RESUMO
AIM: To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats. METHODS: Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity. RESULTS: In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive. CONCLUSION: This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.