Insulin effects on ouabain binding in A6 renal cells.
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.
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Laboratory of Physiology, KULeuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
Erlij
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Van Driessche
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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
D007328:Insulin; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase