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

Background: Platelet-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. Objective: The aim of present study was to evaluate the effect of PAF on NO production in primary cultured human vascular endothelial cells. Methods: Human 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. Results: Significant 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 mu M PAF. PAF-induced NO production was abolished by pretreatment of HUVECs with a NOS inhibitor N-G-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. Conclusions: These results provide direct evidence that PAF cause intracellular NO production via activation of PAF receptors in human vascular endothelial cells (c) 2008 Elsevier Ltd. All rights reserved.