Autoradiographic visualization and characterization of [3H]ouabain binding to the Na+, K+-ATPase of rat brain and pineal

Mary Lou Caspers, Rochelle D. Schwartz, Rodrigo Labarca, Steven M. Paul

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Ouabain binds to the catalytic subunit of Na+, K+-ATPase and specific [3H]ouabain binding can be used as a measure of the number of active enzyme molecules present in a given tissue. Specific [3H]ouabain binding can be demonstrated in frozen, cryostat sections from rat brain and pineal and these sites have the characteristics of Na+, K+-ATPase. Incubations carried out in the absence of ATP or the presence of excess unlabeled ouabain reduces specific binding by ≥98%. The addition of K+ or omission of Mg2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [3H]ouabain binding with IC50 values of 0.73, 0.48 and 1.4 μM, respectively. Scatchard analyses of specific [3H]ouabain binding in brain sections shows a single class of non-interacting binding sites with an apparent affinity (Kd) of 339 nM and a maximal binding capacity (Bmax) of 34.9 pmol/mg protein. [3H]Ouabain binding is unevenly distributed throughout the brain with the olfactory nuclei, superior colliculus, dentate gyrus, pontine nuclei and pineal gland having a relatively high density of binding sites. The outer layers (1-3) of the cerebral cortex show more labeling than the inner layers (4-6) and most other brain areas have intermediate levels of [3H]ouabain binding sites, whereas white matter has virtually no specific binding. Computer-assisted densitometry was used to measure changes in specific [3H]ouabain binding after kainic acid injection into the caudate nucleus. An initial increase in [3H]ouabain binding was observed at 1 and 24 h after lesioning and a decrease in [3H]ouabain binding was evident by 9 days after lesioning. This technique should prove useful for studying the regulation of Na+, K+- ATPase in discrete brain regions under various experimental conditions.

Original languageEnglish
Pages (from-to)335-342
Number of pages8
JournalBrain Research
Volume409
Issue number2
DOIs
StatePublished - Apr 21 1987
Externally publishedYes

Keywords

  • Autoradiography
  • K-ATPase
  • Na
  • Pineal gland
  • [H]Ouabain binding

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