Role of calcium and protein kinase C in the activation of phospholipase D by angiotensin II in vascular smooth muscle cells.

We previously showed that cultured rat aortic vascular smooth muscle cells (VSMC) possess an AT1 angiotensin (Ang) receptor coupled to the activation of a phospholipase D (PLD). AT1 receptors in VSMC are also coupled to the activation of a phosphoinositide-specific phospholipase C (PLC), mobilization of intracellular Ca2+, and activation of protein kinase C (PKC). To determine whether PLD stimulation by Ang II is the result of PLC activation and the subsequent elevation of cytosolic free Ca2+ and PKC activation, we investigated the role of Ca2+ and PKC in the activation of PLD. Chelation of extracellular Ca2+ by EGTA, blockade of voltage-sensitive Ca2+ channels, or chelation of intracellular Ca2+ with BAPTA partially attenuated PLD activation and Ca2+ mobilization in response to Ang II. However, the simultaneous chelation of extracellular Ca2+ with EGTA and intracellular Ca2+ with BAPTA completely attenuated both PLD activation and Ca2+ accumulation. Ca2+ ionophores mimicked Ang II and the combined effects of Ang II and ionophore resulted in no further stimulation of PLD activity above that observed in the presence of either agonist alone. Although the putative PLC inhibitor U73122 blocked the activation of PLD by Ang II, it also may inhibit PLD activation directly, since it attenuated both Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA)-mediated increases in PLD activity. PMA also activated PLD in VSMC in a dose-dependent manner; however, Ang II and PMA stimulation were additive. Down-regulation of PKC via exposure to phorbol dibutyrate almost completely blocked PMA-induced stimulation of PLD while it had no effect on Ang II- or Ca(2+)-ionophore-mediated increases in PLD activity. The PKC inhibitor staurosporine augmented basal PLD activity and partially inhibited PMA stimulation of PLD while it had little effect on Ang II-induced increases in PLD activity. Thus, optimal Ang II stimulation of PLD is dependent on the availability of both intracellular and extracellular Ca2+ and independent of PMA-mediated effects. Furthermore, these data suggest that Ang II stimulation of PLD may occur subsequent to activation of PLC, since Ang II activates PLC and PLC is shown to be responsible for increases in intracellular Ca2- in response to Ang II.