The mechanism of the neurotoxic action of nitric oxide (NO) is not well understood yet. To clarify this question we studied the effects of the NO donor S-nitrosocysteine (SNOC) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and mitochondrial potential in cultured rat cerebellar granule cells loaded with fura-2 AM or/and rhodamine-123. Cells were obtained from humanely killed rats. Application of 0.3-1.0 mM SNOC to resting cells induced a concentration- and time-dependent mitochondrial depolarization (MD) followed by [Ca²⁺]_i increase. Antagonists of NMDA and non-NMDA receptor channels (25 µM memantine and 75 µM CNQX) did not prevent these effects of SNOC. Removal of external Ca²⁺ caused only a partial mitochondrial repolarization and an incomplete (25-50 % decrease of fura-2 ratio) [Ca²⁺]_i recovery. Application of 1 mM Ni²⁺ also failed to suppress the effects of SNOC. A 20 min SNOC (1.0 mM) application to cells in culture led to > 75 % decrease in ATP content. In young cells (6-8 DIV) 100 µM glutamate (Glu) induced only small MD. Addition of 1 mM SNOC during Glu (in Mg²⁺-free and 10 µM glycine-containing solution) exposure caused an additional profound MD accompanied by a secondary [Ca²⁺]_i increase. After 5 min 100 µM Glu challenge, application of SNOC (1 mM) to nerve cells in Ca²⁺-free medium induced strong MD followed in 75 % of cells (n = 34) by [Ca²⁺]_i increase due to release of Ca²⁺ from mitochondria. Oligomycin (2.5 µg ml^-1) prevented this effect in all the cells. After a 40 min pretreatment of cell culture with cyclosporine A (1-5 µM), the number of cells exhibiting [Ca²⁺]_i response to SNOC was decreased to 35 % (n = 54). In conclusion, our data show that: (1) SNOC induces the deterioration of [Ca²⁺]_i homeostasis mainly due to mitochondrial dysfunction (MD and ATP depletion), and (2) Ca²⁺ release from mitochondria caused by SNOC following a Glu challenge is probably mediated by PTP opening.

This work was supported by INTAS, The Physiological Society and the Russian Foundation for Basic Research.