title: Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes.
abstract: In this study the possible role of Na+ influx, arachidonate mediators and alpha-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) x 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 kappa 0.5. 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]P(i), Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that comigrates with Na+/K(+)-ATPase alpha-subunit immunoreactivity in two-dimensional gel electrophoresis. The alpha-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]P(i), the immunoprecipitated alpha-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 alpha-subunit. The EC50 for glucagon-stimulated phosphorylation of the alpha-subunit (approximately 1 x 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 alpha-subunit.
authors.affiliation: Department of Cellular and Molecular Physiology, College of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Lynch
authors.forename: K M
authors.initials: KM
authors.lastname: McCall
authors.forename: Y C
authors.initials: YC
authors.lastname: Ng
authors.forename: S A
authors.initials: SA
authors.lastname: Hazen
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology*; D007120:Immunochemistry; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000493:pharmacokinetics; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
chemical_list: D016718:Arachidonic Acid; D010042:Ouabain; D005934:Glucagon; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium