Glucagon stimulation of hepatic Na+-pump activity and α-subunit phosphorylation in rat hepatocytes

Abstract

In this study the possible role of Na+ influx, arachidonate mediators and α-subunit phosphorylation in the stimulatory response of hepatic Na+/K+-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4(±1.3)×10-10 M. Analysis of the K+-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent K0.5. Unlike monensin, glucagon stimulation of Na+/K+-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K+-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome P-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb+ uptake, hepatocytes were metabolically radiolabelled with [32P]Pi. Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that co-migrates with Na+/K+-ATPase α-subunit immunoreactivity in two-dimensional gel electrophoresis. The α-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K+-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]Pi, the immunoprecipitated α-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197±21% (n = 6). Similar results were observed with a rabbit anti-peptide serum (‘anti-LEAVE’ serum) prepared against an amino acid sequence in the α-subunit. The EC50 for glucagon-stimulated phosphorylation of the α-subunit (1×10-10 M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K+-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na+-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the α-subunit.