Distinct Functions of Gq and G11 Proteins in Coupling α1-Adrenoreceptors to Ca2+ Release and Ca2+ Entry in Rat Portal Vein Myocytes

Abstract

In this study, we identified the subunit composition of G q and G 11 proteins coupling ␣ 1 -adrenoreceptors to increase in cytoplasmic Ca 2؉ concentration ([Ca 2؉ ] i ) in rat portal vein myocytes maintained in short-term primary culture. We used intranuclear antisense oligonucleotide injection to inhibit selectively the expression of subunits of G protein. Increases in [Ca 2؉ ] i were measured in response to activation of ␣ 1 -adrenoreceptors, angiotensin AT 1 receptors, and caffeine. Antisense oligonucleotides directed against the mRNAs coding for ␣ q , ␣ 11 , ␤ 1 , ␤ 3 , ␥ 2 , and ␥ 3 subunits selectively inhibited the increase in [Ca 2؉ ] i activated by ␣ 1 -adrenoreceptors. A corresponding reduction of the expression of these G protein subunits was immunochemically confirmed. In experiments performed in Ca 2؉ -free solution only cells injected with anti-␣ q antisense oligonucleotides displayed a reduction of the ␣ 1 -adrenoreceptor-induced Ca 2؉ release. In contrast, in Ca 2؉ -containing solution, injection of anti-␣ 11 antisense oligonucleotides suppressed the ␣ 1 -adrenoreceptor-induced stimulation of the store-operated Ca 2؉ influx. Agents that specifically bound G␤␥ subunits (anti-␤ com antibody and overexpression of a ␤-adrenergic receptor kinase carboxyl-terminal fragment) had no effect on the ␣ 1 -adrenoreceptor-induced signal transduction. Taken together, these results suggest that ␣ 1 -adrenoreceptors utilize two different G␣ subunits to increase [Ca 2؉ ] i . G␣ q may activate phosphatidylinositol 4,5-bisphosphate hydrolysis and induce release of Ca 2؉ from intracellular stores. G␣ 11 may enhance the Ca 2؉ -activated Ca 2؉ influx that replenishes intracellular Ca 2؉ stores.In vascular smooth muscle, activation of ␣ 1 -adrenoreceptors stimulates phospholipase C-␤ which hydrolyzes phosphatidylinositol-4,5-bisphosphate to yield diacylglycerol and inositol 1,4,5-trisphosphate. In portal vein myocytes, the ␣ 1A -adrenoreceptors are coupled to phospholipase C-␤ through G proteins which have been identified to be G q and/or G 11 , on the basis of intracellular applications of an anti-G␣ q /␣ 11 antibody. Inositol 1,4,5-trisphosphate subsequently releases Ca 2ϩ from the intracellular store. Diacylglycerol in concert with cellular Ca 2ϩ activates protein kinase C which, in turn, stimulates Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (1-2). In addition, depletion of the intracellular store by norepinephrine promotes a sustained Ca 2ϩ entry through dihydropyridine-resistant Ca 2ϩ channels by an unknown mechanism (3). Both norepinephrine-induced Ca 2ϩ release and Ca 2ϩ entry lead to a biphasic rise of the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ). 1 Although the G protein subtypes are currently defined by their ␣ subunits, of which 23 (including splice variants) are known, a functionally active heterotrimeric G protein includes an ␣, ␤, and ␥ subunit. Up to now, 5 different ␤ and 11 different ␥ subunits have been identified (4). Thus, a great number of heterotrimers composed of specific ␣, ␤, and...

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