title: Localization and characterization of binding sites with high affinity for [3H]ouabain in cerebral cortex of rabbit brain using quantitative autoradiography.
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.
authors.affiliation: Veterans Administration Medical Center, Neurochemistry Laboratory, Seattle, WA 98108.
authors.forename: M C
authors.initials: MC
authors.lastname: Antonelli
authors.forename: D G
authors.initials: DG
authors.lastname: Baskin
authors.forename: M
authors.initials: M
authors.lastname: Garland
authors.forename: W L
authors.initials: WL
authors.lastname: Stahl
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D004923:Erythrosine / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
chemical_list: D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D008274:Magnesium; D004923:Erythrosine