, 旗手千鶴, 三輪聡一
第88回日本薬理学会年会, 英語, 口頭発表（一般）
2015年03月18日 - 2015年03月20日

ラット骨格筋細胞においてエンドセリン-1によって惹起されるインスリン抵抗性の分子メカニズムの解明．　　　　　　　　　　　　　　　
堀之内孝広, 星暁壮, 原田拓弥, サリタ・カルキ, 東恒仁, 寺田晃士, 眞井洋輔, ネパル・プラハ, 堀口美香, 三輪聡一
第88回日本薬理学会年会, 英語, 口頭発表（一般）
2015年03月18日 - 2015年03月20日

肺高血圧症治療薬の最近の進歩：エンドセリンシステムからみた作用機序　　　　　　　　　　　　　　　
堀之内孝広, 寺田晃士, 東恒仁, 星暁壮, サリタ・カルキ, 三輪聡一
第88回日本薬理学会年会, シンポジウム・ワークショップパネル（指名）
2015年03月18日 - 2015年03月20日, ［招待講演］

エンドセリン-1によって誘発されるPDGF受容体のトランス活性化を介したERK1/2のリン酸化機構．　　　　　　　　　　　　　　　
堀之内孝広, 星暁壮, 原田拓弥, Karki Sarita, 東恒仁, 寺田晃士, Nepal Prabha, 堀口美香, 三輪聡一
第24回日本循環薬理学会, 2014年12月05日, 日本語, 口頭発表（一般）

タバコ煙による細胞傷害の定量的検出方法の確立と細胞傷害抑制物質の探索．　　　　　　　　　　　　　　　
旗手千鶴, 東恒仁, Elmeligy Enas, 眞井洋輔, 堀之内孝広, 寺田晃士, 星暁荘, Nepal Prabha, Karki Sarita, 堀口美香, 原田拓弥, 三輪聡一
第65回日本薬理学会北部会, 日本語, 口頭発表（一般）
2014年09月26日 - 2014年09月27日

エンドセリンB型受容体の細胞内動態制御におけるユビキチン化の役割．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, 堀口美香, Nepal Prabha, Karki Sarita, 旗手千鶴, 原田拓弥, 眞井洋輔, 星暁荘, 三輪聡一
第65回日本薬理学会北部会, 日本語, 口頭発表（一般）
2014年09月26日 - 2014年09月27日

骨格筋細胞におけるインスリン誘発性グルコース取り込み促進に対するエンドセリン-1の抑制作用機序の解明．　　　　　　　　　　　　　　　
堀之内孝広, 星暁荘, 原田拓弥, Karki Sarita, 寺田晃士, 東恒仁, 比嘉綱己, 眞井洋輔, Nepal Prabha, 堀口美香, 旗手千鶴, 三輪聡一
第65回日本薬理学会北部会, 日本語, 口頭発表（一般）
2014年09月26日 - 2014年09月27日

Role of phosphorylation in cytoplasmic carboxyl-terminal tail of endothelin type B receptor in ET-1 induced receptor desensitization.　　　　　　　　　　　　　　　
Nepal P, Terada K, Horinouchi T, Horiguchi M, Higashi T, Hatate C, Hoshi A, Karki S, Harada T, Mai Y, Miwa S
第28回北海道薬物作用談話会, 2014年07月19日, 英語, 口頭発表（一般）

タバコ煙抽出液で同定された細胞傷害活性因子群の心血管系に対する影響の解析と傷害活性除去法開発の試み．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 眞井 洋輔, 東 恒仁, 比嘉 綱己, 旗手 千鶴, 寺田 晃士
第29回喫煙科学研究財団助成研究発表会, 2014年07月15日, 日本語, 口頭発表（一般）

Molecular mechanism for negative regulation of insulin-stimulated glucose uptake by endothelin-1 in skeletal muscle cells.　　　　　　　　　　　　　　　
Horinouchi T
1st Actelion Academic Forum in Pulmonary Hypertension, 2014年07月05日, 英語, 口頭発表（招待・特別）
［招待講演］

肺高血圧症治療薬の現状と今後の展望－作用機序と創薬を中心に－　　　　　　　　　　　　　　　
堀之内 孝広
第5回東京肺高血圧症研究会, 2014年06月14日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

エンドセリンB型受容体のトラフィッキングにおける受容体ユビキチン化の役割．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, Nepal Prabha, 堀口美香, 旗手千鶴, 星暁壮, 眞井洋輔, 原田拓弥, 三輪聡一
第87回日本薬理学会年会, 英語, 口頭発表（一般）
2014年03月19日 - 2014年03月21日

タバコ煙ガス相中の細胞傷害成分の同定と細胞傷害メカニズムの解析．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 寺田晃士, 星暁壮, 堀口美香, Nepal Prabha, 原田拓弥, 旗手千鶴, 三輪聡一
第87回日本薬理学会年会, 英語, 口頭発表（一般）
2014年03月19日 - 2014年03月21日

ラット骨格筋細胞においてエンドセリン-1により活性化されるシグナル伝達機構の解明．　　　　　　　　　　　　　　　
星暁壮, 堀之内孝広, 原田拓弥, 比嘉綱己, 東恒仁, 寺田晃士, 眞井洋輔, ネパル・プラハ, 堀口美香, 旗手千鶴, 三輪聡一
第87回日本薬理学会年会, 英語, 口頭発表（一般）
2014年03月19日 - 2014年03月21日

ユビキチン化によるエンドセリン受容体の細胞内トラフィッキング制御　　　　　　　　　　　　　　　
三輪聡一, 寺田晃士, 東恒仁, Nepal Prabha, 堀之内孝広
第87回日本薬理学会年会, 2014年03月19日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

エンドセリン受容体シグナルソーム形成からみた治療戦略
堀之内孝広, 寺田晃士, 東恒仁, 星暁壮, 三輪聡一
第87回日本薬理学会年会, 2014年03月19日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

エンドセリン-1によるインスリン-Aktシグナル伝達の抑制性制御におけるGタンパク質共役型受容体キナーゼ2の関与．　　　　　　　　　　　　　　　
堀之内孝広, 原田拓弥, 星暁壮, 東恒仁, 寺田晃士, 比嘉綱己, 眞井洋輔, Nepal Prabha, 堀口美香, 旗手千鶴, 三輪聡一
第23回日本循環薬理学会, 2013年12月06日, 日本語, 口頭発表（一般）

ニコチン及びタール除去タバコ煙抽出物中に含まれる細胞傷害因子の性状解析．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 寺田晃士, 旗手千鶴, 星暁壮, Prabha Nepal, 堀口美香, 三輪聡一
第64回日本薬理学会北部会, 2013年09月13日, 日本語, 口頭発表（一般）

エンドセリンB型受容体のET-1刺激誘発的な細胞内移行とシグナルの制御におけるユビキチン修飾の役割．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, Nepal Prabha, 堀口美香, 旗手千鶴, 星暁壮, 三輪聡一
第64回日本薬理学会北部会, 2013年09月13日, 日本語, 口頭発表（一般）

骨格筋細胞におけるエンドセリン受容体を介したインスリン・シグナルの抑制性制御機構の解明．　　　　　　　　　　　　　　　
堀之内孝広, 原田拓弥, 東恒仁, 寺田晃士, 比嘉綱己, 眞井洋輔, 星暁壮, Nepal Prabha, 堀口美香, 旗手千鶴, 三輪聡一
第64回日本薬理学会北部会, 2013年09月13日, 日本語, 口頭発表（一般）

Endothelin-1 activates extracellular signal-regulated kinases 1 and 2 through transactivation of platelet-derived growth factor receptor in skeletal muscle cells.　　　　　　　　　　　　　　　
Higashi T, Horinouchi T, Harada T, Higa T, Terada K, Hoshi A, Mai Y, Horiguchi M, Nepal P, Hatate C, Miwa S
13th International Conference on Endothelin, 英語, ポスター発表
2013年09月08日 - 2013年09月11日

Negative regulation of endothelin type A receptor-operated TRPC6 channel by adenylate cyclase-cAMP-protein kinase A signaling pathway.　　　　　　　　　　　　　　　
Horinouchi T, Higa T, Higashi T, Terada K, Mai Y, Harada T, Hoshi A, Horiguchi M, Nepal P, Hatate C, Miwa S
13th International Conference on Endothelin, 英語, ポスター発表
2013年09月08日 - 2013年09月11日

Ubiquitin modification plays an important role in ET-1-dependent endothelin type B receptor trafficking.　　　　　　　　　　　　　　　
Terada K, Horinouchi T, Higashi T, Nepal P, Horiguchi M, Hatate C, Hoshi A, Mai Y, Miwa S
13th International Conference on Endothelin, 英語, 口頭発表（一般）
2013年09月08日 - 2013年09月11日

Molecular mechanism for suppression of insulin signaling by endothelin-1 in skeletal muscle cells.　　　　　　　　　　　　　　　
Horinouchi T, Harada T, Higa T, Higashi T, Terada K, Hoshi A, Mai Y, Horiguchi M, Nepal P, Hatate C, Miwa S
13th International Conference on Endothelin, 英語, 口頭発表（一般）
2013年09月08日 - 2013年09月11日

Gタンパク質共役型受容体シグナリングの多様性：β3-アドレナリン受容体及びエンドセリンA型受容体に関する検討．　　　　　　　　　　　　　　　
堀之内孝広, 佐藤将哲, 東恒仁, 寺田 晃士, Roger James Summers, 三輪聡一
第17回活性アミンに関するワークショップ, 2013年08月24日, 日本語, 口頭発表（一般）

タバコ煙抽出液で同定された細胞傷害活性因子群の心血管系に対する影響の解析と傷害活性除去法開発の試み．　　　　　　　　　　　　　　　
堀之内孝広,三輪聡一,眞井洋輔,東恒仁,比嘉綱己,旗手千鶴,寺田晃士
第28回喫煙科学研究財団助成研究発表会, 2013年07月22日, 日本語, 口頭発表（一般）

