Binding of [3H]ouabain to endothelial cells derived from various vascular beds.
Binding experiments were performed with [3H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [3H]ouabain binding to endothelial cells from pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (KD) of 3.29 +/- 0.31 nmol/l and a binding capacity (Bmax) of 5.22 +/- 0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [3H]ouabain revealed much lower affinity (KD 95 +/- 15 nmol/l, Bmax 2.08 +/- 0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na+/K+-ATPase (proscillaridin A greater than ouabain greater than digoxin greater than g-strophanthidin greater than digoxigenin greater than dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K+ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na+/K+-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg2+-ATPase activity.
Freissmuth
M
M
Institute of Pharmacology, University of Vienna, Austria.
Nees
S
S
Böck
M
M
Schütz
W
W
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006168:Guinea Pigs; D010042:Ouabain / Q000378:metabolism*; D011869:Radioligand Assay; D011955:Receptors, Drug / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine
D011955:Receptors, Drug; C028996:cardiac glycoside receptors; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase