[³H]ouabain binding to cultured endothelial cells: Effect of cholesterol enrichment

Binding experiments were performed with [³H]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 dissociation constant (K_D) of 11.7±2.1nM (n=4) in K⁺-free buffer for specific [³H] ouabain binding on these cells. External K⁺ inhibited this binding in a dose-dependent manner. The mean value of Bmax is equivalent to about 4×10⁵ sites per cell, comparable with that of smooth muscle cell. These data demonstrated the presence of specific [³H]ouabain binding linked to Na⁺/K⁺ pump, consistent with the observations of ouabain-sensitive ⁸⁶Rb uptake in huvEC. Effect of cholesterol enrichment was also studied. Incubation in media supplemented with cholesterol-phospholipid liposomes of molar ratio of 2:1 for 18 hours reduced the Bmax by 31% (P<0.05) without significantly changed the value of K_D. This reduction of [³H]ouabain binding appeared to be specific for cholesterol since liposome made with pure phospholipid did not alter binding. Recent findings indicate that cholesterol-enrichment and plasma lipoproteins enhance vascular contractile response, our results suggest that the cholesterol-enrichment of endothelial cells may also indirectly affect the vascular response via disturbing the function of Na⁺/K⁺ pump.