Dose-dependence of antiapoptotic and toxic action of ouabain in neurons of primary cultures of rat cortex

Abstract

Effects of 0.01 nM–1 nM ouabain on neuronal survival in excitotoxic stress and ouabain self toxic action in concentrations from 10 nM to 30 μM were studied. Neuronal viability was evaluated by measuring Bcl-2 protein expression and using vital staining test allowing recognition of live, necrotic and apoptotic cells. Excitotoxic stress was induced by 240-min treatment with agonists of ionotropic glutamate receptors (NMDA or kainate). Experiments were performed on rat primary neuronal cultures of 7–14 DIV (days in vitro). Thirty μM NMDA induced apoptosis in 45 ± 9% (n = 5), and 30 μM kainate, in 52 ± 5% (n = 5) of neurons. An antiapoptotic effect of ultra low (0.01 nM–1 nM) ouabain concentrations was found to restore Bcl-2 expression and to bring apoptosis level back to control values (about 10%, n = 5). Since in this concentration range ouabain is not able to inhibit NKA, we conclude that neuroprotection discloses the signaling function of NKA. Whereas ouabain self toxic action in higher concentrations (10 nM–30 μM, during 240 min) resulted in necrotic death of 45% neurons (apoptosis remained as under the control conditions), the large portion of neurons were unaffected. The relatively low threshold concentration of ouabain toxic action (10 nM) is consistent with the sensitivity to ouabain of α3-isoform of NKA. Thus, ouabain was found to have a bimodal effect, including antiapoptotic action in excitotoxic stress in the concentration range from 0.01 nM to 1 nM, and self toxic action at larger concentrations. Self toxicity of ouabain is initiated through inhibition of NKA pumping function. Neuronal heterogeneity with respect to ouabain toxic action is probably related with the different expression of α1 and α3-isoforms of NKA in pyramid neurons and interneurons.