Inhibition of Na +,K +-ATPase activity in cultured rat cerebellar granule cells prevents the onset of apoptosis induced by low potassium

In cerebellar granule cells in culture, lowering of extracellular [K +] results in apoptotic death (D'Mello, S.R., Galli, C., Ciotti, T. and Calissano, P., Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP, Proc. Natl. Acad. Sci. USA, 90 (1993) 10989-10993). In this model, we studied the influence of Na +,K +-ATPase inhibition on apoptosis. We demonstrate that cell death (93±2 vs. 46±1.6%) as well as fragmentation of nuclear DNA induced by low extracellular potassium were prevented by addition of ouabain (0.1 mM), a specific inhibitor of the Na +,K +-ATPase. Blockade of glutamatergic N-methyl- d-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors by 5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801; 20 μM) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) did not inhibit the protective effect of ouabain. 24 h treatment with ouabain also decreased cell death induced by Fe 2+/ascorbic acid (74±2% to 49±3%). We speculate that ouabain pretreatment enhances the resistance against low [K +]-induced apoptosis independent of glutamate-receptor activation. Since this effect can be mimicked by a free-radical generating system, we suggest an antioxidative effect underlying ouabain-induced neuroprotection.