Cholesterol modulation of transmembrane cation transport systems in human erythrocytes

The aim of this study was to investigate whether in vitro cholesterol enrichment of human erythrocytes affects transmembrane cation transport systems by changes induced in the membrane microviscosity of these cells. Human erythrocytes in suspension were incubated with cholesterol-lecithin dispersions to obtain an enrichment of their membrane cholesterol. The ouabain-sensitive Na+ efflux, the Na+, Li+(-)countertransport activity, the Na+, K+(-)cotransport activity, the basal transmembrane leakage of Na+ and K+, and the enzymatic activity of ATPases were determined in these cholesterol-rich cells and compared with control cells. Membrane core and surface microviscosity was also measured in the control and cholesterol-enriched cells, using the fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium (TMA)-DPH, respectively. The cholesterol content of the erythrocytes incubated in the presence of cholesterol-rich dispersions increased gradually over time. A 47% increase membrane cholesterol content was obtained after 16 h of incubation, while no change in the erythrocyte phospholipid content was found. High membrane cholesterol in the human erythrocyte phospholipid content was found. High membrane cholesterol in the human erythrocyte, obtained by in vitro enrichment of the cells with cholesterol-lecithin dispersion, inhibited in intact cell suspensions the ouabain-sensitive Na+ efflux, an estimate of the Na+(-)pump activity, and in isolated erythrocyte membranes the enzymatic activity of Na+, K+(-)ATPase, and Mg2+(-)ATPase. The dissociation constant for internal sodium and the maximal rate of ouabain-sensitive Na+ efflux is decreased in cholesterol-rich erythrocytes compared to control cells. The elevated erythrocyte membrane cholesterol content was also accompanied by a decrease in the Na+,K+(-)cotransport activity, the Na+, Li+(-)countertransport activity, and the transmembrane basal leakage of Na+ and K+. Microviscosity, measured in the erythrocyte membrane core with the fluorescence probe DPH, was increased in the cholesterol-rich cells compared to the control cells. However, the membrane surface microviscosity, measured with the probe TMA-DPH, was not different between the control cell and the cholesterol-rich cells. The present data show that enrichment of the human erythrocyte membrane with cholesterol results in an increase of membrane core microviscosity, resulting in an inhibition of transmembrane cation transport systems in erythrocytes in suspensions and of erythrocyte membrane Na+,K+(-)ATPase, Ca2+(-)ATPase, and Mg2+(-)ATPase.