GLUCAGON STIMULATION OF HEPATIC NA-PUMP ACTIVITY AND ALPHA-SUBUNIT PHOSPHORYLATION IN RAT HEPATOCYTES()

In this study the possible role of Na+ influx, arachidonate mediators and alpha-subunit phosphorylation in the stimulatory response of hepat ic Na+/K+-ATPase to glucagon was examined. Glucagon stimulation of oua bain-sensitive Rb-86(+) uptake in freshly isolated rat hepatocytes rea ched maximal levels in less than 1 min after hormone addition and was half-maximal (EC(50)) at a concentration of 2.4(+/- 1.3) x 10(-10) M. Analysis of the K+-dependence of this response indicates an effect on the apparent V-max. for K+ with no significant change in the apparent K-0.5. Unlike monensin, glucagon stimulation of Na+/K+-ATPase-mediated transport activity was not associated with an increase in Na-22(+) in flux. This indicates that the stimulation of Na+/K+-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonat e mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive Rb-86(+) uptake was signifi cantly affected by supramaximal concentrations of cyclo-oxygenase, lip oxygenase, cytochrome P-450 or phospholipase A(2) inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the s timulation of ouabain-sensitive Rb-86(+) uptake, hepatocytes were meta bolically radiolabelled with [P-32]P-i. Glucagon stimulated incorporat ion of P-32 into a 95 kDa phosphoprotein that comigrates with Na+/K+-A TPase alpha-subunit immunoreactivity in two-dimensional gel electropho resis. The alpha-subunit could be immunoprecipitated from detergent-so lubilized particulate fractions of hepatocytes using an anti-(rat kidn ey Na+/K+-ATPase) serum. When hepatocytes were metabolically radi alph a olabelled with [P-32]P-i, the immunoprecipitated alpha-subunit conta ined (32)p. Glucagon increased the incorporation of P-32 into the immu no-precipitated subunit by 197+/-21% (n = 6). Similar results were obs erved with a rabbit anti-peptide serum ('anti-LEAVE' serum) prepared a gainst an amino acid sequence in the alpha-subunit. The EC(50) for glu cagon-stimulated phosphorylation of the alpha-subunit (similar to-1x10 (-10) M) was very close to that for glucagon stimulation of ouabain-se nsitive Rb-86(+) uptake. In conclusion, it appears that glucagon stimu lation of hepatic Na+/K+-ATPase-mediated transport activity is not sec ondary to increases in Na+ influx or changes in the levels of an arach idonate mediator. The data provide support for the hypothesis that glu cagon stimulation of Na+-pump activity in hepatocytes may be related t o protein kinase-mediated changes in the phosphorylation state of the alpha-subunit.