Abstract
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 µM, 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 GTPγS also induced a gradual decrease in I~Na~ amplitude, while the G‐protein antagonist GDPβS 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. J. Cell. Physiol. 213: 151–160, 2007. © 2007 Wiley‐Liss, Inc.