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

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 Mg 2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [ 3H]ouabain binding with IC 50 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( K d) of 339 nM and a maximal binding capacity ( B max) 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.