In cultured vascular smooth muscle cells (VSMCs), activation of phospholipase D (PLD) by angiotensin II (Ang II) represents a major source of sustained generation of second messengers. Understanding the molecular mechanisms controlling activation of this pathway is essential to clarify the Complexities of Ang II signaling, but the most proximal mechanisms coupling AT1 receptors to PLD have not been defined. Here we examine the role of heterotrimeric G proteins in AT1 receptor-PLD coupling. In alpha-toxin permeabilized VSMCs, GTPγS 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 AT1 receptor-PLD coupling requires pertussis toxin-insensitive heterotrimeric G proteins. Ang II-stimulated PLD activity was significantly inhibited in VSMCs electroporated with anti-Gβ antibody (56 ± 5%) and in bells overexpressing the Gβγ-binding region of the carboxyl terminus of beta-adrenergic receptor kinasel (79 ± 8%), suggesting a critical role for Gβγ in PLD activation by Ang II. This effect may be mediated by pp60(c-src) because in beta-adrenergic receptor kinasel overexpressing cells, pp60(c-src) activation was inhibited, and in normal cells anti-pp60(c-src) antibody inhibited Ang II-stimulated PLD activity. Gα12 may also contribute to AT1 receptor-PLD coupling because electroporation of anti-Ga12 antibody significantly inhibited PLD activity, whereas anti-Gα1 and Gα(q/11) antibodies had no effect. Furthermore, electropotation 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 Gβγ as well as Gα12 subunits mediate AT1 receptor coupling to tonic PLD activation via pp60(c- src)-dependent mechanisms, and that RhoA is involved in these signaling pathways in rat VSMCs. These results may provide 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.
ASJC Scopus subject areas
- Molecular Medicine