Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons

Energy deficiency and dysfunction of the Na⁺, K⁺-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na⁺, K⁺-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na⁺, K⁺-ATPase induces apoptosis by depleting cellular K⁺ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca²⁺ and Na⁺. The Na⁺, K⁺-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca²⁺ and Na⁺ increase, K⁺ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K⁺ medium, but not Ca²⁺ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K⁺, Ca²⁺, or Na⁺ channels or high K⁺ medium each attenuated the ouabain-induced cell death; combined inhibition of K⁺ channels and Ca²⁺ or Na⁺ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifeclipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na⁺, K⁺-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K⁺ and accumulation of Ca²⁺ and Na⁺, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.