Agonist-induced activation of phospholipase D in bovine pulmonary artery endothelial cells: Regulation by protein kinase C and calcium

Regulation of phospholipase D (PLD) activity was investigated in cultured monolayers of bovine pulmonary artery endothelial cells (BPAECs). Agonists such as bradykinin, histamine, vasopressin, α-thrombin, and adenosine triphosphate (ATP) stimulated up to 15-fold accumulation of phosphatidylethanol (PEt) in the presence of ethanol through PLD-catalyzed phosphatidyltransferase activity. To examine mechanisms of PLD regulation, we investigated the role of protein kinase C (PKC) and Ca²⁺ fluxes in agonist-induced PLD activation. The PKC activator 12-0-tetradecanoylphorbol-13-acetate (TPA, 100 nmol/L) produced up to a 25-fold increase in PEt formation in a time-and dose-dependent manner. PEt production was also stimulated by other cell-permeant PKC activators such as 1,2 dioctanoylglycerol and 1-oleyl-2-acetylglycerol, whereas inactive phorbol derivatives 4-α-phorbol-12,13-didecanoate and 4-β-phorbol showed no effect. The effect of TPA on PEt accumulation was inhibited by the PKC inhibitors staurosporine (5 (μmol/L, 95% inhibition) and sphingosine (10 μmol/L, 50% inhibition). TPA-induced PEt accumulation was almost completely abolished (>95% inhibition) by PKC down-regulation accomplished by long-term treatment with 100 nmol/L TPA. In contrast, bradykinin- or ATP-Induced phosphorus 32-labeled PA and [³²P]-labeled PEt formation was only partially blocked (70% inhibition) by either staurosporine (10 (μmol/L) or PKC down-regulation, suggesting that part of agonist-stimulated PLD activity may occur in the absence of PKC activation. An increase in Ca²⁺₁ appears to be involved in agonist-induced PLD activation as bradykinin-, ATP-, or Ca²⁺ ionophore-induced [³²P]. PEt production was attenuated by either depletion of extra-cellular Ca²⁺ with EGTA or chelation of intracellular Ca²⁺ by BAPTA. TPA-mediated PEt accumulation was not affected by EGTA treatment, whereas BAPTA reduced TPA-mediated PEt formation by 50%. These results suggest that direct PKC activation is a potent stimulus for PLD activity and that the major pathway for agonist-induced PLD activation involves PKC activation and is dependent on an increase in intracellular Ca²⁺. Further, these studies suggest that agonist-induced PLD activation may also involve a PKC-independent mechanism.