Binding of [3H]ouabain to endothelial cells derived from various vascular beds

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Abstract
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 form 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>ouabain>digoxin>g-strophanthidin>digoxigenin>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.

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