Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries.
We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.
Zhang
David X
DX
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Zou
Ai-Ping
AP
Li
Pin-Lan
PL
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D002518:Ceramides / Q000502:physiology*; D003331:Coronary Vessels / Q000503:physiopathology*; D004730:Endothelium, Vascular / Q000503:physiopathology*; D004789:Enzyme Activation / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002518:Ceramides; D007527:Isoenzymes; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1