title: Angiotensin II receptor coupling to phospholipase D is mediated by the betagamma subunits of heterotrimeric G proteins in vascular smooth muscle cells.
abstract: 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, GTPgammaS 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-Gbeta antibody (56 +/- 5%) and in cells overexpressing the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1 (79 +/- 8%), suggesting a critical role for Gbetagamma in PLD activation by Ang II. This effect may be mediated by pp60(c-src), because in beta-adrenergic receptor kinase1 overexpressing cells, pp60(c-src) activation was inhibited, and in normal cells anti-pp60(c-src) antibody inhibited Ang II-stimulated PLD activity. Galpha12 may also contribute to AT1 receptor-PLD coupling because electroporation of anti-Galpha12 antibody significantly inhibited PLD activity, whereas anti-Galphai and Galphaq/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 Gbetagamma as well as Galpha12 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.
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, USA. mfukai@emory.edu
authors.forename: M
authors.initials: M
authors.lastname: Ushio-Fukai
authors.forename: R W
authors.initials: RW
authors.lastname: Alexander
authors.forename: M
authors.initials: M
authors.lastname: Akers
authors.forename: P R
authors.initials: PR
authors.lastname: Lyons
authors.forename: B
authors.initials: B
authors.lastname: Lassègue
authors.forename: K K
authors.initials: KK
authors.lastname: Griendling
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000502:physiology*; D020742:rhoA GTP-Binding Protein
chemical_list: D011945:Receptors, Angiotensin; D000804:Angiotensin II; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016392:Proto-Oncogene Proteins pp60(c-src); D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein