[(3)H]OUABAIN BINDING TO CULTURED ENDOTHELIAL-CELLS - EFFECT OF CHOLESTEROL ENRICHMENT

Binding experiments were performed with [H-3]ouabain on cultured human umbilical vein endothelial cells (huvEC). Saturation studies yielded a binding capacity (Bmax) of 820+/-81 fmole/mg pr.(n=4) and dissociati on constant (K-D) of 11.7+/-2.1nM (n=4) in K+-free buffer for specific [H-3] ouabain binding on these cells. External K+ inhibited this bind ing in a dose-dependent manner. The mean value of Bmax is equivalent t o about 4x10(5) sites per cell, comparable with that of smooth muscle cell. These data demonstrated the presence of specific [H-3]ouabain bi nding linked to Na+/K+ pump, consistent with the observations of ouaba in-sensitive Rb-86 uptake in huvEC. Effect of cholesterol enrichment w as also studied. Incubation in media supplemented with cholesterol-pho spholipid liposomes of molar ratio of 2:1 for 18 hours reduced the Bma x by 31% (P<0.05) without significantly changed the value of KD. This reduction of [H-3]ouabain binding appeared to be specific for choleste rol since liposome made with pure phospholipid did not alter binding. Recent findings indicate that cholesterol-enrichment and plasma lipopr oteins enhance vascular contractile response, our results suggest that the cholesterol-enrichment of endothelial cells may also indirectly a ffect the vascular response via disturbing the function of Na+/K+ pump .