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

Abstract We previously showed that cultured rat aortic vascular smooth muscle cells (VSMC) possess an AT 1 angiotensin (Ang) receptor coupled to the activation of a phospholipase D (PLD). AT 1 receptors in VSMC are also coupled to the activation of a phosphoinositide-specific phospholipase C (PLC), mobilization of intracellular Ca 2+ , 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 Ca 2+ and PKC activation, we investigated the role of Ca 2+ and PKC in the activation of PLD. Chelation of extracellular Ca 2+ by EGTA, blockade of voltage-sensitive Ca 2+ channels, or chelation of intracellular Ca 2+ with BAPTA partially attenuated PLD activation and Ca 2+ mobilization in response to Ang II. However, the simultaneous chelation of extracellular Ca 2+ with EGTA and intracellular Ca 2+ with BAPTA completely attenuated both PLD activation and Ca 2+ accumulation. Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ in response to Ang II.