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

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 (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.