LYSOPHOSPHATIDYLCHOLINE STIMULATES PHOSPHOLIPASE-D IN HUMAN CORONARY ENDOTHELIAL-CELLS - ROLE OF PKC

Lysophosphatidylcholine (lyso PC) mediates multiple potentially athero genic effects on endothelial cells, although the cellular mechanism of these effects remains unclear. Phospholipase D (PLD) has been recogni zed as a novel second-messenger system that may regulate cellular func tion. 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. Stimulati on of PLD activity by lyse PC was concentration dependent (half-maximu m effective concentration of 7.6 mu M) and was not mimicked by phospha tidylcholine (20 mu M). Because PLD can be regulated by protein kinase s, the effect of several protein kinase inhibitors on lyso PC-stimulat ed PLD activity was tested. The protein kinase A inhibitor H-89 (300 n M) and the tyrosine kinase inhibitors genistein (30 mu M) and tyrphost in 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-dibutyra te (PDBu, 300 nM)-stimulated PLD activity, suggesting that this agent may not inhibit PKC in these cells. In contrast, the selective PKC inh ibitors GF-109203X (0.3-10 mu M) and chelerythrine (1-30 mu M) concent ration dependently inhibited lyso PC (20 mu M)-stimulated PLD activity and blocked PDBu (300 nM)-stimulated PLD activity. Together, these da ta document that lyso PC stimulated PLD in human endothelial cells, po ssibly by a PKC-dependent mechanism, and provide evidence that PLD act ivation in human endothelium is a novel and important mechanism by whi ch lyse PC mediates its cellular and possibly atherogenic effects.