The effects of anesthetics and ethanol on cannabinoid receptor function.

代谢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，如毒蕈碱M受体（M_1R）和5-羟色胺（5-HT） 2A型受体已被报道为麻醉剂的靶点。然而，关于麻醉剂对g_i偶联受体的影响，我们所知甚少。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i偶联受体(毒蕈碱m_2受体（M_2R）和大麻素1受体（CB1R）功能的方法。我们测定了乙酰胆碱（ACh）诱导的Ca^<2+>激活的Cl^-电流在共表达g_i偶联M_2R和嵌合G α_<qi5>的爪蟾卵母细胞中。虽然ACh在单独表达M_2R的卵母细胞中不产生电流，但在G α_< 5>共表达M_2R的卵母细胞中产生强大的Cl^-电流。由G α_<qi5>介导的乙酰胆碱诱导的Cl^-电流的EC_<50>为0.2 μmol/l，是由内源性表达的G α_i释放的G β γ亚基激活的乙酰胆碱诱导的G蛋白激活的内校正K^+电流的2.2倍。其他g_i偶联的生长抑素2,5 - ht_ <1A>和δ阿片受体，当与G α_<qi5>在卵母细胞中共表达时，也会引起强大的Ca^<2+>激活的Cl^-电流。在共表达M_2R和G α_< 5>的卵母细胞中，挥发性麻醉剂氟烷抑制M_2R诱导的Cl^-电流呈浓度依赖性，IC_<50>为1.1 μmol/l，表明氟烷在临床相关浓度下抑制M_2R诱导的细胞反应。蛋白激酶C抑制剂GF109203X可使表达M_2R和G_<qi5>的卵母细胞在1 μmol/l ACh诱导下的初始Cl^-电流增强3.5倍。然而，在用GF109203X预处理的卵母细胞中，卤烷诱导的对乙酰胆碱引起的Cl^-电流的抑制率没有改变。这些发现表明，氟烷通过作用于PKC活性以外的位点来抑制m_2r诱导的信号传导。综上所述，利用表达G α_<qi5>的卵母细胞，将有助于研究麻醉或镇痛药物对非洲爪蟾卵母细胞表达系统中gi偶联受体功能的影响。嵌合G α_q蛋白G α_<qi5>含有G α_i的羧基末端5个氨基酸，使g_i偶联受体能够偶联到gq偶联受体介导的下游途径，如磷脂酶c的激活。我们在共表达g_i偶联CB1R和嵌合G α_<qi5>的爪虫卵母细胞中测定了阿南达胺诱导的Ca^<2+>激活的Cl^-电流。虽然anandamide在单独表达CB1R的卵母细胞中没有产生电流，但在Gα_< q5 >共表达CB1R的卵母细胞中产生了强大的Cl^-电流。在共表达CB1R和G α_<qi5>的卵母细胞中，挥发性麻醉剂氟烷抑制CB1R诱导的Cl^-电流，表明氟烷在临床相关浓度下抑制CB1R诱导的细胞反应。这些发现表明氟烷抑制了cir诱导的信号传导。综上所述，利用表达G α_<qi5>的卵母细胞，将有助于研究麻醉或镇痛药物对爪蟾卵母细胞表达系统中g_i偶联受体功能的影响。尽管人们对中枢神经系统中的离子通道作为麻醉药的靶点给予了很多关注，但一些研究表明，gpcr也是麻醉药的靶点。gi偶联受体也可能是麻醉药的靶点。更多关于Gi偶联受体的信息可能有助于阐明gpcr在麻醉剂作用机制中的作用。少

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