エンドセリン-1による骨格筋インスリン・シグナルの抑制性制御機構の解明．　　　　　　　　　　　　　　　
原田拓弥, 堀之内孝広, 東恒仁, 寺田晃士, 比嘉綱己, 眞井洋輔, 星暁壮, Nepal Prabha, 堀口美香, 旗手千鶴, 三輪聡一
第27回北海道薬物作用談話会, 2013年07月20日, 日本語, 口頭発表（一般）

ニコチン及びタール除去タバコ煙水抽出物はCa2+依存的にPKCを活性化することで細胞傷害を引き起こす．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 堀之内孝広, 寺田晃士, 旗手千鶴, 三輪聡一
第86回日本薬理学会年会, 英語, 口頭発表（一般）
2013年03月21日 - 2013年03月23日

エンドセリンB型受容体の動態制御におけるユビキチン化修飾の役割．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, 旗手千鶴, 三輪聡一
第86回日本薬理学会年会, 英語, 口頭発表（一般）
2013年03月21日 - 2013年03月23日

小胞体Ca2+センサーSTIM1の新規スプライスバリアントSTIM1Lの同定とその薬理学的特徴．　　　　　　　　　　　　　　　
堀之内孝広, 東恒仁, 寺田晃士, 比嘉綱己, 眞井洋輔, 旗手千鶴, ネパル・プラハ, 堀口美香, 原田拓弥, 三輪聡一
第86回日本薬理学会年会, 英語, 口頭発表（一般）
2013年03月21日 - 2013年03月23日

アデニル酸シクラーゼ-cAMP-プロテインキナーゼA系によるエンドセリン受容体活性化型TRPC6チャネルを介した受容体作動性Ca2+流入の抑制性制御．　　　　　　　　　　　　　　　
堀之内孝広, 寺田晃士, 東恒仁, 比嘉綱己, 原田拓弥, 三輪聡一
第42回日本心脈管作動物質学会, 日本語, 口頭発表（一般）
2013年02月08日 - 2013年02月09日

Functional role of adenylate cyclase-cAMP-protein kinase A signaling pathway in regulation of endothelin type A receptor-operated Ca2+ entry via TRPC6 channel.　　　　　　　　　　　　　　　
Horinouchi T, Higa T, Terada K, Higashi T, Hatate C, Miwa S
BPS Winter Meeting 2012., 2012年12月, British Pharmacological Society., 英語, ポスター発表
2012年12月18日 - 2012年12月20日, London, グレートブリテン・北アイルランド連合王国(英国)

ユビキチン化によるエンドセリン受容体の動態制御機構の解析．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, 旗手千鶴, 三輪聡一
第22回日本循環薬理学会, 2012年11月30日, 日本語, 口頭発表（一般）

小胞体Ca2+センサーSTIM1とその新規スプライスバリアントによるTRPC3及びTRPC6チャネルを介したエンドセリンA型受容体作動性Ca2+流入の抑制性制御．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 東恒仁, 寺田晃士, 眞井洋輔, 原田拓弥, 旗手千鶴, 三輪聡一
第22回日本循環薬理学会, 2012年11月30日, 日本語, 口頭発表（一般）

循環動態改善薬の最前線－基礎から臨床応用へ－　　　　　　　　　　　　　　　
堀之内 孝広
第32回日本小児循環動態研究会学術集会, 2012年11月18日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

肺高血圧症の発症・進展に寄与するTRPC6チャネルの抑制性機能制御機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 東恒仁, 寺田晃士, 旗手千鶴, 三輪聡一
第1回日本肺循環学会学術集会, 2012年09月22日, 日本語, 口頭発表（一般）

エンドセリンB型受容体の動態制御におけるユビキチン化の役割の解析．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 東恒仁, 旗手千鶴, 三輪聡一
第63回日本薬理学会北部会, 2012年09月14日, 日本語, 口頭発表（一般）

ニコチン及びタール除去タバコ煙抽出物の細胞傷害作用に関与するPKCアイソザイムの同定と活性化機構の解明．　　　　　　　　　　　　　　　
東恒仁, 眞井洋輔, 寺田晃士, 旗手千鶴, 堀之内孝広, 三輪聡一
第63回日本薬理学会北部会, 2012年09月14日, 日本語, 口頭発表（一般）

小胞体Ca2+センサーSTIM1の新規バリアントの同定とその分子薬理学的特性の解析．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 東恒仁, 寺田晃士, 旗手千鶴, 三輪聡一
第63回日本薬理学会北部会, 2012年09月14日, 日本語, 口頭発表（一般）

ニコチン及びタール除去タバコ煙抽出液の細胞傷害作用に関与するPKCアイソザイムの同定とその活性化機構の解明．　　　　　　　　　　　　　　　
眞井洋輔, 東恒仁, 寺田晃士, 旗手千鶴, 堀之内孝広, 三輪聡一
第26回北海道薬物作用談話会, 2012年07月28日, 日本語, 口頭発表（一般）

タバコ煙抽出液で同定された細胞傷害活性因子群の心血管系に対する影響の解析と傷害活性除去法開発の試み．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 眞井洋輔, 東恒仁, 比嘉綱己, 旗手千鶴, 寺田晃士
第27回喫煙科学研究財団助成研究発表会, 2012年07月18日, 日本語, 口頭発表（一般）

エンドセリン受容体活性化型TRPC6チャネルの機能制御機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 青柳裕之, 東恒仁, 寺田晃士, 旗手千鶴, 三輪聡一
第8回TRPチャネル研究会, 日本語, 口頭発表（一般）
2012年06月14日 - 2012年06月15日

ニコチン・タール除去タバコ煙水抽出液によるNADPHオキシダーゼ依存性細胞傷害の分子メカニズム．　　　　　　　　　　　　　　　
眞井洋輔, 堀之内孝広, 朝野拓史, 澤田修, 西屋禎, 南雅文, 片山貴博, 寺田晃士, 三輪聡一
第85回日本薬理学会年会, 英語, 口頭発表（一般）
2012年03月14日 - 2012年03月16日

エンドセリン受容体の動態制御におけるユビキチン修飾の役割．　　　　　　　　　　　　　　　
寺田晃士, 堀之内孝広, 三輪聡一
第85回日本薬理学会年会, 英語, 口頭発表（一般）
2012年03月14日 - 2012年03月16日

エンドセリンA型受容体刺激によって誘発されるTRPC3及びTRPC6チャネルを介した受容体作動性Ca2+流入の制御機構．　　　　　　　　　　　　　　　
比嘉綱己, 堀之内孝広, 青柳裕之, 寺田晃士, 西屋禎, 三輪聡一
第85回日本薬理学会年会, 英語, 口頭発表（一般）
2012年03月14日 - 2012年03月16日

PKAリン酸化によるエンドセリンA型受容体作動性TRPC6チャネルの抑制性制御機構　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 青柳裕之, 寺田晃士, 西屋禎, 三輪聡一
第85回日本薬理学会年会, 英語, 口頭発表（一般）
2012年03月14日 - 2012年03月16日

プロテインキナーゼAによるTRPC6チャネルを介したエンドセリンA型受容体作動性Ca2+流入の抑制性制御の分子機構．　　　　　　　　　　　　　　　
比嘉綱己, 堀之内孝広, 青柳裕之, 西屋禎, 寺田晃士, 三輪聡一
第21回日本循環薬理学会, 2011年12月02日, 日本語, 口頭発表（一般）

エンドセリンA型受容体作動性TRPC6チャネルの抑制性機能制御に関与するPKAリン酸化部位の同定．　　　　　　　　　　　　　　　
堀之内孝広, 寺田晃士, 比嘉綱己, 青柳裕之, 西屋禎, 三輪聡一
第39回薬物活性シンポジウム, 日本語, 口頭発表（一般）
2011年11月21日 - 2011年11月22日

Phos-tag biotinを用いたエンドセリンA型受容体作動性TRPC6チャネルのPKAによるリン酸化部位の同定．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 青柳裕之, 西屋禎, 寺田晃士, 三輪聡一
第62回日本薬理学会北部会, 日本語, 口頭発表（一般）
2011年09月29日 - 2011年09月30日

誘導型NO合成酵素のユビキチン・プロテアソーム依存性分解制御機構．　　　　　　　　　　　　　　　
松本一馬, 西屋禎, 前川聡, 堀之内孝広, 藤室雅弘, 小笠原康悦, 上原孝, 三輪聡一
第25回北海道薬物作用談話会, 2011年07月23日, 日本語, 口頭発表（一般）

タバコ煙抽出液で同定された細胞傷害活性因子群の心血管系に対する影響の解析と傷害活性除去法開発の試み．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 眞井洋輔, 澤田修, 西屋禎
第26回喫煙科学研究財団助成研究発表会, 2011年07月22日, 日本語, 口頭発表（一般）

Regulatory mechanisms of human endothelin type A receptor-operated Ca2+ influx via TRPC6 channel.　　　　　　　　　　　　　　　
Horinouchi T, Higa T, Aoyagi H, Nishiya T, Miwa S
The 6th International Symposium on Receptor Mechanisms, Signal Transduction and Drug Effects., 英語, ポスター発表
2011年04月01日 - 2011年04月02日

SPRY domainとSOCS boxタンパク質による誘導型一酸化窒素合成酵素の分解制御機構．　　　　　　　　　　　　　　　
西屋禎, 松本一馬, 前川聡, 堀之内孝広, 藤室雅弘, 小笠原康悦, 上原孝, 三輪聡一
第84回日本薬理学会年会, 英語, その他
2011年03月22日 - 2011年03月24日

アデニル酸シクラーゼ/cAMP/PKA系によるエンドセリンA型受容体作動性TRPC6チャネルの負の制御機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 西屋禎, 青柳裕之, 鈴木浩之, 西本新, 三輪聡一
第84回日本薬理学会年会, 英語, その他
2011年03月22日 - 2011年03月24日

エンドセリンA型受容体作動性TRPC6チャネルの多様な機能制御機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 西屋禎, 青柳裕之, 鈴木浩之, 西本新, 三輪聡一
第40回日本心脈管作動物質学会, 日本語, 口頭発表（一般）
2011年02月04日 - 2011年02月05日

cAMP/PKA系によるエンドセリンA型受容体作動性TRPC6チャネルの抑制性機能制御．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 青柳裕之, 鈴木浩之, 西屋禎, 西本新, 三輪聡一
第61回日本薬理学会北部会, 2010年09月10日, 日本語, 口頭発表（一般）

