High affinity and low affinity ouabain binding sites in the rat heart

Ventricular muscle of rat heart has two classes of receptors which are responsible for the positive inotropic effect of ouabain. Low affinity receptors are apparently related to Na⁺, K⁺-ATPase. To determine if high affinity receptors are also sarcolemmal Na⁺, K⁺-ATPase of muscle cells, their characteristics were examined. Binding of [³H]ouabain to the high affinity binding site required ATP in the presence of Mg²⁺ and Na⁺, was stimulated by Na⁺ in the presence of Mg²⁺ and ATP, and was inhibited by K⁺. Digoxin, digitoxin and cassaine all inhibited [³H]ouabain binding to the high affinity site. Cassaine was about an order of magnitude less potent than the glycosides. These results indicate similarities in high affinity ouabain binding sites in ventricular muscle of rat heart and Na⁺, K⁺-ATPase obtained from other sources. Destruction od sympathetic nerve terminals with 6-hydrodopamine failed to affect the high affinity ouabain binding sites indicating that high affinity sites do not represent the Na⁺, K⁺-ATPase in sympathetic nerve terminals. Labeling of Na⁺, K⁺-ATPase from [γ-³²P]ATP indicates that high affinity ouabain binding sites account for 25% of the total enzyme molecules present in ventricular muscle of rat heart.