Characterization of the (Na+ (+) K+)-ATPase from 3T3-F442A fibroblasts and adipocytes. Isozymes and insulin sensitivity.

Abstract

The (Na+ + K+)-ATPase from 3T3-F442A fibroblasts and adipocytes was characterized by immunoblotting, ouabain-sensitive rubidium uptake, sodium affinity, and Northern analysis. Using an antibody that cross-reacts with all three forms of the catalytic subunit of the enzyme, it was found that only the alpha 1 isozyme was present in both fibroblasts and adipocytes. This result was confirmed using an antibody specific for the alpha 2 isoform. Additionally, the ouabain dependence of Rb+ uptake in both cell types gave KI values of 0.7-1.0 X 10(-4) M, a concentration that is characteristic for the alpha 1 isoform. For both fibroblasts and adipocytes, the dependence of rubidium uptake activity on sodium concentration was characterized by K0.5 values of 9.4 and 6.2 mM, respectively, which is also diagnostic for the alpha 1 subunit in vivo. Although in fibroblasts there was no detectable message for the alpha 2 isozyme, the 3.4-kilobase message for this isozyme was present in adipocytes; this discrepancy is discussed. The (Na+ (+) K+)-ATPase was activated in fibroblasts and adipocytes by insulin at half-maximal concentrations of 11 nM and about 100 pM, respectively. Glucose uptake was also stimulated at similar concentrations of the hormone. In fibroblasts, insulin caused an increase in sodium uptake which was not inhibited by 1 mM amiloride. From these data, the presence of an insulin-sensitive sodium channel is hypothesized.