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 mu M) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 mu M) did not inhibit the protective effect of ouabain. 24 h treatment with ouabain also decreased cell death induced by Fe2+/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. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.