Cholesterol enrichment inhibits Na +/K + pump in endothelial cells

Abstract

We have previously shown that cholesterol enrichment reduces 3H-ouabain binding in cultured vascular endothelial cells. The present study aimed to determine the effect of cholesterol enrichment on ouabain-sensitive 86Rb (as a substitute for K +) influx, i.e. K+ transport via the pump, and to examine whether cellular K+ content was affected in human umbilical vein endothelial cells. 86Rb influx was inhibited by both ouabain and bumetanide in a dose-dependent manner. Consistent with an earlier report [1], inhibition achieved was greater for ouabain (∼ 70% at mM range) than for bumetanide (∼ 55% at 0.1 mM), indicating that K + influx via Na +/K + pump was greater than that via Na +K +Cl − cotransport in these cells. After incubation of 18 h or more with cholesterol-enriched liposomes (2:1 cholesterol to phospholipid ratio), a significant reduction (>20%) of the ouabain-sensitive K + influx and an increase in cellular cholesterol content were observed. The inhibitory effect was observed only at liposome concentrations above 2 mg/ml. Following 18 h incubation with 2 mg/ml cholesterol-enriched liposomes, cellular K + content was significantly decreased. The phospholipid liposome treatment did not alter K + content, suggesting that the inhibitory effect on Na +/K + pump and the cellular K + content reduction was not due to liposome fusion alone or to the phospholipid present. These findings indicate that cholesterol enrichment inhibits Na +/K + pump and thus reduces cellular K + content in endothelial cells, and may play a role in the widely observed abnormal endothelium-dependent vascular response induced by hypercholesterolemia.