Molecular mechanism of ETAR-operated Ca2+ entry via TRPC channels and visualization of homo- and heteromeric TRPC3/6 interaction in living cells using BiFC.　　　　　　　　　　　　　　　
Horinouchi T, Higa T, Aoyagi H, Asano H, Nishiya T, Nishimoto A, Miwa S
16th World Congress of Basic and Clinical Pharmacology., 2010年07月, International Union of Basic and Clinical Pharmacology (IUPHAR)., 英語, ポスター発表
2010年07月17日 - 2010年07月23日, Copenhagen, デンマーク王国, ［国際会議］

Molecular mechanisms of endothelin type A receptor-operated Ca2+ entry via TRPC channels.　　　　　　　　　　　　　　　
Horinouchi T
7th Scientific Symposium, 2010年07月03日, 英語, 口頭発表（招待・特別）
［招待講演］

タバコ煙中に含まれる動脈硬化促進因子の単離・同定に関する研究．　　　　　　　　　　　　　　　
野矢洋一, 朝野拓史, 堀之内孝広, 三輪聡一, 関興一, 久下裕司
日本薬学会第130年会, 日本語, 口頭発表（一般）
2010年03月28日 - 2010年03月30日

幼若期ストレス負荷は恐怖条件付けにおける海馬5-HT1A受容体機能を減弱させる．　　　　　　　　　　　　　　　
松崎広和, 泉剛, 松本眞知子, 山口拓, 吉田隆行, 堀之内孝広, 朴秀賢
第83回日本薬理学会年会, 英語
2010年03月16日 - 2010年03月18日

受容体結合蛋白質Jab1によるエンドセリン受容体の発現レベル調節．　　　　　　　　　　　　　　　
西本新, 西屋禎, 堀之内孝広, 三輪聡一
第83回日本薬理学会年会, 英語, 口頭発表（一般）
2010年03月16日 - 2010年03月18日

蛍光タンパク質再構成法によるエンドセリンA型受容体作動性TRPC3/6チャネルの分子間相互作用の可視化．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西屋禎, 西本新, 三輪聡一
第83回日本薬理学会年会, 英語, ポスター発表
2010年03月16日 - 2010年03月18日

TRPC3及びTRPC6チャネルを介したエンドセリンA型受容体作動性Ca2+流入の分子機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第83回日本薬理学会年会, 英語, 口頭発表（一般）
2010年03月16日 - 2010年03月18日

受容体結合蛋白質Jab1によるエンドセリン受容体の発現レベル調節機構．　　　　　　　　　　　　　　　
西本新, 西屋禎, 堀之内孝広, 三輪聡一
第19回日本循環薬理学会, 2009年11月27日, 日本語, 口頭発表（一般）

エンドセリンA型受容体作動性Ca2+流入に関与するTRPCチャネルの活性化機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第19回日本循環薬理学会, 2009年11月27日, 日本語, 口頭発表（一般）

受容体結合蛋白質Jab1によるエンドセリンA型受容体の発現レベル調節機構とその病態的意義．　　　　　　　　　　　　　　　
三輪聡一, 西本新, 西屋禎, 堀之内孝広
第37回薬物活性シンポジウム, 日本語, 口頭発表（一般）
2009年10月09日 - 2009年10月10日

エンドセリンA型受容体作動性TRPCチャネルの活性制御機構．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第37回薬物活性シンポジウム, 日本語, 口頭発表（一般）
2009年10月09日 - 2009年10月10日

受容体結合蛋白質Jab1によるエンドセリン受容体の発現レベル調節機構．　　　　　　　　　　　　　　　
西本新, 西屋禎, 堀之内孝広, 三輪聡一
第60回日本薬理学会北部会, 2009年09月26日, 日本語, 口頭発表（一般）

蛍光タンパク質再構成法を用いたエンドセリンA型受容体作動性TRPCチャネルの分子間相互作用の可視化．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第60回日本薬理学会北部会, 2009年09月26日, 日本語, 口頭発表（一般）

Regulation of cell surface endothelin type A receptor level by a novel receptor-interacting protein, Jab1.　　　　　　　　　　　　　　　
Nishimoto A, Lu L, Hayashi M, Nishiya T, Horinouchi T, Miwa S
The 36th Congress of the International Union of Physiological Sciences., 2009年07月30日, 口頭発表（一般）

エンドセリンA型受容体作動性Ca2+流入に関与するTRPCチャネルの同定と機能解析．　　　　　　　　　　　　　　　
堀之内孝広, 比嘉綱己, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第2回北大若手研究者交流会, 2009年07月17日, 日本語, 口頭発表（一般）

タバコ煙中に含まれる血管壁構成細胞障害因子の性状解析．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 西屋禎, 朝野拓史, 藤井敏輔
第24回喫煙科学研究財団助成研究発表会, 2009年07月16日, 日本語, 口頭発表（一般）

エンドセリンA型受容体作動性Ca2+流入に関与するTRPCチャネルの同定．　　　　　　　　　　　　　　　
比嘉綱己, 堀之内孝広, 朝野拓史, 西本新, 西屋禎, 三輪聡一
第23回北海道薬物作用談話会, 2009年07月11日, 日本語, 口頭発表（一般）

エンドセリンA型受容体(ETAR)結合蛋白質Jab1は、ETARのユビキチン化とリソソームでの分解を促進し、その細胞膜表面のレベルと細胞内シグナル伝達を制御する．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第82回日本薬理学会年会, 英語
2009年03月16日 - 2009年03月18日

受容体結合蛋白質によるエンドセリン受容体の発現レベル調節．　　　　　　　　　　　　　　　
西本新, 西屋禎, 堀之内孝広, 三輪聡一
第82回日本薬理学会年会, 英語
2009年03月16日 - 2009年03月18日

心筋β-アドレナリン受容体発現量に対するβ-アドレナリン受容体遮断薬長期投与の影響：組織片を用いた受容体結合実験法による定量的解析．　　　　　　　　　　　　　　　
堀之内孝広, 森島繁, 田中高志, 鈴木史子, 田中芳夫, 小池勝夫, 三輪聡一, 村松郁延
第82回日本薬理学会年会, 英語, ポスター発表
2009年03月16日 - 2009年03月18日

リガンド依存性に活性化されるβ3-アドレナリン受容体シグナリングの多様性．　　　　　　　　　　　　　　　
堀之内孝広, 佐藤将哲, Hutchinson Dana.S, Evans Bronwyn.A, 田中芳夫, 小池勝夫, 三輪聡一, Summers, Roger.J
第82回日本薬理学会年会, 英語, 口頭発表（一般）
2009年03月16日 - 2009年03月18日

たばこ煙が中枢神経系の細胞に与える影響．　　　　　　　　　　　　　　　
藤井俊輔, 朝野拓史, 片山貴博, 堀之内孝広, 西屋禎, 西本新, 南雅文, 三輪聡一
第1回北大若手研究者交流会, 2008年11月29日, 日本語, ポスター発表

エンドセリンA型受容体を介したシグナル伝達機構の多様性とCa2+シグナリング．　　　　　　　　　　　　　　　
堀之内孝広, 西本新, 西屋禎, 村松郁延, 三輪聡一
第1回北大若手研究者交流会, 2008年11月29日, 日本語, ポスター発表

新規エンドセリンA型受容体(ETAR)結合蛋白質Jab1によるETARレベルの調節．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第18回日本循環薬理学会, 2008年11月21日, 日本語, 口頭発表（一般）

エンドセリンA型受容体により誘発される持続性Ca2+流入の分子機構．　　　　　　　　　　　　　　　
堀之内孝広, 西屋禎, 西本新, 森島繁, 村松郁延, 三輪聡一
第18回日本循環薬理学会, 2008年11月21日, 日本語, 口頭発表（一般）

エンドセリンA型受容体(ETAR)結合蛋白質Jab1によるETAR蛋白質分解制御機構の解明．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第59回日本薬理学会北部会, 2008年09月27日, 日本語, 口頭発表（一般）

エンドセリンA型受容体を介した細胞内Ca2+濃度上昇反応に関与するシグナルカスケードの多様性．　　　　　　　　　　　　　　　
三宅由美恵, 堀之内孝広, 西屋禎, 西本新, 三輪聡一
第59回日本薬理学会北部会, 2008年09月27日, 日本語, 口頭発表（一般）

エンドセリンA型受容体を介して活性化されるCa2+シグナリングの多様性．　　　　　　　　　　　　　　　
三宅由美恵, 堀之内孝広, 西屋禎, 西本新, 三輪聡一
第22回北海道薬物作用談話会, 2008年07月26日, 日本語, 口頭発表（一般）

タバコ煙のニューロンおよびグリアに対する影響．　　　　　　　　　　　　　　　
藤井俊輔, 朝野拓史, 片山貴博, 堀之内孝広, 西屋禎, 西本新, 南雅文, 三輪聡一
第22回北海道薬物作用談話会, 2008年07月26日, 日本語, 口頭発表（一般）

タバコ煙中に含まれる血管壁構成細胞障害因子の性状解析．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 西屋禎, 朝野拓史, 藤井敏輔
第23回喫煙科学研究財団助成研究発表会, 2008年07月, 日本語, 口頭発表（一般）

エンドセリンA型受容体を介したCa2+シグナリングの多様性とTRPCチャネル．　　　　　　　　　　　　　　　
堀之内孝広, 三宅由美恵, 西屋禎, 西本新, 三輪聡一
第4回TRPチャネル研究会, 2008年06月06日, 日本語, 口頭発表（一般）

α1A-アドレナリン受容体を介して活性化されるCa2+シグナリングの分子メカニズム．　　　　　　　　　　　　　　　
三宅由美恵, 堀之内孝広, 西屋禎, 西本新, 三輪聡一
第81回日本薬理学会年会, 2008年03月, 英語

エンドセリンA型受容体結合蛋白質によるエンドセリンA型受容体蛋白質の分解制御機構の解明．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第81回日本薬理学会年会, 2008年03月, 英語

Gタンパク質共役型受容体を介して引き起こされる持続性Ca2+流入の分子メカニズム．　　　　　　　　　　　　　　　
堀之内孝広, 三宅由美恵, 西屋禎, 西本新, 森島繁, 村松郁延, 三輪聡一
第81回日本薬理学会年会, 2008年03月, 英語

MyD88の細胞局在とシグナル伝達におけるTIRドメインとそれ以外の領域の機能的差異．　　　　　　　　　　　　　　　
西屋禎, 梶田絵美, 堀之内孝広, 西本新, 三輪聡一
日本薬学会第128年会, 2008年03月, 日本語

エンドセリンA型受容体(ETAR)の蛋白質分解における新規結合蛋白質Jab1の役割．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第17回日本循環薬理学会, 2007年11月, 日本語, 口頭発表（一般）

Gタンパク質共役型受容体を介した持続性Ca2+濃度上昇反応の分子メカニズム．　　　　　　　　　　　　　　　
堀之内孝広, 三宅由美恵, 西屋禎, 西本新, 森島繁, 村松郁延, 三輪聡一
第17回日本循環薬理学会, 2007年11月, 日本語, 口頭発表（一般）

MyD88の細胞局在及びシグナル伝達におけるTIRドメインとnon-TIR領域の機能的差異．　　　　　　　　　　　　　　　
西屋禎, 梶田絵美, 堀之内孝広, 西本新, 三輪聡一
第58回日本薬理学会北部会, 2007年09月, 日本語, 口頭発表（一般）

タバコ煙による細胞傷害の性状解析．　　　　　　　　　　　　　　　
朝野拓史, 堀之内孝広, 西本新, 西屋禎, 三輪聡一
第58回日本薬理学会北部会, 2007年09月, 日本語, 口頭発表（一般）

エンドセリンA型受容体の蛋白質分解における新規結合蛋白質Jab1の役割．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 三輪聡一
第58回日本薬理学会北部会, 2007年09月, 日本語, 口頭発表（一般）

有機カチオントランスポーターOCTN2のバリアント体の単離と機能解析．　　　　　　　　　　　　　　　
前川聡, 森大輔, 西屋禎, 堀之内孝広, 西本新, 梶田絵美, 三輪聡一
第58回日本薬理学会北部会, 2007年09月, 日本語, 口頭発表（一般）

α1A-アドレナリン受容体を介した細胞内Ca2+濃度上昇反応の分子メカニズム．　　　　　　　　　　　　　　　
三宅由美恵, 堀之内孝広, 西屋禎, 西本新, 三輪聡一
第58回日本薬理学会北部会, 2007年09月, 日本語, 口頭発表（一般）

タバコ煙中に含まれる血管壁構成細胞障害因子の性状解析．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 朝野拓史, 西屋禎
第22回喫煙科学研究財団助成研究発表会, 2007年07月, 日本語, 口頭発表（一般）

エンドセリンA型受容体を介して活性化されるCa2+シグナリングの分子メカニズム．　　　　　　　　　　　　　　　
堀之内孝広, 三宅由美恵, 西屋禎, 西本新, 三輪聡一
第3回TRPチャネル研究会, 2007年07月, 日本語, 口頭発表（一般）

有機カチオントランスポーターOCTN2のスプライスバリアント(OCTN2VT)の単離と機能解析．　　　　　　　　　　　　　　　
前川聡, 森大輔, 西屋禎, 堀之内孝広, 西本新, 梶田絵美, 三輪聡一
第21回北海道薬物作用談話会, 2007年07月, 日本語, 口頭発表（一般）

タバコ煙中に含まれる血管壁構成細胞障害因子の性状解析．　　　　　　　　　　　　　　　
朝野拓史, 堀之内孝広, 西屋禎, 西本新, 三輪聡一
第21回北海道薬物作用談話会, 2007年07月, 日本語, 口頭発表（一般）

新規エンドセリンA型受容体結合蛋白質Jab1は、その受容体発現レベルとシグナル伝達を制御する．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 梶田絵美, 三輪聡一
日本適応医学会第11回学術集会, 2007年06月, 日本語, 口頭発表（一般）

循環系調節因子としてのエンドセリンにより活性化される細胞内カルシウムシグナルとその調節系．　　　　　　　　　　　　　　　
三輪聡一, 堀之内孝広, 西本新, 西屋禎
日本適応医学会第11回学術集会, 2007年06月, 日本語, 口頭発表（一般）

自律神経機能異常に起因する疾患の新規創薬ターゲット分子：β3-アドレナリン受容体の分布・機能・細胞内シグナリングと創薬研究．　　　　　　　　　　　　　　　
堀之内孝広
第15回カテコールアミンと神経疾患研究会, 2007年04月, 日本語, 口頭発表（一般）

MyD88の細胞局在、シグナル伝達、およびToll様受容体との相互作用に関与する分子内領域の解析．　　　　　　　　　　　　　　　
梶田絵美, 西屋禎, 西本新, 堀之内孝広, 三輪聡一
第80回日本薬理学会年会, 2007年03月, 英語

新規エンドセリンA型受容体結合蛋白質Jab 1は、その受容体発現レベルとシグナル伝達を制御する．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 梶田絵美, 三輪聡一
第80回日本薬理学会年会, 2007年03月, 英語

エンドセリンA受容体刺激によって活性化されるCa2+シグナリングにおけるNa+/Ca2+交換機構の役割．　　　　　　　　　　　　　　　
堀之内孝広, 魯凌云, 西屋禎, 西本新, 梶田絵美, 三輪聡一
第80回日本薬理学会年会, 2007年03月, 英語

Molecular mechanisms for Ca2+ signaling activated by Endothelin-1.　　　　　　　　　　　　　　　
Miwa S, Horinouchi T, Nishimoto A, Nishiya T
5th United States-Japan Workshop on Cellular and Molecular Aspects of Vascular Smooth Muscle Function., 2007年01月, 英語, 口頭発表（招待・特別）
アメリカ合衆国, ［招待講演］, ［国際会議］

新規エンドセリン受容体結合蛋白質Jab1によるエンドセリン誘導性ERKリン酸化の調節．　　　　　　　　　　　　　　　
三輪聡一, 魯凌云, 西本新, 西屋禎, 堀之内孝広, 梶田絵美
第16回日本循環薬理学会, 2006年12月, 日本語, 口頭発表（一般）

Tissue segment binding assay, a novel technique, for studying plasma membrane β-adrenoceptors using intact cardiac tissues.　　　　　　　　　　　　　　　
Horinouchi T., Miwa S., Muramatsu I.
The 23rd Annual Meeting of International Society for Heart Research Japanese Section., 2006年12月, 英語, 口頭発表（一般）

JAB1, a novel endothelin receptor type A-interacting protein, is involved in the regulation of ET-1-induced ERK1/2 phosphorylation.　　　　　　　　　　　　　　　
Lu L, Nishimoto A, Nishiya T, Horinouchi T, Kajita E, Miwa S
第57回日本薬理学会北部会, 2006年09月, 英語, 口頭発表（一般）

組織片を用いた新規結合実験法による眼内β-アドレナリン受容体の薬理学的解析．　　　　　　　　　　　　　　　
堀之内孝広, 村松郁延, 田中芳夫, 小池勝夫, 三輪聡一
第115回日本薬理学会関東部会, 2006年09月, 日本語, 口頭発表（一般）

新規エンドセリン受容体会合蛋白質であるJab1のエンドセリン誘導性ERKリン酸化への関与．　　　　　　　　　　　　　　　
西本新, 魯凌云, 西屋禎, 堀之内孝広, 梶田絵美, 三輪聡一
第34回薬物活性シンポジウム, 2006年09月, 日本語, 口頭発表（一般）

Functional C-terminal domains of the mouse β3-adrenoceptor for regulating G protein coupling.　　　　　　　　　　　　　　　
Horinouchi T, Sato M, Hutchinson D.S, Evans B.A, Tanaka Y, Koike K, Summers R.J
The 18th Japan-Korea Joint Seminar on Pharmacology., 2006年09月, 英語, 口頭発表（一般）

タバコ煙中に含まれる血管壁構成細胞障害因子の性状解析．　　　　　　　　　　　　　　　
堀之内孝広, 三輪聡一, 朝野拓史, 西屋禎
第21回喫煙科学研究財団助成研究発表会, 2006年07月, 日本語, 口頭発表（一般）

Development of tissue segment binding method to evaluate plasma membrane β-adrenoceptors in rat hearts.　　　　　　　　　　　　　　　
Horinouchi T, Miwa S, Muramatsu I
2nd. World Congress of the International Academy of Cardiovascular Science., 2006年07月, 英語, 口頭発表（一般）

Multiple signalling pathways of the mouse β3-adrenoceptor stably expressed in CHO-K1 cells.　　　　　　　　　　　　　　　
Sato M, Horinouchi T, Hutchinson D.S, Evans B.A, Summers R.J
15th International Congress of Pharmacology., 2006年07月, International Union of Basic and Clinical Pharmacology (IUPHAR)., 英語, 口頭発表（一般）
中華人民共和国, ［国際会議］

モルモット膀胱及び尿道に対するdistigmineの作用．　　　　　　　　　　　　　　　
関谷更沙, 大河夏美, 青木智子, 秋山理恵, 小野友子, 芝野真理, 堀之内孝広, 田中芳夫, 小池勝夫
第79回日本薬理学会年会, 2006年03月, 英語, 口頭発表（一般）

β3-アドレナリン受容体シグナリングにおける細胞内C末端ドメインの役割．　　　　　　　　　　　　　　　
堀之内孝広, 佐藤将哲, Hutchinson DS, Evans BA, 田中芳夫, 小池勝夫, Summers RJ
第79回日本薬理学会年会, 2006年03月, 英語, 口頭発表（一般）

モルモットの膀胱運動に対する臭化ジスチグミンの作用とその機序に関する検討．　　　　　　　　　　　　　　　
関谷更沙, 大河夏美, 青木智子, 秋山理恵, 小野友子, 芝野真理, 堀之内孝広, 田中芳夫, 小池勝夫
日本薬学会第124年会, 2006年03月, 日本語

