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 microM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine < 20 microM. Veratridine neurotoxicity was due to the influx of Na(+); a medium low in Na(+) (36 mM) completely abolished its neurotoxicity, whereas a Ca(2+)-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 Ca(2+) levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na(+) effect. Elevated K(+) effectively blocked veratridine neurotoxicity in a Ca(2+)-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 (IC(50) = 0.5 microM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.
Koike
T
T
Molecular Neurobiology Laboratory, Graduate School of Science, Hokkaido University, Sapporo, Japan. tatsurok@ccms1.hucc.hokudai.ac.jp
Tanaka
S
S
Oda1
T
T
Ninomiya
T
T
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015640:Ion Channel Gating; D008854:Microscopy, Electron; D009336:Necrosis; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D017783:Superior Cervical Ganglion / Q000378:metabolism / Q000473:pathology*; D014701:Veratridine / Q000633:toxicity
D002614:Chelating Agents; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D017923:Sodium-Hydrogen Exchangers; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D014701:Veratridine; D012964:Sodium; D011188:Potassium; D002118:Calcium