Lysophosphatidylcholine stimulates phospholipase D in human coronary endothelial cells: Role of PKC

Lysophosphatidylcholine (lyso PC) mediates multiple potentially atherogenic effects on endothelial cells, although the cellular mechanism of these effects remains unclear. Phospholipase D (PLD) has been recognized as a novel second-messenger system that may regulate cellular function. The purpose of this study was to determine the effect of lyso PC on PLD activity in human coronary artery endothelial cells (HCAEC) by measuring [H-3]phosphatidylethanol production in cells labeled with [H-3]myristic acid. After incubation with lyso PC (20 mu M) for 40 min, PLD activity was markedly stimulated from five- to sixfold. Stimulation of PLD activity by lyse PC was concentration dependent (half-maximum effective concentration of 7.6 mu M) and was not mimicked by phosphatidylcholine (20 mu M). Because PLD can be regulated by protein kinases, the effect of several protein kinase inhibitors on lyso PC-stimulated PLD activity was tested. The protein kinase A inhibitor H-89 (300 nM) and the tyrosine kinase inhibitors genistein (30 mu M) and tyrphostin A25 (100 mu M) had no effect on the stimulation of PLD by lyso PC (20 mu M). The protein kinase C (PKC) inhibitor calphostin C (10-300 nM) affected neither lyso PC (20 mu M)-nor 4 beta-phorbol 12,13-dibutyrate (PDBu, 300 nM)-stimulated PLD activity, suggesting that this agent may not inhibit PKC in these cells. In contrast, the selective PKC inhibitors GF-109203X (0.3-10 mu M) and chelerythrine (1-30 mu M) concentration dependently inhibited lyso PC (20 mu M)-stimulated PLD activity and blocked PDBu (300 nM)-stimulated PLD activity. Together, these data document that lyso PC stimulated PLD in human endothelial cells, possibly by a PKC-dependent mechanism, and provide evidence that PLD activation in human endothelium is a novel and important mechanism by which lyse PC mediates its cellular and possibly atherogenic effects.