Na(+)-K(+)-ATPase in adipocyte differentiation in culture

Differentiation of 3T3-L1 cells from a fibroblast to an adipocyte phenotype results in an approximately 50% decline in Na(+)-K(+)-ATPase activity and ouabain-sensitive 86Rb uptake. Kinetic analysis revealed a K 1/2 for Na+ of approximately 14 mM, a Km for ATP of approximately 0.4 mM, and maximal activation by sodium dodecyl sulfate at a 0.05 (wt/wt) detergent/protein ratio in both mature fibroblasts and adipocytes. Both fibroblasts and adipocytes exhibited Na(+)-K(+)-ATPase activity with an inhibition constant (Ki) for ouabain of approximately 10(-4) M. In addition, adipocytes exhibited a second component representing 30% of total activity with a Ki of approximately 5 x 10(-7) M. The emergence of biphasic ouabain inhibition kinetics in adipocytes raised the possibility of a change in alpha-subunit isoform composition with cytodifferentiation. This inference was evaluated by isoform-specific mRNA analysis (Northern blots) and by alpha-isoform-specific immunoassays (Western blots). Northern blots revealed a modest decrease in mRNA alpha 1, a striking increase in mRNA alpha 2, and a significant loss of mRNA beta content with differentiation of fibroblasts to adipocytes. By immunoassay, fibroblasts exhibited the alpha 1-isoform. Adipocytes exhibited an admixture of alpha 1- and alpha 2-isoforms, with alpha 2 being the more abundant isoform. There was no one-to-one correspondence either between the mRNA isoform and alpha-subunit abundances or between alpha-subunit abundances and enzymatic activity, suggesting that regulation occurs at multiple levels in this system. Findings indicate, however, that a shift in alpha-isoform composition accompanied by a change in ouabain inhibition kinetics occurs with cytodifferentiation.