Temporal dispersion of activation of phospholipase C-β1 and -γ isoforms by angiotensin II in vascular smooth muscle cells -: Role of αq/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 (AT(1)) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT(1) Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-gamma and G protein-dependent PLC-beta activation pathways have been reported. The expression of PLC-beta 1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric Gr proteins involved in AT(1) R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-beta 1, -gamma 1, and -delta 1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly inhibited by electroporation of antibodies against PLC-beta 1, but not by anti-PLC-gamma and -delta antibodies. Electroporation of anti-G alpha(q/11), and -G alpha(12) antibodies also showed significant inhibition of the Ang II-induced IP3 generation at 15 s, while anti-G alpha(i) and G alpha(13) antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-G beta antibody and cells stably transfected with the plasmid encoding the G beta gamma-binding region of the carboxyl terminus of beta-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-gamma tyrosine phosphorylation in response to Ang II. Moreover, electroporation of anti-PLC-gamma antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-gamma contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT(1) receptors sequentially couple to PLC-beta 1 via a heterotrimeric G protein and to PLC-gamma via a downstream tyrosine kinase; 2) the initial AT(1) receptor-PLC-beta 1 coupling is mediated by G alpha(q/11)beta gamma and G alpha(q/11)beta gamma; 3) G beta gamma acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT(1) receptors to PLC-beta 1 and PLC-gamma, as well as dual coupling of AT(1) receptors to distinct Ga proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.