Insulin Stimulates Transepithelial Sodium Transport by Activation of a Protein Phosphatase That Increases Na-K Atpase Activity in Endometrial Epithelial Cells

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na⁺ transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na⁺ transport two- to threefold by increasing Na⁺-K⁺ ATPase transport activity and basolateral membrane K⁺ conductance without increasing the apical membrane amiloride-sensitive Na⁺ conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na⁺ absorption with a further increase in Na⁺-K⁺ ATPase transport activity and an increase in apical membrane amiloride-sensitive Na⁺ conductance. The effect of insulin on the Na⁺-K⁺ ATPase was the result of an increase in V_max for extracellular K⁺ and intracellular Na⁺, and an increase in affinity of the pump for Na⁺. Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of α-1 and α-2 isoforms, but not the α-3 isoform of Na⁺-K⁺ ATPase, which did not change in the presence of insulin. Insulin-stimulated Na⁺ transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)₃], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na⁺ transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3–kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3–kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na⁺-K⁺ ATPase activation.