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The effects of anesthetics and ethanol on cannabinoid receptor function.

麻醉剂和乙醇对大麻素受体功能的影响。

基本信息

批准号：
14571479
负责人：
KAMOCHI Masayuki
金额：
$ 2.24万
依托单位：
University of Occupational and Environmental Health, Japan
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571479/
关键词：
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

项目摘要

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

代谢型G蛋白偶联受体最近被认为是麻醉药和镇痛药的靶点。特别是G_q偶联受体如M_1 R和5-羟色胺（5-HT）2A型受体已被报道为麻醉药的靶点。然而，关于麻醉药对G_i偶联受体的影响，人们所知甚少。本文报道了一种分析表达嵌合G α蛋白的非洲爪蟾卵母细胞中G_i偶联受体（M_2受体（M_2 R）和大麻素1受体（CB_1 R））功能的方法，并在同时表达M_2 R和嵌合G α蛋白的非洲爪蟾卵母细胞中测定了乙酰胆碱（ACh）诱导的Ca^2+激活的Cl^-电流<qi5>。ACh在单独表达M_2R的卵母细胞中不诱导任何电流，但在同时表达M_2R和G α_的卵母细胞中可诱导强的Cl^-电流<qi5>。ACh<50>诱导的G α介导的Cl^-电流的<qi5>EC为0.2 μmol/l，是ACh诱导的G α介导的Cl ^-电流的2.2倍。 ...更多信息非洲爪蟾卵母细胞内源性G α_i释放的G β γ亚基激活乙酰胆碱诱导的G蛋白激活内向整流K^+电流在卵母细胞中，当与G α共表达时，其他G_i偶联的生长抑素2型、5-HT_<1A>和δ-阿片受体<qi5>也能引起强的Ca^2+激活的Cl^-电流。在共表达M_2R和G α_的卵母细胞中<qi5>，挥发性麻醉剂氟烷以浓度依赖方式抑制M_2 R诱导的Cl_2-电流，IC<50>_1为1.1 μmol/l，表明氟烷在临床相关浓度下抑制M_2 R诱导的细胞反应。蛋白激酶C抑制剂GF 109203 X可使表达M_2R和G_2的卵母细胞中1 μmol/lACh诱导的初始Cl ~-电流增强3.5倍<qi5>。然而，在用GF 109203 X预处理的卵母细胞中，氟烷对ACh诱发的Cl^-电流的抑制率没有改变。这些结果提示氟烷可能通过作用于PKC活性以外的位点抑制M_2 R诱导的信号转导。这些结果表明，利用表达G α_的卵母<qi5>细胞研究麻醉剂或镇痛剂对非洲爪蟾卵母细胞表达系统中G_i偶联受体功能的影响是有意义<qi5>的。我们在共表达G_i偶联CB 1 R和嵌合G α_的非洲爪蟾卵母细胞中测定了花生四烯酸胺诱导的Ca^2+激活的Cl^-电流<qi5>。尽管大麻素在单独表达CB 1 R的卵母细胞中不诱导任何电流，但在共表达CB 1 R和Gα的卵母细胞中引起了强的Cl^-电流<qi5>。在共表达CB 1 R和G α_的卵母细胞中<qi5>，挥发性麻醉剂氟烷抑制CB 1 R诱导的Cl^-电流，表明氟烷在临床相关浓度下抑制CB 1 R诱导的细胞反应。这些发现表明氟烷抑制CBIR诱导的信号传导。这些结果表明，使用表达G α的卵母细胞<qi5>将有助于研究麻醉剂或镇痛剂对非洲爪蟾卵母细胞表达系统中G_i偶联受体功能的影响。尽管中枢神经系统中的离子通道作为麻醉剂的作用靶点已受到广泛关注，但一些研究表明，GPCR也是麻醉剂的作用靶点。Gi偶联受体也可能是麻醉剂的靶点。对Gi偶联受体的进一步研究将有助于阐明GPCRs在麻醉药作用机制中的作用。少