Identification of plasma membrane β-adrenoceptors in rat hearts by tissue segment binding method.　　　　　　　　　　　　　　　
Horinouchi T, Morishima S, Tanaka T, Suzuki F, Tanaka Y, Koike K, Summers R.J, Muramatsu I
Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists, and Australasian Pharmaceutical Science Association., 2005年12月, Australasian Pharmacology, Clinical Pharmacology, Toxicology or Clinical Toxicology (ASCEPT)., 英語, 口頭発表（一般）
Melbourne, オーストラリア連邦

Multiple signaling pathways of the mouse β3-adrenoceptor stably expressed in CHO-K1 cells.　　　　　　　　　　　　　　　
Sato M, Horinouchi T, Hutchinson D.S, Evans B.A, Summers R.J
Molecular Pharmacology of G protein-Coupled Receptors 2005., 2005年12月, Australasian Pharmacology, Clinical Pharmacology, Toxicology or Clinical Toxicology (ASCEPT)., 英語, 口頭発表（一般）
Melbourne, オーストラリア連邦

ノルアドレナリンの気管平滑筋弛緩作用に2種類のβ1-アドレナリン受容体が関与する可能性について．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 小池勝夫
第47回日本平滑筋学会総会, 2005年07月, 日本語

平滑筋の機能調節と臨床応用．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 小池勝夫
第47回日本平滑筋学会総会, 2005年07月, 日本語

内因性カテコラミンによるモルモット気管平滑筋の弛緩反応に関与するβ-アドレナリン受容体（β-AR）サブタイプに関する検討．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 小池勝夫
第112回日本薬理学会関東部会, 2005年06月, 日本語, 口頭発表（一般）

ノルアドレナリンの気管平滑筋弛緩作用に新しいβ-アドレナリン受容体サブタイプが関与する可能性について．　　　　　　　　　　　　　　　
嶺村彩, 八巻史子, 堀之内孝広, 田中芳夫, 小池勝夫
第78回日本薬理学会年会, 2005年03月, 英語

モルモット気管平滑筋の弛緩反応に関与するβ-アドレナリン受容体 (β-AR) サブタイプについて：ノルアドレナリンとサルブタモールによる弛緩反応の比較．　　　　　　　　　　　　　　　
八巻史子, 山下庸子, 堀之内孝広, 田中芳夫, 小池勝夫
第111回日本薬理学会関東部会, 2004年10月, 日本語, 口頭発表（一般）

眼のβ-アドレナリン受容体について −組織片を用いた新しい結合実験法による薬物受容体の解析−　　　　　　　　　　　　　　　
堀之内孝広, 村松郁延, 田中芳夫, 小池勝夫
第24回日本眼薬理学会, 2004年10月, 日本語, 口頭発表（一般）

ノルアドレナリンならびにアドレナリンによる気管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプの比較検討．　　　　　　　　　　　　　　　
嶺村彩, 八巻史子, 山下庸子, 堀之内孝広, 田中芳夫, 小池勝夫
生体機能と創薬シンポジウム2004, 2004年09月, 日本語

膀胱機能障害と新しい治療薬開発の可能性．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 小池勝夫
生体機能と創薬シンポジウム2004, 2004年09月, 日本語

IP受容体ならびにβ-アドレナリン受容体を介した平滑筋の弛緩反応におけるcAMP非依存性機構について：MaxiKチャネルの役割とその活性化機序．　　　　　　　　　　　　　　　
八巻史子, 山下庸子, 堀之内孝広, 田中芳夫, 小池勝夫
第46回日本平滑筋学会総会, 2004年07月, 日本語, 口頭発表（一般）

IP受容体ならびにβ-アドレナリン受容体を介した組織cAMP量の上昇を伴わない平滑筋の弛緩反応機構について：MaxiKチャネルの役割とその活性化機序．　　　　　　　　　　　　　　　
八巻史子, 山下庸子, 堀之内孝広, 田中芳夫, 小池勝夫
第110回日本薬理学会関東部会, 2004年06月, 日本語, 口頭発表（一般）

β-アドレナリン受容体 (β-AR) を介したGsタンパクの活性化による細胞内cAMP量の上昇を伴わない気管平滑筋の弛緩反応について．　　　　　　　　　　　　　　　
田中芳夫, 山下庸子, 八巻史子, 堀之内孝広, 小池勝夫
日本薬学会第124年会, 2004年03月, 日本語

糖尿病モデルマウスを用いた消化管運動障害の検討：ニコチン受容体結合能の変化．　　　　　　　　　　　　　　　
千倉知子, 小池勝夫, 田中芳夫, 堀之内孝広, 小野景義, 中村智徳, 矢野眞吾
日本薬学会第124年会, 2004年03月, 日本語

モルモット食道におけるβ-アドレナリン受容体のmRNA発現解析.　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 辻谷典彦, 小池勝夫
第77回日本薬理学会年会, 2004年03月, 英語

β-アドレナリン受容体 (β-AR) を介したモルモット気管平滑筋の弛緩反応には，GsタンパクにcAMPの上昇を伴わない細胞内機序が関与する．　　　　　　　　　　　　　　　
田中芳夫, 山下庸子, 八巻史子, 堀之内孝広, 小池勝夫
第77回日本薬理学会年会, 2004年03月, 英語

泌尿器疾患とその治療薬開発への新しいアプローチ −高血圧症との関連−．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 小池勝夫
第77回日本薬理学会年会, 2004年03月, 英語

cAMP-非依存性の-MaxiK channel活性化機構について: G蛋白を介する活性化調節と血管平滑筋の緊張性制御における役割．　　　　　　　　　　　　　　　
八巻史子, 田中芳夫, 堀之内孝広, 重信弘毅, 小池勝夫
第31回薬物活性シンポジウム, 2003年11月, 日本語, 口頭発表（一般）

消化管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプの分布及びβ3-アドレナリン受容体のユニークな抑制性緊張制御機構の解明．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 小池勝夫
第31回薬物活性シンポジウム, 2003年11月, 日本語, 口頭発表（一般）

β2-アドレナリン受容体を介した気管平滑筋の弛緩反応機構におけるcAMPの役割について．　　　　　　　　　　　　　　　
山下庸子, 八巻史子, 堀之内孝広, 田中芳夫, 小池勝夫
第109回日本薬理学会関東部会, 2003年10月, 日本語, 口頭発表（一般）

β2-アドレナリン受容体刺激に伴う気管平滑筋の弛緩反応機構：イソプレナリンによるcAMPを介さないモルモット気管筋の弛緩機序とマキシKチャネル活性化の関連性について．　　　　　　　　　　　　　　　
山下庸子, 八巻史子, 堀之内孝広, 田中芳夫, 小池勝夫
第45回日本平滑筋学会総会, 2003年07月, 日本語

カテコラミン刺激により誘発される消化管平滑筋弛緩反応に関与するβ-アドレナリン受容体サブタイプとその部位差．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 小池勝夫
第45回日本平滑筋学会総会, 2003年07月, 日本語

モルモット消化管平滑筋の弛緩反応に関与するβ-アドレナリン受容体サブタイプの分布とmRNA発現の比較．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 辻谷典彦, 小池勝夫
第108回日本薬理学会関東部会, 2003年06月, 日本語, 口頭発表（一般）

Physiological significance of Gs protein triggered, cAMP-independent functional coupling between prostacyclin (IP) receptor and large-conductance Ca2+-sensitive K+ (MaxiK) channel in the regulation of vascular smooth muscle tone.　　　　　　　　　　　　　　　
Yamaki F, Tanaka Y, Horinouchi T, Shigenobu K, Koike K
The 4th International Symposium on Receptor Mechanisms, Signal Transduction and Drug Effects., 2003年04月, 英語, 口頭発表（一般）

Discrepancy between function and mRNA expression of β1-, β2- and β3-adrenoceptors in guinea-pig gastrointestinal smooth muscles.　　　　　　　　　　　　　　　
Horinouchi T, Tanaka Y, Koike K
The 4th International Symposium on Receptor Mechanisms, Signal Transduction and Drug Effects., 2003年04月, 英語, 口頭発表（一般）

イソプレナリンによる弛緩反応におけるβ3-アドレナリン受容体の寄与について：ラット胸部大動脈及び腹部大動脈での比較．　　　　　　　　　　　　　　　
松下真由美, 堀之内孝広, 田中芳夫, 水流弘通, 小池勝夫
日本薬学会第123年会, 2003年03月, 日本語

カテコラミンによる気管平滑筋弛緩作用に関与するβ-アドレナリン受容体（β-AR）について：ノルアドレナリンとイソプレナリンの作用の相違．　　　　　　　　　　　　　　　
山下庸子, 堀之内孝広, 田中芳夫, 小池勝夫
日本薬学会第123年会, 2003年03月, 日本語

Pharmacological characterization of α1-adrenoceptor in mouse iliac artery.　　　　　　　　　　　　　　　
Shibano M, Horinouchi T, Tanaka Y, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

IP receptor-activated, cAMP-independent vasorelaxant mechanisms: a substantial role of Gs protein-mediated, MaxiK channel activation.　　　　　　　　　　　　　　　
Yamaki F, Horinouchi T, Tanaka Y, Shigenobu K, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

Effects of MaxiK channel activators on the rhythmic contraction and action potential generated spontaneously in guinea-pig urinary bladder smooth muscle.　　　　　　　　　　　　　　　
Okamoto T, Imai T, Horinouchi T, Tanaka H, Mitani A, Nakahara T, Tanaka Y, Ishii K, Shigenobu K, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

Pharmacological characterization of β-adrenoceptor which mediates noradrenaline (NA)-elicited tracheal smooth muscle relaxation.　　　　　　　　　　　　　　　
Yamashita Y, Horinouchi T, Tanaka Y, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

Functional coupling between Q-type Ca2+ channels and tetraethylammonium (TEA)- sensitive K+ channels in the sympathetic neurotransmission of rabbit facial vein.　　　　　　　　　　　　　　　
Tanaka Y, Akutsu A, Tanaka H, Horinouchi T, Tsuru H, Shigenobu K, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

Different contribution of classical β- and atypical β-adrenoceptors in vasodilation to isoprenaline of rat thoracic and abdominal aortae: analysis using by propranolol and bupranolol in vitro.　　　　　　　　　　　　　　　
Matsushita M, Horinouchi T, Tanaka Y, Tsuru H, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

