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Analysis of the effects of halothane on Gi-coupled muscarinic M2 receptor signaling in Xenopus oocytes using a chimeric G alpha protein.

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(1) receptors (M(1)R) 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 G(i)-coupled receptors in Xenopus oocytes expressing a chimeric G alpha protein. A chimeric G alpha(q) protein G alpha(qi5), which contains carboxy-terminus five amino acids of G alpha(i), enables G(i)-coupled receptors to couple to Gq-coupled receptor-mediated downstream pathways such as activation of phospholipase C. We determined acetylcholine (ACh)-induced Ca(2+)-activated Cl(-) currents in Xenopus oocytes coexpressing G(i)-coupled muscarinic M(2)receptors (M(2)R) with the chimeric G alpha(qi5). Although ACh did not induce any currents in oocytes expressing M(2)R alone, it caused robust Cl(-) currents in oocytes coexpressing M(2)R with G alpha(qi5). The EC(50) of the ACh-induced Cl(-) current mediated through G alpha(qi5) was 0.2 micromol/l, which was 2.2 times higher than that of the ACh-induced G protein-activated inwardly rectifying K(+) currents activated by G beta gamma subunits liberated from endogenously expressed G alpha(i) in Xenopus oocytes. Other G(i)-coupled somatostatin type 2, 5-HT(1A) and delta-opioid receptors, when coexpressed with G alpha(qi5) in oocytes, also caused robust Ca(2+)-activated Cl(-) currents. In oocytes coexpressing M(2)R and G alpha(qi5), a volatile anesthetic halothane inhibited M(2)R-induced Cl(-) currents in a concentration-dependent manner with the IC(50) of 1.1 mmol/l, suggesting that halothane inhibits M(2)R-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 micromol/l ACh in oocytes expressing M(2)R 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(2)R-induced signaling by acting at sites other than PKC activity. Collectively these findings suggest that the use of oocyte expressing G alpha(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.

journal_name

Pharmacology

journal_title

Pharmacology

authors

Minami K,Uezono Y,Shiraishi M,Okamoto T,Ogata J,Horishita T,Taniyama K,Shigematsu A

doi

10.1159/000080106

subject

Has Abstract

pub_date

2004-11-01 00:00:00

pages

205-12

issue

eissn

0031-7012

issn

1423-0313

pii

80106

journal_volume

pub_type

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Analysis of the effects of halothane on Gi-coupled muscarinic M2 receptor signaling in Xenopus oocytes using a chimeric G alpha protein.