Liu Zheng, Fei Xiao-Wei, Fang Yan-Jia, Shi Wen-Jie, Zhang Yu-Qiu, Mei Yan-Ai
Institute of Brain Science, School of Life Sciences, Fudan University, Shanghai, PR China.
J Neurochem. 2008 Sep;106(6):2463-75. doi: 10.1111/j.1471-4159.2008.05562.x. Epub 2008 Jul 8.
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 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS 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 Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) 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.
在本报告中，研究了C(6)-神经酰胺对培养的大鼠小脑颗粒细胞电压门控内向钠电流（I(Na)）、两种主要钾电流[外向整流延迟钾电流（I(K)）和外向瞬态钾电流（I(A)）]以及细胞死亡的影响。在0.01 - 100微摩尔浓度范围内，神经酰胺对I(Na)产生剂量依赖性且可逆的抑制作用，而不改变稳态激活和失活特性。用C(2)-神经酰胺处理对I(Na)产生类似的抑制作用。然而，二氢-C(6)-神经酰胺未能调节I(Na)。细胞内注入钙螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸可消除C(6)-神经酰胺对I(Na)的作用，但咖啡因可模拟其作用。用兰尼碱受体阻滞剂阻断肌浆网中钙的释放会导致I(Na)幅度逐渐增加，并消除神经酰胺对I(Na)的作用。相反，肌醇1,4,5-三磷酸敏感钙受体的阻滞剂不影响C(6)-神经酰胺的作用。细胞内应用GTPγS也会导致I(Na)幅度逐渐降低，而GDPβS可消除C(6)-神经酰胺对I(Na)的作用。此外，应用磷脂酶C（PLC）阻滞剂后，C(6)-神经酰胺对I(Na)的作用被消除，而钙调蛋白抑制剂可使其作用大大降低。荧光染色显示，C(6)-神经酰胺降低细胞活力，用河豚毒素阻断I(Na)不能模拟C(6)-神经酰胺的作用，用丹曲林抑制细胞内钙释放也不能降低C(6)-神经酰胺诱导的细胞死亡。因此，我们认为通过兰尼碱敏感钙受体增加PLC依赖性钙释放可能是C(6)-神经酰胺诱导I(Na)抑制的原因，这似乎与C(6)-神经酰胺诱导的颗粒神经元死亡无关。
