Inhibition of α2/α3 sodium pump isoforms potentiates glutamate neurotoxicity

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na⁺, K⁺-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (α1) and high (α2/α3) affinity for ouabain, we were able to inhibit selectively the α2 (principally glial) and α3 (neuronal) catalytic subunits without affecting the α1 isoform. Brief exposures (5-60 min) to high ouabain concentrations (1-10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the α2/α3 isoforms (by 1 μM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [³H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reduction in α2/α3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude glutamate excitotoxicity. Since the distribution of NaP is highly heterogenous in the nervous system, with similar cell types varying greatly in isoform expression, constitutive levels of this isoenzyme could constitute a major factor in the survival of stimulated neurons. Further, factors which directly affect pump activity, such as activation of protein kinase A, may modulate excitotoxicity.