Production and metabolism of ceramide in normal and ischemic-reperfused myocardium of rats

Ceramide has been shown to be a key signaling molecule involved in the apop totic effect of tumor necrosis factor alpha (TNF- alpha) and other cytokine s. Given the importance of cytokines such as TNF-alpha in myocardial ischem ia-reperfusion injury, we hypothesize that ceramide is increased during isc hemia or reperfusion, and that the activity of enzymes responsible for its production or breakdown should be increased and/or decreased, respectively. Therefore, in the present study, we characterized the enzymatic activities responsible for ceramide production and metabolism in the myocardium of ra ts, and determined the contribution of these enzymes to altered ceramide le vels during myocardial ischemia and reperfusion. The basal ceramide concent ration in the myocardium of rats was 34.0 pmol/mg tissue. As determined by the conversion of C-14-sphingomyelin into ceramide and C-14-choline phospha te, both neutral (N-) and acidic (A-) SMase were detected in the myocardium , with a conversion rate of 0.09 +/- 0.008 and 0.32 +/- 0.05 nmol/min per m g protein, respectively. The activity of A-SMase (78 % of total cellular ac tivity) was significantly higher in microsomes than in cytosol, while the a ctivity of N-SMase was similar in both fractions. Ceramidase, a ceramide-me tabolizing enzyme, was also detected in the myocardium of rats. It metaboli zed ceramide into sphingosine at a rate of 9.94 +/- 0.42 pmol/min per mg pr otein. In anesthetized rats, 30 min of ischemia had no apparent effect on c eramide concentrations in the myocardium, while 30 min of ischemia followed by 3 h of reperfusion resulted in a significant increase in ceramide by 48 %. The activities of bath N- and A-SMase were reduced by 44 % and 32 %, re spectively, in the myocardium subjected to ischemia followed by reperfusion , but unaltered in the ischemic myocardium. It was also found that myocardi al ischemia followed by reperfusion produced a marked inhibition of ceramid ase (by 29 %). These results demonstrate that the myocardium of rats expres ses N- and A-SMase and ceramidase, which contribute to the production and m etabolism of ceramide, respectively. Tissue ceramide concentrations increas ed in reperfused myocardium. These increases in ceramide were not associate d with enhanced SMase activity, but rather with reduced ceramidase activity .