[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 (KD) 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 x 10(5) 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 KD. 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.
Lau
Y T
YT
Department of Physiology, Chang Gung Medical College, Kweisan, Taiwan, ROC.
Chen
J K
JK
Hsu
M M
MM
Yu
H M
HM
D001665:Binding Sites; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D010042:Ouabain / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects; D014471:Umbilical Veins
D008081:Liposomes; D010042:Ouabain; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium