Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na+/Ca2+ exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 muM). The sphingolipids reduced exchange-mediated Ba2+ influx by 50-70% and also inhibited the Ca2+ efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta (241-680) and Delta (680-685) deletion mutants of the Na+/Ca2+ exchanger were not inhibited by ceramide; these mutants show defects in both Naf-dependent and Ca2+-dependent regulatory behavior Another mutant, which was defective only in Na+-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca2+ stores. Sphingosine (2.5 muM) also inhibited the Ca2+ influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba2+ influx in the Delta (241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca2+-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca2+.