PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells

In this report, the effects of C-6-ceramide on the voltage-gated inward Na+ currents (I-Na), two types of main K+ current [outward rectifier delayed K+ current (I-K) and outward transient K+ current (I-A)], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 mu M, ceramide produced a dose-dependent and reversible inhibition of IN, without alteration of the steady-state activation and inactivation properties. Treatment with C-2-ceramide caused a similar inhibitory effect on I-Na. However, dihydro-C-6-ceramide failed to modulate I-Na. The effect of C-6-ceramide on I-Na was abolished by intracellular infusion of the Ca2+-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca2+ from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I-Na amplitude and eliminated the effect of ceramide on I-Na. In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca2+ receptor did not affect the action Of C-6-ceramide. Intracellular application of GTP gamma S also induced a gradual decrease in I-Na amplitude, while GDP beta S eliminated the effect of C-6-ceramide on I-Na. Furthermore, the C-6-ceramide effect on I-Na was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C-6-ceramide decreased cell viability and blocking I-Na by tetrodotoxin did not mimic the effect of C-6-ceramide, and inhibiting intracellular Ca2+ release by dantrolene could not decrease the C-6-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca2+ release through the ryanodine-sensitive Ca2+ receptor may be responsible for the C-6-ceramide-induced inhibition of I-Na, which does not seem to be associated with C-6-ceramide-induced granule neuron death.