Wotta D R, Birnbaum A K, Wilcox G L, Elde R, Law P Y
Department of Pharmacology, University of Minnesota, Medical School, Minneapolis 55455, USA.
Brain Res Mol Brain Res. 1997 Feb;44(1):55-65. doi: 10.1016/s0169-328x(96)00189-1.
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).
