| Abstrakt: |
In this report, the effects of C6-ceramide on the voltage-gated inward Na+ currents ( INa), two types of main K+ current [outward rectifier delayed K+ current ( IK) and outward transient K+ current ( IA)], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01–100 μM, ceramide produced a dose-dependent and reversible inhibition of INa without alteration of the steady-state activation and inactivation properties. Treatment with C2-ceramide caused a similar inhibitory effect on INa. However, dihydro-C6-ceramide failed to modulate INa. The effect of C6-ceramide on INa 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 INa amplitude and eliminated the effect of ceramide on INa. In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca2+ receptor did not affect the action of C6-ceramide. Intracellular application of GTPγS also induced a gradual decrease in INa amplitude, while GDPβS eliminated the effect of C6-ceramide on INa. Furthermore, the C6-ceramide effect on INa was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C6-ceramide decreased cell viability and blocking INa by tetrodotoxin did not mimic the effect of C6-ceramide, and inhibiting intracellular Ca2+ release by dantrolene could not decrease the C6-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca2+ release through the ryanodine-sensitive Ca2+ receptor may be responsible for the C6-ceramide-induced inhibition of INa, which does not seem to be associated with C6-ceramide-induced granule neuron death. [ABSTRACT FROM AUTHOR] |
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