GI-ALPHA(2) MEDIATES THE INHIBITORY REGULATION OF ADENYLYLCYCLASE IN-VIVO - ANALYSIS IN TRANSGENIC MICE WITH GI-ALPHA(2) SUPPRESSED BY INDUCIBLE ANTISENSE RNA

The role of the GTP-binding regulatory protein (G-protein) G(ialpha2) in vivo was explored using transgenic mice in which the a-subunit of G(ialpha2) was suppressed by antisense RNA. Rat hepatoma FTO-2B cells provide an ideal test system for constructs employing the expression vector pPCK-AS, designed to express antisense RNA at birth under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cells transfected with the expression vector containing a sequence antisense to G(ialpha2) (pPCK-ASG(ialpha2) displayed expression of RNA antisense to G(ialpha2) that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to G(ialpha2) and suppression of the expression of G(ialpha2), but not G(sa) and G(ialpha3), was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of G(ialpha2) in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of G(ialpha2) in white adipocytes of transgenic mice resulted in 3.1-fold elevation of basal cyclic AMP accumulation. Cyclic AMP accumulation in response to stimulation by epinephrine (10 muM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the G(ialpha2)-deficient cells. The inhibitory adenylylcyclase pathway, in sharp contrast, was severely blunted in response to challenge by the inhibitory A1-purinergic agonist, (-)R-N6-phenylisopropyladenosine. These studies illuminate a critical role of G(ialpha2) in the inhibitory adenylylcyclase signaling pathway in vivo. (C) 1993 Wiley-Liss, Inc.