Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

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Abstract
[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8–12 nM for specific high‐affinity binding of [3H]ouabain were found by both methods. High‐affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1,3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50= 0.7–0.9 mM), N‐ethylmaleimide, 5,5′‐dithiobis(2‐nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+‐ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.