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 andN-hexanoylsphingosine (5–15 μm). The sphingolipids reduced exchange-mediated Ba2+ influx by 50–70% and also inhibited the Ca2+ efflux mode of exchange activity. The biologically inactive ceramide analogN-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Δ(241–680) and Δ(680–685) deletion mutants of the Na+/Ca2+ exchanger were not inhibited by ceramide; these mutants show defects in both Na+-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 μm) 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 Δ(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+.