Ceramide reduces endothelium-dependent vasodilation by increasing superoxide production in small bovine coronary arteries

Ceramide serves as a second messenger in a variety of mammalian cells. Litt le is known regarding the role of ceramide in the regulation of vascular en dothelial function. The present study was designed to determine whether cer amide affects endothelium-dependent vasodilation in coronary arteries and t o explore the mechanism of action of ceramide. In isolated and pressurized small bovine coronary arteries, cell-permeable C-2-ceramide (10(-5) mol/L) markedly attenuated vasodilator responses to bradykinin and A23187 (by 40% and 60%, respectively). In the presence of NG-nitro-L-arginine methyl ester , ceramide produced no further inhibition on the vasodilation induced by th ese vasodilators. Ceramide had no effect on DETA NONOate-induced vasodilati on. By use of a fluorescence NO indicator (4,5-diaminofluorescein diacetate ), intracellular NO was measured in the endothelium of freshly isolated sma ll coronary arteries. It was found that ceramide significantly inhibited br adykinin-induced NO increase within endothelial cells. However. it had no e ffect on the activity of arterial or endothelial NO synthase. Pretreatment of the arteries with sodium dihydroxybenzene disulfonate (Tiron, 10(-3) mol /L), a cell-permeable superoxide scavenger, or polyethylene glycol superoxi de dismutase (100 U/mL) largely restored the inhibitory effects of ceramide on the vasodilation and NO increase induced by bradykinin or A23187, Moreo ver, ceramide time-dependently increased intracellular superoxide (O-2(-.)) in the endothelium, as measured by a fluorescent O-2(-.) indicator, dihydr oethidium. These results demonstrate that ceramide inhibits endothelium-dep endent vasodilation in small coronary arteries by decreasing NO in vascular endothelial cells and that this decrease in NO is associated with increase d O-2(-.). but not with the inhibition of NO synthase activity within these cells.