Angiotensin II receptor coupling to phospholipase D is mediated by the beta gamma subunits of heterotrimeric G proteins in vascular smooth muscle cells

In cultured vascular smooth muscle cells (VSMCs), activation of phospholipa se D (PLD) by angiotensin II (Ang II) represents a major source of sustaine d generation of second messengers. Understanding the molecular mechanisms c ontrolling activation of this pathway is essential to clarify the complexit ies of Ang II signaling, but the most proximal mechanisms coupling AT(1) re ceptors to PLD have not been defined. Here we examine the role of heterotri meric G proteins in AT(1) receptor-PLD coupling. In alpha-toxin permeabiliz ed VSMCs, GTP gamma S enhanced Ang Ii-stimulated PLD activation. In intact cells, Ang II activation of PLD was pertussis toxin-insensitive and was not additive with sodium fluoride, a cell-permeant activator of heterotrimeric G proteins, indicating that AT(1) receptor-PLD coupling requires pertussis toxin-insensitive heterotrimeric G proteins. Ang II-stimulated PLD activit y was significantly inhibited in VSMCs electroporated with anti-G beta anti body (56 +/- 5%) and in cells overexpressing the G beta gamma-binding regio n of the carboxyl terminus of beta-adrenergic receptor kinase1 (79 +/- 8%), suggesting a critical role for G beta gamma in PLD activation by Ang II. T his effect may be mediated by pp60(c-src), because in beta-adrenergic recep tor kinase1 overexpressing cells, pp60(c-src) activation was inhibited, and in normal cells anti-pp60(c-src) antibody inhibited Ang It-stimulated PLD activity. G alpha(12) may also contribute to AT(1) receptor-PLD coupling be cause electroporation of anti-G alpha(12) antibody significantly inhibited PLD activity, whereas anti-G alpha(i) and Ga alpha(q/11) antibodies had no effect. Furthermore, electroporation of anti-RhoA antibody also attenuated Ang Ii-induced PLD activation, suggesting a role for small molecular weight G protein RhoA in this response. Thus, we provide evidence here that G bet a gamma as well as G alpha(12), subunits mediate AT(1) receptor coupling to tonic PLD activation via pp60(c-src)-dependent mechanisms, and that RhoA i s involved in these signaling pathways in rat VSMCs. These results may prov ide insight into the molecular mechanisms underlying the highly organized, complex, chronic signaling programs associated with vascular smooth muscle growth and remodeling in response to Ang II.