title: Insulin effects on ouabain binding in A6 renal cells.
abstract: The effects of insulin on the Na+-K+-ATPase pump of the basolateral membrane of tight epithelia were evaluated by measuring transepithelial transport and [3H]ouabain binding in cultured A6 kidney cells. [3H]Ouabain binding in epithelia incubated in either K+-containing or K+-free solutions was measured. Insulin induced increases in transepithelial sodium transport, as measured by the short-circuit current (Isc), and in the initial rate of [3H]ouabain binding determined when the preparation was bathed in K+-containing solutions. However, when initial [3H]ouabain binding in tissues incubated in K+-free solutions was measured the stimulation of the initial rate of [3H]ouabain binding caused by insulin was markedly reduced. Incubating the apical side of the epithelium with either amiloride or Na+-free solutions also reduced or abolished the increase in the initial rate of [3H]ouabain binding caused by insulin. Equilibrium binding measurements showed that insulin did not increase the maximum number of [3H]ouabain-binding sites in tissues incubated with either normal K+ or K+-free solutions. These results indicate that the increase in the initial rate of [3H]ouabain binding under transporting conditions is due to an effect on the binding kinetics of ouabain, probably related to an increased rate of Na+ entry, rather than to an increase in the number of Na+-K+-ATPases in the basolateral membrane. Cycloheximide inhibited both the increase in Isc and the increase in the initial rate of [3H]ouabain binding caused by insulin in epithelia incubated in K+-containing solutions. However, cycloheximide was without effect on the initial rate of [3H]ouabain binding in insulin-treated tissues incubated in K+-free solution. This finding suggests that the cycloheximide-sensitive step of the action of insulin is related to Na+ delivery to the pump.
authors.affiliation: Laboratory of Physiology, KULeuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: P D
authors.initials: PD
authors.lastname: Smet
authors.forename: D
authors.initials: D
authors.lastname: Erlij
authors.forename: W
authors.initials: W
authors.lastname: Van Driessche
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D007328:Insulin / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects*; D010042:Ouabain / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects*; D014982:Xenopus laevis
chemical_list: D007328:Insulin; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase