CHOLESTEROL ENRICHMENT INHIBITS NA+/K+ PUMP IN ENDOTHELIAL-CELLS

We have previously shown that cholesterol enrichment reduces H-3-ouabain binding in cultured vascular endothelial cells. The present study aimed to determine the effect of cholesterol enrichment on ouabain-sensitive Rb-86 (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. Rb-86 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 (similar to 70% at mM range) than for bumetanide (similar to 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.