Evidence that human endothelial cells express different isoforms of Na,K-ATPase.

Abstract

The catalytic alpha and smaller beta subunits of the plasma membrane Na,K-ATPase occur in various molecular forms (alpha1, alpha2, alpha3, beta1 and beta2). The alpha isoforms of the enzyme have varying affinities for ouabain and exist in different tissues with particular distribution patterns. To document the existence of isoforms of the Na,K-ATPase in cultured human umbilical vein endothelial cells. Microsomal fractions were prepared by differential ultracentrifugation from primary cultures of human umbilical vein endothelial cells and from such cells obtained after three passages. Na,K-ATPase activity was assayed using the coupled assay method and sensitivity to ouabain was determined in the presence of varying concentrations of ouabain. Specific antibodies for the various Na,K-ATPase isoforms were used to label these different proteins by immunocytochemistry in endothelial cells and by Western blotting in isolated membranes. In plotting the dose-response curves for Na,K-ATPase activity in response to ouabain we assumed the existence of two independent sites exhibiting different affinities for ouabain (in the micromol/l and the nmol/l ranges). The contribution of low-affinity sites was threefold that of high-affinity sites. After three passages in culture, a specific increase in Na,K-ATPase activity of the high-affinity sites was observed compared with that of cells from primary cultures. Confocal microscopy revealed the existence of beta1, beta2, and alpha1 subunit proteins in human umbilical endothelial cells. Staining for alpha3 isoform was less pronounced and no obvious alpha2 was detected. These findings suggest that human umbilical vein endothelial cells contain beta1, beta2, a large amount of alpha1 isoform with an apparently low affinity for ouabain, and a lesser amount of high-affinity sites, which may correspond to the alpha3 protein.