Gastrointestinal smooth muscle β3-adrenoceptor: its distribution and coupling with a unique transduction system.　　　　　　　　　　　　　　　
Horinouchi T, Tanaka Y, Tsujitani M, Koike K
第76回日本薬理学会年会, 2003年03月, 英語

膀胱平滑筋の自発性収縮調節におけるBKチャネルの役割．　　　　　　　　　　　　　　　
田中芳夫, 堀之内孝広, 田中光, 重信弘毅, 小池勝夫
第30回薬物活性シンポジウム, 2002年11月, 日本語

モルモット胃底におけるβ３-アドレナリン受容体を介した弛緩反応に関与する細胞内情報伝達系の解析と電位依存性K+ channelの役割．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 小池勝夫
第30回薬物活性シンポジウム, 2002年11月, 日本語

交感神経電気刺激により誘発されるウサギ顔面静脈弛緩反応とTEAによる増強効果の薬理学的解析．　　　　　　　　　　　　　　　
田中芳夫, 阿久津あや, 堀之内孝広, 田中光, 水流弘通, 重信弘毅, 小池勝夫
第107回日本薬理学会関東部会, 2002年10月, 日本語, 口頭発表（一般）

β3-アドレナリン受容体刺激により誘発されるcyclic AMPの上昇を介さない胃底平滑筋の弛緩反応機構の解析：電位依存性K+チャネルの関与について．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 辻谷典彦, 小池勝夫
第107回日本薬理学会関東部会, 2002年10月, 日本語, 口頭発表（一般）

モルモット膀胱平滑筋の自発性収縮の性質とBKチャネルの役割の検討．　　　　　　　　　　　　　　　
田中芳夫, 今井利安, 岡本貴男, 堀之内孝広, 田中光, 小池勝夫, 重信弘毅
第44回日本平滑筋学会総会, 2002年07月, 日本語

ラット腹部大動脈におけるatypical β-アドレナリン受容体を介した弛緩反応の特徴．　　　　　　　　　　　　　　　
松下真由美, 堀之内孝広, 田中芳夫, 小池勝夫
第44回日本平滑筋学会総会, 2002年07月, 日本語

Characterization of β3-adrenoceptor-mediated relaxation in the guinea pig duodenum: Stereoselectivity, β-adrenoceptor alkylating and structure-activity relationship.　　　　　　　　　　　　　　　
Horinouchi T, Koike K
14th International Congress of Pharmacology., 2002年07月, International Union of Basic and Clinical Pharmacology (IUPHAR)., 英語, ポスター発表
San Francisco, アメリカ合衆国, ［国際会議］

膀胱平滑筋の自発性収縮の発生と活動電位の関連性．　　　　　　　　　　　　　　　
田中芳夫, 岡本貴男, 今井利安, 堀之内孝広, 田中光, 小池勝夫, 重信弘毅
第106回日本薬理学会関東部会, 2002年06月, 日本語, 口頭発表（一般）

モルモット胃底及び十二指腸におけるβ3-アドレナリン受容体を介したcyclic AMP非依存性弛緩反応について．　　　　　　　　　　　　　　　
堀之内孝広, 田中芳夫, 辻谷典彦, 小池勝夫
第106回日本薬理学会関東部会, 2002年06月, 日本語, 口頭発表（一般）

IP受容体刺激に伴うMaxiK channel活性化を介する血管弛緩反応機構の解析．　　　　　　　　　　　　　　　
田中芳夫, 八巻史子, 加賀桃子, 堀之内孝広, 田中光, 小池勝夫, Ligia Toro, 重信弘毅
日本薬学会第122年会, 2002年03月, 日本語

ラット腹部大動脈におけるatypical β-アドレナリン受容体に関する研究．　　　　　　　　　　　　　　　
松下真由美, 堀之内孝広, 小池勝夫
日本薬学会第122年会, 2002年03月, 日本語

モルモット胃底のβ３-アドレナリン受容体における(±)-terbutaline 及び (±)-fenoterolを用いた構造−活性相関に関する研究．　　　　　　　　　　　　　　　
堀之内孝広, 中川佳子, 若林麻希子, 小池勝夫
日本薬学会第122年会, 2002年03月, 日本語

Partial agonistic activity of labetalol on 3-adrenoceptors in the guinea pig gastric fundus.　　　　　　　　　　　　　　　
Horinouchi T, Asai S, Fukushima M, Koike K
第75回日本薬理学会年会, 2002年03月, 英語

選択的β３-アドレナリン受容体拮抗薬SR59230Aのβ３-アドレナリン受容体活性作用について．　　　　　　　　　　　　　　　
堀之内孝広, 小池勝夫
第29回薬物活性シンポジウム, 2001年11月, 日本語, 口頭発表（一般）

ウサギ瞳孔括約筋における基礎研究 －β-アドレナリン受容体を中心とした薬物反応－．　　　　　　　　　　　　　　　
堀之内孝広, 小池勝夫
第21回日本眼薬理学会・第13回国際眼研究会議日本部会合同会議, 2001年09月, 日本語, 口頭発表（一般）

β３-アドレナリン受容体に対するarylethanolamine類の構造活性相関に関する研究．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第43回日本平滑筋学会総会, 2001年07月, 日本語, 口頭発表（一般）

IP受容体刺激を介する血管弛緩反応におけるMaxiK channelの役割とその活性化機序について．　　　　　　　　　　　　　　　
八巻史子, 田中芳夫, 堀之内孝広, 田中光, 小池勝夫, Toro Ligia, 重信弘毅
第104回日本薬理学会関東部会, 2001年06月, 日本語, 口頭発表（一般）

Agonistic activity of SR59230A, a selective β3-adrenoceptor antagonist, at β3-adrenoceptors in the guinea pig gastric fundus and duodenum.　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第104回日本薬理学会関東部会, 2001年06月, 日本語, 口頭発表（一般）

α1-アドレナリン受容体の性質．　　　　　　　　　　　　　　　
山本佳久, 堀之内孝広, 小池勝夫
日本薬学会第121年会, 2001年03月, 日本語

Further characterization of β3-adrenoceptors in the guinea pig gastric fundus: Stereoselectivity and β-adrenoceptor alkylating.　　　　　　　　　　　　　　　
Koike K, Horinouchi T
第74回日本薬理学会年会, 2001年03月, 英語

Structure-activity relationship of arylethanolamine and aryloxypropanolamine drugs at β3-adrenoceptor on the guinea pig gastric fundus.　　　　　　　　　　　　　　　
Horinouchi T, Koike K
第74回日本薬理学会年会, 2001年03月, 英語, 口頭発表（一般）

薬物の化学構造と薬物受容体　　　　　　　　　　　　　　　
堀之内孝広, 小池勝夫
第28回薬物活性シンポジウム, 2000年11月, 日本語, 口頭発表（一般）

モルモット胃底のβ3-アドレナリン受容体に対する(±)-propranolol及び(±)-pindololの作用態度の違いについて．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第103回日本薬理学会関東部会, 2000年10月, 日本語, 口頭発表（一般）

モルモット胃底におけるβ3-アドレナリン受容体に対するカルテオロールの部分活性作用について．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第42回日本平滑筋学会総会, 2000年07月, 日本語, 口頭発表（一般）

モルモット十二指腸に存在するatypical β-アドレナリン受容体に対するカルテオロールの部分活性作用について．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第102回日本薬理学会関東部会, 2000年06月, 日本語, 口頭発表（一般）

モルモット胃底におけるatypical β-アドレナリン受容体を介した弛緩反応に対するSR59230Aの作用．　　　　　　　　　　　　　　　
堀之内孝広, 山本佳久, 小池勝夫
日本薬学会第120年会, 2000年03月, 日本語

Carteolol is a partial agonist on β2-adrenoceptors and atypical β-adrenoceptors in guinea pig gastrointestine.　　　　　　　　　　　　　　　
Horinouchi T, Tsujitani M, Koike K
第73回日本薬理学会年会, 2000年03月, 英語

モルモット消化管に存在するatypical β-アドレナリン受容体の薬理学的解析．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第27回薬物活性シンポジウム, 1999年10月, 日本語, 口頭発表（一般）

モルモット胃底に存在するatypical β-アドレナリン受容体について －モルモット十二指腸との比較－．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第101回日本薬理学会関東部会, 1999年10月, 日本語, 口頭発表（一般）

モルモット十二指腸縦走筋におけるatypical β-アドレナリン受容体薬理学的特徴．　　　　　　　　　　　　　　　
堀之内孝広, 辻谷典彦, 小池勝夫
第100回日本薬理学会関東部会, 1999年06月, 日本語, 口頭発表（一般）

Effect of bupranolol for BRL37344 and noradrenaline-induced relaxation mediating β3-adrenoceptor in rat esophageal muscularis mucosae.　　　　　　　　　　　　　　　
Koike K, Horinouchi T, Ichino T
第72回日本薬理学会年会, 1999年03月, 英語

モルモット盲腸紐におけるイソプレナリン及びサルブタモールによるβ2-及びβ3-受容体を介した弛緩反応について．　　　　　　　　　　　　　　　
堀之内孝広, 小池勝夫
第40回日本平滑筋学会総会, 1998年07月, 日本語, 口頭発表（一般）

β2- and β3-adrenoceptors mediated relaxation induced by various β-adrenergic drugs in guinea pig taenia caecum.　　　　　　　　　　　　　　　
Koike K, Horinouchi T
13th International Congress of Pharmacology., 1998年07月, International Union of Basic and Clinical Pharmacology (IUPHAR)., 英語, ポスター発表
Munich, ドイツ連邦共和国, ［国際会議］

β2- and β3-adrenoceptor-mediated relaxation of guinea pig taenia caecum induced by isoprenaline and salbutamol.　　　　　　　　　　　　　　　
Koike K, Horinouchi T, Ichino T
第71回日本薬理学会年会, 1998年03月, 英語

モルモット盲腸紐におけるCGP12177による弛緩反応及び組織cAMP量に対するブプラノロールの影響について．　　　　　　　　　　　　　　　
小池勝夫, 堀之内孝広, 高柳一成
第92回日本薬理学会関東部会, 1995年06月, 日本語, 口頭発表（一般）

