Effect of ceramide on K-Ca channel activity and vascular tone in coronary arteries

A sphingomyeIin metabolite, ceramide, serves as a second messenger in a var iety of mammalian cells. Little is known regarding the production and actio ns of this novel intracellular signaling lipid molecule in the vasculature. The present study was designed to test the hypothesis that a ceramide-medi ated signaling pathway is present in coronary arterial smooth muscle and th at ceramide serves as an inhibitor of the large-conductance Ca2+-activated potassium (K-Ca) channels and mediates vasoconstriction in coronary circula tion. We found that C-2-ceramide produced a concentration-dependent decreas e in K-Ca channel activity in vascular smooth muscle cells from small bovin e coronary arteries. The average channel activity of the K-Ca channels in c ell-attached patches decreased from 0.046+/-0.01 to 0.008+/-0.001 at a C-2- ceramide concentration of 10 mu mol/L. In inside-out patches, C-2-ceramide (1 mu mol/L) reduced the average channel activity of the K-Ca channels from 0.06+/-0.007 to 0.016+/-0.004. Dithiothreitol, an inhibitor of acidic sphi ngomyelinase (1 mmol/L), increased the average channel activity of the K-Ca channels in cell-attached patches from 0.05+/-0.02 of control to 0.26+/-0. 04, a 5-fold increase that was reversed by addition of 1 mu mol/L ceramide. Glutathione, an inhibitor of neutral sphingomyelinase, was without effect. C-2-ceramide significantly reduced the diameter of isolated perfused small coronary arteries in a concentration-dependent manner. Addition of 1 mu mo l/L C-2-ceramide decreased average arterial diameter by 28%. When C-14-sphi ngomyelin was incubated with coronary arterial homogenates at pH 7.4 and pH 5.0, C-14-choline phosphate and ceramide were produced. The conversion rat es of C-14-sphingomyelin into C-14-choline phosphate and ceramide were 65.1 +/-1.0 fmol/min per milligram protein at pH 7.4 and 114.6+/-8.3 fmol/min pe r milligram protein at pH 5.0. We conclude that both acidic and neutral sph ingomyelinases are present in the bovine coronary arteries and that ceramid e inactivates the K-Ca channel in arterial smooth muscle cells and hence ex erts a tonic vasoconstrictor action in coronary microcirculation.