Localization and characterization of binding sites with high affinity for [3H]ouabain in cerebral cortex of rabbit brain using quantitative autoradiography.
[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.
Antonelli
M C
MC
Veterans Administration Medical Center, Neurochemistry Laboratory, Seattle, WA 98108.
Baskin
D G
DG
Garland
M
M
Stahl
W L
WL
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
D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D008274:Magnesium; D004923:Erythrosine