C(6)-ceramide inhibited Na(+) currents by intracellular Ca(2+) release in rat myoblasts

Ceramides are novel second messengers that may mediate signaling leading to apoptosis and the regulation of cell cycle progression. Moreover, ceramide analogs have been reported to directly modulate K(+) and Ca(2+) channels in different cell types. In this report, the effect of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)) in cultured rat myoblasts was investigated using whole-cell current recording and a fluorescent Ca(2+) imaging experiment. At concentrations of 1-100 microM, ceramide produced a dose-independent and reversible inhibition of I(Na). Ceramide also significantly shifted the steady-state inactivation curve of I(Na) by 16 mV toward the hyperpolarizing potential, but did not alter the steady-state activation properties. C(2)-ceramide caused a similar inhibitory effect on I(Na) amplitude. However, dihydro-C(6)-ceramide, the inactive analog of ceramide, failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent BAPTA, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with xestospongin C or heparin, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, ruthenium red, which is a blocker of the ryanodine-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide on I(Na). Intracellular application of the G-protein agonist GTPgammaS also induced a gradual decrease in I(Na) amplitude, while the G-protein antagonist GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Calcium imaging showed that C(6)-ceramide could give rise to a significant elevation of intracellular calcium. Our data show that increased calcium release through the IP(3)-sensitive Ca(2+) receptor, which probably occurred through the G-protein and phospholipase C pathway, may be responsible for C(6)-ceramide-induced inhibition of the I(Na) of rat myoblasts.