Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'- monophosphate

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether α-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [³²P(i)] and PLD activity determined by formation of the PLD metabolite [³²P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. α-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10^-6 to 10^-10) with maximal PLD stimulation achieved with 10 nmol/L α-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active ζ-thrombin (10 nmol/L) and γ-thrombin (1 μmol/L), but not inactive DIP-α-thrombin (1 μmol/L) also increased PLD activity. The role of Ca²⁺ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca²⁺ ionophores A23187 and ionomycin (1 μmol/L, threefold to fourfold increase in PEt). α-Thrombin-stimulated PEt formation was abolished (>90% inhibition) with chelation of intracellular calcium (Ca(i)/²⁺) by pretreatment with BAPTA-AM (25 μmol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca(E)/²⁺) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced α-thrombin-induced PEt formation in a dose-dependent manner (10 μmol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of α-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered α-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 μg/mL, 60 minutes) consistently augmented α-thrombin- stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that α-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, α-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca²⁺ secretagogue IP₃ and DAG. This increase in PLC activity resulting in elevated [Ca(i)/²⁺] and PKC translocation and activation is required for the coupling of the thrombin receptor to phospholipid hydrolysis by PLD. Thrombin-stimulated PA formation catalyzed by PLD and subsequent DAG production represents an important and heretofore unrecognized pathway of thrombin-induced signal transduction in human endothelium.