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 p rovide an ideal test system for constructs employing the expression ve ctor pPCK-AS, designed to express antisense RNA at birth under the con trol 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 antisens e to G(ialpha2) that, like transcription of the PEPCK gene, was induci ble by cyclic AMP. Expression of RNA antisense to G(ialpha2) and suppr ession 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 t ransgene displayed suppression of G(ialpha2) in liver and fat, two tar gets for tissue-specific expression of the PEPCK gene. The loss of G(i alpha2) in white adipocytes of transgenic mice resulted in 3.1-fold el evation of basal cyclic AMP accumulation. Cyclic AMP accumulation in r esponse to stimulation by epinephrine (10 muM) was normal in adipocyte s of transgenic mice, demonstrating no alteration in the stimulatory a denylylcyclase capacity in the G(ialpha2)-deficient cells. The inhibit ory adenylylcyclase pathway, in sharp contrast, was severely blunted i n 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 v ivo. (C) 1993 Wiley-Liss, Inc.