Protein kinase M zeta regulation of Na/K ATPase: A persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures

Abstract In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia–hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia–hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMζ), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the α1 and α2 isoforms of the Na/K ATPase. Similarly, active PKMζ levels were increased at 24 h, but not 2 h, after preconditioning. PKMζ overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMζ regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit α1, dihydroouabain to inhibit α2/3 and a ζ-pseudosubstrate peptide to inhibit PKMζ. These experiments showed that PKMζ regulated both the activity and surface expression of the α1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and ζ-pseudosubstrate peptide were used to determine if PKMζ or the α1 and α2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMζ levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMζ and Na/K ATPase in ischemic preconditioning.