D-Ala-L-Leu-D-Asp-L-Arg-Phe-D-Glu (iso)-D-Ala, cyclic form

We investigated the mechanism by which GABA-B receptors enhance the Gs-coupled receptor-mediated cAMP production in Xenopus oocytes expressing poly (A)+ RNA derived from rat brain cortex. We expressed the cystic fibrosis transmembrane conductance regulator gene (CFTR) as a reporter for cAMP changes in oocytes. The GABA-B agonist (-)baclofen enhanced the adrenergic beta 2 agonist isoproterenol- or vasoactive intestinal peptide (VIP)-induced CFTR currents, whereas (-)baclofen alone did not cause any currents. The (-)baclofen-enhanced currents were inhibited by the GABA-B antagonist 2-OH saclofen. The enhancement by (-)baclofen was further augmented by coexpressing adenylyl cyclase (AC) type II, an isotype activated by G beta gamma and G alpha s, but not by coexpressing AC type III, an isotype insensitive to G beta gamma. Moreover, pretreatment of the oocytes with pertussis toxin (PTX) abolished the enhanced effect of (-)baclofen. These results indicate that upon GABA-B activation, the G beta gamma released from PTX-sensitive G-proteins activates the AC type II (or IV), and this process requires the G alpha s activation by Gs-coupled receptors.

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.

The objective of this study was to characterize the signaling mechanisms of the mu-opioid receptor in its coupling to the cystic fibrosis transmembrane conductance regulator (CFTR) when coexpressed in Xenopus oocytes. Because oocytes do not contain endogenous cAMP-regulated ion channels, the cAMP-modulated CFTR was coexpressed with receptors as a 'reporter' channel. Agonist treatment of oocytes coexpressing mu-opioid receptors, beta2-adrenergic receptors and CFTR produced Cl- currents in a dose-related manner and immunocytochemical analysis confirmed receptor expression. These data suggest that opioid agonists could activate adenylyl cyclase in this system to elevate cAMP levels. Heterotrimeric G protein betagamma-subunits acting on adenylyl cyclase type II would increase cAMP levels. The probable presence of adenylyl cyclase type II and other components of opioid signal transduction such as G(i alpha2), were demonstrated by RT-PCR. However, measurement of cAMP levels in individual oocytes by radioimmunoassay showed that opioid agonist application to oocytes expressing mu-opioid receptors, beta2-adrenergic receptors and CFTR did not increase cAMP levels, whereas application of the beta2-adrenergic agonist, isoproterenol, or IBMX alone did increase cAMP levels. Opioid-induced CFTR activation was not affected by either application of the broad spectrum kinase inhibitor, H7, nor by application of the specific PKA inhibitor, KT5720. Injection of free betagamma-subunits, which could activate the endogenous type II cyclase, was unable to produce measurable currents in oocytes expressing the CFTR. These studies indicate that opioid activation of the CFTR is not mediated through a cAMP/PKA pathway, by either betagamma-subunit activation of an adenylyl cyclase type II or promiscuous coupling to G(s alpha).