Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells.
Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.
Gutiérrez-Martín
Yolanda
Y
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias and Escuela de Ingenierías Agrarias, Universidad de Extremadura, Badajoz, Spain.
Martín-Romero
Francisco Javier
FJ
Henao
Fernando
F
Gutiérrez-Merino
Carlos
C
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006706:Homeostasis / Q000187:drug effects* / Q000502:physiology; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D030421:Peroxynitrous Acid / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D020746:Calcium Channels, L-Type; D030421:Peroxynitrous Acid; C002385:linsidomine; D008981:Molsidomine; D002118:Calcium