Platelet-activating factor (PAF) increases NO production in human endothelial cells—real-time monitoring by DAR-4M AM

BackgroundPlatelet-activating factor (PAF) is a potent inflammatory lipid mediator that increases vascular permeability and vasodilation. Several studies have addressed the effect of PAF on nitric oxide (NO) production from microvessels in vivo.ObjectiveThe aim of present study was to evaluate the effect of PAF on NO production in primary cultured human vascular endothelial cells.MethodsHuman umbilical vein endothelial cells (HUVECs) were loaded with diaminorhodamine-4M acetoxymethyl ester (DAR-4MAM), and the cells were stimulated with PAF. Intracellular NO production was monitored as increase in fluorescence intensity. Also, NO production was visualized at cellular levels using DAR-4M AM and fluorescence imaging.ResultsSignificant increases in NO production in HUVECs were soon after the PAF stimulation, reaching a plateau after 10 min of the stimulation. The increase of NO production at 10 min after the stimulation was statistically significant (p<0.05) for 0.01–10 μM PAF. PAF-induced NO production was abolished by pretreatment of HUVECs with a NOS inhibitor NG-monomethyl-l-arginine (l-NMMA) or PAF receptor antagonist BN 52021. LysoPAF, the inactive metabolite of PAF, did not exert a significant effect on intracellular NO levels.ConclusionsThese results provide direct evidence that PAF cause intracellular NO production via activation of PAF receptors in human vascular endothelial cells