Sodium overload through voltage-dependent Na+ channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na+ channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 mu M) The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine <20 mu M. Veratridine neurotoxicity was due to the influx of Na+; a medium low in Na+ (36 mM) completely abolished its neurotoxicity, whereas a Ca2+-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca2+ levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na+ effect. Elevated K+ effectively blocked veratridine neurotoxicity in a Ca2+-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na+/H+ exchanger (IC50 = 0.5 mu M). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bisbenzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death. (C) 2000 Elsevier Science Inc.