Signal transduction pathway involved in β3-adrenoceptor-mediated relaxation in guinea pig taenia caecum.　　　　　　　　　　　　　　　
Horinouchi T, Koike K, Takayanagi I
第68回日本薬理学会年会, 1995年03月, 英語

Involvement of β3-adrenoceptor in the relaxation response in guinea-pig taenia caecum.　　　　　　　　　　　　　　　
Koike K, Takayanagi I, Muramatsu M, Ohki S, Horinouchi T
第67回日本薬理学会年会, 1994年04月, 英語, 口頭発表（一般）

臨床薬理学　　　　　　　　　　　　　　　
天使大学
2024年04月 - 現在

薬理学Ⅰ　　　　　　　　　　　　　　　
北海道大学
2013年04月 - 現在

統合・呼吸器学　　　　　　　　　　　　　　　
北海道大学
2012年04月 - 現在

薬理学実習　　　　　　　　　　　　　　　
北海道大学
2006年04月 - 現在

薬理学Ⅱ　　　　　　　　　　　　　　　
北海道大学
2013年04月 - 2024年03月

保健薬理学　　　　　　　　　　　　　　　
北海道大学
2019年04月 - 2023年03月

看護薬理学　　　　　　　　　　　　　　　
天使大学
2017年04月 - 2022年03月

薬理学　　　　　　　　　　　　　　　
名寄市立大学
2017年04月 - 2020年03月

臨床薬理学　　　　　　　　　　　　　　　
日本医療大学
2017年04月 - 2019年03月

薬理学　　　　　　　　　　　　　　　
日本医療大学
2017年04月 - 2019年03月

基礎情報伝達学　　　　　　　　　　　　　　　
北海道大学
2010年04月 - 2013年03月

薬理学　　　　　　　　　　　　　　　
北海道大学
2006年03月 - 2013年03月

看護薬理学　　　　　　　　　　　　　　　
天使大学
2009年04月 - 2012年03月

統合薬理学　　　　　　　　　　　　　　　
北海道大学
2006年04月 - 2010年03月

看護薬理学　　　　　　　　　　　　　　　
千葉県立若葉看護高等学校
2003年04月 - 2003年09月

看護薬理学　　　　　　　　　　　　　　　
千葉県立若葉看護高等学校
2001年04月 - 2001年09月

加熱式たばこ主流煙水抽出物の調製法バリデーションと血管壁構成細胞に対する作用解析
特定研究助成
2022年04月 - 2025年03月
堀之内孝広
公益財団法人喫煙科学研究財団, 北海道大学, 研究代表者, 競争的資金, 2022T005

たばこ煙ガス相水抽出物による血管内皮機能障害機序の薬理学的解析－紙巻きたばこと加熱式たばこの比較研究－　　　　　　　　　　　　　　　
一般研究助成
2019年04月 - 2022年03月
堀之内孝広
公益財団法人喫煙科学研究財団, 北海道大学, 研究代表者, 競争的資金, 2019G011

エンドセリン受容体関連分子のカルボニル化修飾に着目した肺高血圧症の病態基盤の解明
科学研究費助成事業 基盤研究(C)
2018年04月01日 - 2021年03月31日
堀之内 孝広
肺動脈性肺高血圧症の病態形成におけるポリアミン系の病態生理的役割を明らかにするため、ポリアミンの代謝産物であるアクロレインが、血管内皮細胞と血管平滑筋細胞に及ぼす影響について検討した。
ヒト血管内皮細胞に、アクロレインを処理したところ、濃度依存的な細胞膜傷害が認められた。そして、アクロレインによる細胞膜傷害は、プロテインキナーゼC阻害薬及び細胞内Ca2+キレート薬によって、有意に抑制された。これらのことから、アクロレインは、プロテインキナーゼC及び細胞内Ca2+依存性に、細胞膜傷害を惹起することが明らかになった。
次に、アクロレインが、プロテインキナーゼCを活性化するか明らかにするため、プロテインキナーゼCの基質であるMARCKS (myristoylated alanine-rich C kinase substrate) のリン酸化レベルを解析した。ヒト血管内皮細胞に、アクロレインを60-240分間処理したところ、MARCKSのリン酸化レベルが顕著に上昇し、この上昇は、プロテインキナーゼC阻害薬によって、完全に抑制された。この結果は、アクロレインによるプロテインキナーゼCの活性化を介した細胞膜傷害を支持するものである。
血管平滑筋細胞においても、アクロレインによるプロテインキナーゼC依存性の細胞膜傷害が認められた。また、アクロレインを前処理した摘出ラット胸部大動脈リング標本において、α1-アドレナリン受容体やエンドセリン受容体を介した血管平滑筋の収縮反応が消失した。これらのことから、アクロレインは、血管組織を傷害し、血管収縮能を喪失させることが明らかになった。
以上の結果から、アクロレインは、血管内皮細胞や血管平滑筋細胞において、プロテインキナーゼCの活性化を介した細胞膜傷害を惹起することによって、血管張力制御機構の破綻をもたらすと考えられた。
日本学術振興会, 基盤研究(C), 北海道大学, 18K06697

骨格筋に発現するGタンパク質共役型受容体キナーゼ2に着目した新たな2型糖尿病発症メカニズムの解明　　　　　　　　　　　　　　　
第30回研究助成
2015年04月 - 2016年03月
堀之内 孝広
公益財団法人 寿原記念財団, 研究代表者, 競争的資金

骨格筋に発現するユニークな機能分子を標的にした新規2型糖尿病発症メカニズムの解明　　　　　　　　　　　　　　　
研究助成金
2013年07月 - 2014年07月
堀之内 孝広
公益財団法人 薬理研究会, 研究代表者, 競争的資金

エンドセリン受容体シグナルソームの形成を介した２型糖尿病発症メカニズムの解明
科学研究費助成事業 基盤研究(C)
2012年04月01日 - 2014年03月31日
堀之内 孝広
【背景・目的】エンドセリン-1(ET-1)は，ヒト骨格筋において，インスリン誘発性のグルコース取り込みを抑制することによって，インスリン抵抗性2型糖尿病の発症・進展に関与する。しかしながら，その詳細なメカニズムは不明である。本研究では，ラット骨格筋細胞(L6細胞)を用いて，ET-1によるインスリン・シグナルの抑制性制御機構について検討した。
【方法】L6細胞におけるAktのリン酸化量の変化は，Western blot法を用いて解析した。ETA受容体(ETAR)及びETB受容体(ETBR)のmRNA発現は，RT-PCR法を用いて検出した。内在性に発現するGタンパク質共役型受容体キナーゼ2(GRK2)の機能を阻害するために，アデノウィルスを用いて，GRK2のC末端領域(GRK2-ct)をL6細胞に過剰発現させた。
【結果】L6細胞において，ヒト骨格筋細胞と同様に，ETAR及びETB受容体ETBRのmRNA発現が検出された。L6細胞において，インスリンは，濃度依存的かつ持続的なAkt(308番目のトレオニン残基・473番目のセリン残基)のリン酸化を引き起こした。インスリンによるAktのリン酸化は，PI3キナーゼ阻害薬によって完全に消失した。インスリンによるAktの持続的なリン酸化は，ET-1の後処理によって抑制され，このET-1の抑制作用は，選択的Gq/11タンパク質阻害薬及び選択的ETAR遮断薬によって解除された。さらに，ET-1によるインスリン・シグナルの抑制は，内在性GRK2の機能を阻害するGRK2-ctを過剰発現させることによって解除された。
【考察】骨格筋細胞において，ET-1によるETARの活性化は，インスリンによって誘発されるPI3キナーゼを介したAktのリン酸化をGRK2依存性に抑制することが示唆された。
日本学術振興会, 基盤研究(C), 北海道大学, 24590309

エンドセリン受容体シグナルソームの形成を介した新たな2型糖尿病発症メカニズムの解明　　　　　　　　　　　　　　　
研究助成金
2012年11月 - 2013年10月
堀之内 孝広
鈴木謙三記念 公益財団法人 医科学応用研究財団, 研究代表者, 競争的資金

骨格筋に発現するエンドセリンA型受容体を介した2型糖尿病発症メカニズムの解明　　　　　　　　　　　　　　　
一般研究助成金
2012年04月 - 2013年03月
堀之内 孝広
公益財団法人 秋山記念生命科学振興財団, 研究代表者, 競争的資金

タバコ煙中傷害因子による細胞傷害の分子機構の解明と無害化方法の開発
科学研究費助成事業
2011年 - 2012年
三輪 聡一, 堀之内 孝広
喫煙は動脈硬化症の危険因子の一つである。申請者らはタバコ主流煙に含まれる細胞傷害成分として複数のカルボニル化合物を同定した。これらの化合物はグリオーマ細胞において、細胞内カルシウム依存的にPKCを活性化し、細胞膜傷害及びアポトーシスを誘導した。更にこれらの化合物は、血管壁構成細胞や血球系細胞においても細胞内カルシウム依存的に細胞傷害を惹起すること、また単球からマクロファージへの分化を促進することを見出した。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 23659129

受容体結合蛋白によるエンドセリンA型およびB型受容体発現レベル調節と病態的意義
科学研究費助成事業
2009年 - 2011年
三輪 聡一, 西屋 禎, 堀之内 孝広, 寺田 晃士, 西本 新
エンドセリン受容体(ET_ARおよびET_BR)のC末端領域に結合する蛋白質としてJab1を同定し、Jab1がエンドセリン(ET)刺激のない状態でユビキチン化を介して受容体の代謝回転を調節することを見出した。さらに、ET刺激によりET_BRのユビキチン化レベルが上昇すること、その上昇がET刺激によるET_BRの急速な細胞内移行に必要であることを明らかにした。以上のことより、エンドセリン受容体レベルの調節にはユビキチン化が重要な役割をもつことが示された。
日本学術振興会, 基盤研究(B), 北海道大学, 21390068

エンドセリン受容体作動性TRPCチャネルの機能制御機構及び病態生理的役割の解明
科学研究費助成事業 若手研究(B)
2009年 - 2011年
堀之内 孝広
本研究では,エンドセリンA型受容体(ET_AR)作動性チャネルとして機能するTRPCチャネルの機能制御機構について検討した。その結果,(1) TRPC3及びTRPC6がET_AR作動性Ca^<2+>チャネルとして機能すること,(2) G_タンパク質,ホスホリパーゼC, Src, PI3キナーゼ,カルモジュリン(CaM)及びCaM/IP_3受容体結合ドメインがTRPC3/6を介した受容体作動性Ca^<2+>流入に関与すること,(3)プロテインキナーゼAによるTRPC6の28番目のセリン残基のリン酸化によって, TRPC6の機能が抑制されること,を明らかにした。
日本学術振興会, 若手研究(B), 北海道大学, 21790236

心血管系疾患の病態形成に関与するエンドセリンの持続性Ca2+流入作用機序の解明　　　　　　　　　　　　　　　
研究助成金
2009年04月 - 2010年03月
堀之内 孝広
財団法人 島原科学振興会, 研究代表者, 競争的資金

エンドセリン系由来の循環器疾患に関与するイオン輸送システムの機能制御機構とその病態生理的意義の解明　　　　　　　　　　　　　　　
第3回 アクテリオン アカデミア プライズ 研究奨励賞助成金
2008年04月 - 2010年03月
堀之内 孝広
アクテリオン・ファーマシューティカル・ジャパン, 研究代表者, 競争的資金

心血管リモデリングに関与するTRPCチャネルの形質膜移行の分子機構の解明　　　　　　　　　　　　　　　
奨励研究助成金
2008年04月 - 2009年03月
堀之内 孝広
財団法人 秋山記念生命科学振興財団, 研究代表者, 競争的資金

心血管系疾患の発症・進展に関与するTRPCチャネルの形質膜移行の分子機構の解明　　　　　　　　　　　　　　　
第10回研究助成
2008年04月 - 2009年03月
堀之内 孝広
財団法人 薬理研究会, 研究代表者, 競争的資金

心血管疾患の発症・進展に関与するTRPCチャネルの形質膜移行の分子機構の解明　　　　　　　　　　　　　　　
第7回循環医学分野萌芽研究助成
2008年04月 - 2009年03月
堀之内 孝広
財団法人 先進医薬研究振興財団, 研究代表者, 競争的資金

血管リモデリングに関与する新規創薬ターゲット分子の解明　　　　　　　　　　　　　　　
研究開発助成事業補助金
2007年04月 - 2008年03月
堀之内 孝広
ノーステック財団, 研究代表者, 競争的資金

エンドセリン受容体により直接活性化される新規シグナル分子群の網羅的検索
科学研究費助成事業
2006年 - 2008年
三輪 聡一, 西屋 禎, 堀之内 孝広, 西本 新
我々は、酵母ツーハイブリッド法を用いて成人ヒト心臓cDNAライブラリーから新規エンドセリンA型受容体C末端領域(ETAR C-tail)結合蛋白質としてJab1を単離・同定した。Jab1はETARのユビキチン化、リソソームでの蛋白質分解を亢進させてETARレベルを調節していることを明らかにした。さらに、アゴニストであるET-1でETARを刺激するとアゴニスト誘導性の蛋白質分解が引き起こされ、ETARレベルは時間依存的に減少していく一方、ETARに結合しているJab1量が増加すること、またETARより代謝回転が速いエンドセリンB型受容体 (ETBR)では、ユビキチン化が亢進し、より多くのJab1と結合していることが明らかになった。これらの結果より、Jab1とエンドセリン受容体との結合様式とエンドセリン受容体の蛋白質分解に強い関連性が認められた。
日本学術振興会, 基盤研究(B), 北海道大学, 18390072

Gタンパク質共役型受容体シグナリングを基盤とした動脈硬化症の発症・進展メカニズムの解明と新規創薬ターゲット分子の網羅的探索　　　　　　　　　　　　　　　
研究助成金
2006年10月 - 2007年09月
堀之内 孝広
鈴木謙三記念 財団法人 医科学応用研究財団, 研究代表者, 競争的資金

Gタンパク質共役型受容体シグナリングの多様性に関する分子薬理学的解析
科学研究費助成事業 若手研究(B)
2006年 - 2007年
堀之内 孝広
G_qタンパク質共役型受容体(G_q protein-coupled receptor:G_qPCR)であるエンドセリンA型受容体(ET_AR)、ET_BR、プリンP_2Y受容体(P_2YR)が活性化されると、G_q-ホスホリパーゼC (PLC)系を介した細胞内Ca∧<2+>イオン濃度([Ca∧<2+>]_i)上昇反応が生じる。しかし、これらG_qPCRを介した[Ca∧<2+>]_i上昇反応のパターンは一様ではなく、ET_AR及びET_BR刺激の場合、持続的な[Ca∧<2+>]_i上昇反応が生じるが、P_2YR刺激の場合、持続的な[Ca∧<2+>]_i上昇反応は生じない。これらの知見は、持続的なCa∧<2+>流入に関与するシグナリングカスケードが、G_qPCRのタイプに依存して、活性化される可能性を示唆している。
本研究では、G_qPCRを介して活性化されるシグナル伝達機構の多様性と普遍性を解明するため、[Ca∧<2+>]_i上昇反応、細胞外酸排出速度上昇反応ならびにp38MAKPのリン酸化量上昇反応を指標として、ET_AR、ET_BR、P_2Y-Rのシグナル伝達機構の解析を行った。
その結果、ET_AR及びET_BRを介した持続的な[Ca∧<2+>]_i上昇反応に、(1)G_q→PLC→TRPCチャネル、及び、(2)G_12タンパク質→p38MAPK→Na∧+/H∧+交換体→Na∧+/Ca∧<2+>交換体といった2つの機構の活性化が必須であることが明らかになった。一方、P_2YR刺激の場合、上述の機構は活性化されず、P_2YRを介して活性化されるシグナル伝達機構として、G_q→PLC→p38MAPK→Na∧+/H∧+交換体が同定された。
今後、このようなGPCRシグナリングの多様性と普遍性を創出する分子メカニズムを解明するため、多数のシグナル分子が集積すると考えられているカベオラ膜脂質マイクロドメインにおいて、本研究で同定されたシグナル分子がどの様に相互作用し、シグナル分子のネットワークを形成しているのか、詳細に解析する予定である。
日本学術振興会, 若手研究(B), 北海道大学, 18790183

研究開発調査助成
2006年12月 - 2006年12月
堀之内 孝広
北海道心臓協会, 研究代表者, 競争的資金

β-アドレナリン受容体シグナリングに関与する細胞内C末端ドメインの役割とその分子メカニズムの解明　　　　　　　　　　　　　　　
内藤記念海外研究留学助成金
2005年01月 - 2006年01月
堀之内 孝広
公益財団法人 内藤記念科学振興財団, 研究代表者, 競争的資金

β-アドレナリン受容体シグナリングにおける細胞内C末端ドメインの役割と分子メカニズムの解明　　　　　　　　　　　　　　　
海外留学助成金
2005年01月 - 2006年01月
堀之内 孝広
かなえ医薬振興財団, 研究代表者, 競争的資金

平滑筋におけるβ3-アドレナリン受容体の薬理学的特徴と新しい抑制性調節機構の解明
科学研究費助成事業 若手研究(B)
2003年 - 2005年
堀之内 孝広
少量の平滑筋組織におけるβ-アドレナリン受容体(β-AR)の定量を可能にするため、極小の組織片を用いた新規受容体結合実験法を確立した。なお、本研究では、この結合実験法を普遍化するため、平滑筋組織の他、β-ARが高密度に発現している心房・肺・眼組織についても検討した。本研究より得られた知見を次に示す。
1.受容体結合実験に使用する組織量
(1)極少量の組織、例えば、ラット一個体分の心房、ウサギ一個体分の眼組織のみで、一例分の飽和実験もしくは置換実験が実施可能。
2.放射性リガンド
(1)水溶性放射性リガンドである[^3H]-CGP12177Aを用いて、細胞膜上のβ-ARのみを特異的に定量可能。
(2)脂溶性放射性リガンドである[^3H]-dihydroalprenololでは、非特異的結合が高いため、定量不可能。
3.組織・β-ARサブタイプによる違い
(1)適用可能な平滑筋:ウサギ結腸紐(β_1/β_2)・膀胱(β_1/β_2)・虹彩(β_2)・毛様体筋(β_2)における[^3H]-CGP12177Aの非特異的結合は低く、また、その特異的結合は、2000pM以下の濃度で飽和した。
(2)適用不可能な平滑筋:低濃度(50-2000pM:β_1/β_2)及び高濃度(1-100nM:β_3)の[^3H]-CGP12177Aを用いて、各サブタイプの定量を試みた。ラット胃底・回腸・膀胱・胸部大動脈・腹部大動脈、ウサギ胸部大動脈・気管支平滑筋、ヒト膀胱では、全結合に対する非特異的結合の割合が高く、特異的結合を良好に分離することは困難であった。
(3)適用可能な平滑筋以外の組織:ウサギ心房(β_1)・肺(β_1/β_2)・毛様体突起(β_2)、ヒト肺(β_1/β_2)における[^3H]-CGP12177Aの非特異的結合は低く、また、その特異的結合は、2000pM以下の濃度で飽和した。
以上の知見から、組織片を用いた受容体結合実験法は、極少量の組織(非特異的結合が著しく高い組織や受容体密度が低い組織を除く)における細胞膜上のβ-ARの定量を可能にすることが示唆された。
日本学術振興会, 若手研究(B), 東邦大学, 15790139

新規β-アドレナリン受容体サブタイプによる血管平滑筋緊張調節制御の可能性：β3-アドレナリン受容体の薬理学的特徴及び新しい細胞内情報伝達機構の解明　　　　　　　　　　　　　　　
第5回研究助成
2003年04月 - 2004年03月
堀之内 孝広
財団法人 薬理研究会, 研究代表者, 競争的資金

血管平滑筋の新しいβ-アドレナリン受容体サブタイプに関する研究：β3-アドレナリン受容体の薬理学的特性とそのユニークな細胞内情報伝達機構について　　　　　　　　　　　　　　　
共同研究補助金
2002年04月 - 2003年03月
堀之内 孝広
東邦大学, 研究代表者, 競争的資金