Characterization of "High-Affinity"[3H]Ouabain Binding in the Rat Central Nervous System

Abstract

The characteristics of [3H] ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (Kd) of 10.4 .+-. 0.9 n M and an estimated binding capacity (Bmax) of 7.6 .+-. 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus and frontal cerebral cortex. [3H] Ouabain binding to rat brain was Na- and ATP dependent and strongly inhibited by K. Proscillaridin A was the most potent cardiac glycoside tested in inhibiting specific. [3H] ouabain binding to brain membranes and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that for inhibition of heart Na, K-ATPase. To assess whether the high-affinity binding sites for [3H] ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H] ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H] ouabain binding to striatal homogenates by 79.6 .+-. 1.6%. Evidently, the high-affinity binding of [3H] ouabain binding sites are localized to neuronal elements. Thus, the high-affinity binding of [3H] ouabain to brain membranes may selectively label a neuronal form or conformation of Na, K-ATPase.