| Project/Area Number |
14571479
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Anesthesiology/Resuscitation studies
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| Research Institution | University of Occupational and Environmental Health, Japan |
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Principal Investigator |
KAMOCHI Masayuki UOEH, University hospital, Associate Professor, 大学病院, 助教授 (90204643)
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| Co-Investigator(Kenkyū-buntansha) |
MINAMI Kouitiro UOEH, School of medicine, assistant professor, 医学部, 講師 (70279347)
UEZONO Yasuhito Nagasaki University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (20213340)
SHIGEMATSU Akio UOEH, President, 学長 (30037428)
HORISHITA Takahumi UOEH, School of medicine, instructor, 医学部, 助手 (40369070)
SHIRAISHI Munehiro UOEH, School of medicine, instructor, 医学部, 助手 (40389458)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Cannabinoid Receptor / Protein G / Xenopus oocyte / Isoflurane / Enflurane / Halothane / Protein Kinase C (PKC) / Protein Kinase C(PKC) / G蛋白質共役型受容体 / Ca^<2+>依存性Cl^-チャネル / 麻酔薬 / Gi / Gq蛋白 |
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| Research Abstract |
Metabotropic G protein-coupled receptors have recently been recognized as targets for anesthetics and analgesics. In particular, G_q-coupled receptors such as muscarinic M, receptors (M_1R) and 5-hydroxytryptamine (5-HT) type 2A receptors have been reported to be targets for anesthetics. Much less is known, however, about the effects of anesthetics on G_i-coupled receptors. Here we report a method to analyze functions of Gi-coupled receptors (muscarinic M_2receptors (M_2R) and cannabinoid 1 receptors (CB1R) in Xenopus oocytes expressing a chimeric G α protein.
We determined acetylcholine (ACh)-induced Ca^<2+> -activated Cl^- currents in Xenopus oocytes coexpressing G_i-coupled M_2R with the chimeric G α_<qi5>. Although ACh did not induce any currents in oocytes expressing M_2R alone, it caused robust Cl^- currents in oocytes coexpressing M_2R with G α_<qi5>. The EC_<50> of the ACh-induced Cl^- current mediated through G α_<qi5> was 0.2 μmol/l, which was 2.2 times higher than that of the
… More
ACh-induced G protein-activated inwardly rectifying K^+ currents activated by G beta gamma subunits liberated from endogenously expressed G α_i in Xenopus oocytes. Other G_i-coupled somatostatin type 2, 5-HT_<1A> and delta-opioid receptors, when coexpressed with G α_<qi5> in oocytes, also caused robust Ca^<2+> -activated Cl^- currents. In oocytes coexpressing M_2R and G α_<qi5>, a volatile anesthetic halothane inhibited M_2R-induced Cl^- currents in a concentration-dependent manner with the IC_<50> of 1.1 μmol/l, suggesting that halothane inhibits M_2R-induced cellular responses at clinically relevant concentrations. Treatment with the protein kinase C inhibitor GF109203X produced a 3.5-fold enhancement of the initial Cl^- currents induced by 1 μmol/l ACh in oocytes expressing M_2R and G_<qi5>. The rate of halothane-induced inhibition of Cl^- currents elicited by ACh, however, was not changed in such oocytes pretreated with GF109203X. These findings suggest that halothane inhibits the M_2R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing G α_<qi5> would be helpful to examine the effects of anesthetics or analgesics on the function of Gi-coupled receptors in the Xenopus oocyte expression system.
A chimeric G α_q protein G α_<qi5>, which contains carboxy-terminus five amino acids of G α_i, enables G_i-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined anandamide-induced Ca^<2+>-activated Cl^- currents in Xenopus oocytes coexpressing G_i-coupled CB1R with the chimeric G α_<qi5>. Although anandamide did not induce any currents in oocytes expressing CB1R alone, it caused robust Cl^-currents in oocytes coexpressing CB1R with Gα_<qi5>. In oocytes coexpressing CB1R and G α_<qi5>, a volatile anesthetic halothane inhibited CB1R-induced Cl^- currents, suggesting that halothane inhibits CB1R-induced cellular responses at clinically relevant concentrations. These findings suggest that halothane inhibits the CBIR-induced signaling. Collectively these findings suggest that the use of oocyte expressing G α_<qi5> would be helpful to examine the effects of anesthetics or analgesics on the function of G_i-coupled receptors in the Xenopus oocyte expression system.
Although much attention has been paid to ion channels in the CNS as targets for anesthetics, several lines of study have shown that GPCRs are also targets for anesthetics. Gi-coupled receptors might also be targets for anesthetics. More information about Gi coupled receptors might help to elucidate the role of GPCRs in the mechanisms of anesthetics. Less
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