Temporal dispersion of activation of phospholipase C-β1 and -γ isoforms by angiotensin II in vascular smooth muscle cells. Role of α(q/11), α₁₂, and βγ G protein subunits

Activation of phospholipase C (PLC) is one of the earliest events in angiotensin II (Ang II) type 1 (AT₁) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT₁ Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-γ and G protein-dependent PLC-β activation pathways have been reported. The expression of PLC-β1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT₁ R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-β1, - γ1, and -δ1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP₃) formation measured at 15 s, which corresponds to the peak response, was significantly in. hibited by electroporation of antibodies against PLC-β1, but not by anti-PLC-γ and -δ antibodies. Electroporation of anti-Gα(q/11) and -Gα₁₂ antibodies also showed significant inhibition of the Ang II- induced IP₃ generation at 15 s, while anti-Gα(i) and Gα₁₃ antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gβ antibody and cells stably transfected with the plasmid encoding the Gβγ-binding region of the carboxyl terminus of β-adrenergic receptor kinase1, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP₃ production after 30 s, a time period which temporally correlated with PLC-γ tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-γ antibody markedly inhibited the IP₃ production measured at 30 s, indicating that tyrosine phosphorylation of PLC-γ contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT₁ receptors sequentially couple to PLC-β1 via a heterotrimeric G protein and to PLC-γ via a downstream tyrosine kinase; 2) the initial AT₁ receptor-PLC-β1 coupling is mediated by Gα(q/11)βγ and Gα₁₂βγ; 3) Gβγ acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT₁ receptors to PLC-β1 and PLC-γ, as well as dual coupling of AT₁ receptors to distinct Gα proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.