Regulation of Human and Pig Renal Na+,K+-ATPase Activity by Tyrosine Phosphorylation of Their a1-Subunits

Abstract Modulation of the physiologically influential Na?,K?-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min–24 h) with those PTP inhibitors (0–100 lM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na,K-ATPase activity in both human and pig samples.The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na,K-ATPase levels.Despite rigorous activity recovery attempts, the PTP inhibitors’ effects were sustained on Na?,K?-ATPase activity. Western blotting experiments revealed the expression of both a1- and b1-subunits in both human and pig tissues. a1-Subunits possessed higher tyrosine phos- phorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both a1- and b1-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we pro-vide evidence that Na?,K?-ATPase can be regulated through tyrosine phosphorylation of primarily their subunits, using PTP inhibitors

Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis

Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na(+)-K(+)-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na(+)-K(+)-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or Tumor necrosis factor--related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H(2)O(2), thapsigargin or UV-C implicating a role for the Na(+)-K(+)-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca(2+) homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca(2+) levels in response to H(2)O(2), thapsigargin or UV-C. FasL-induced alterations in Ca(2+) were not abolished in Ca(2+)-free medium but incubation of cells with BAPTA-AM inhibited both Ca(2+) perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na(+)-K(+)-ATPase activity during apoptosis is linked to perturbations in cell Ca(2+) homeostasis that modulate apoptosis induced by the activation of Fas by FasL