Effect of Na⁺, K⁺‐ATPase modifiers on high‐affinity ouabain binding determined by quantitative autoradiography

Abstract

There is growing evidence on the existence of endogenous ouabain-like factors that modulate Na+, K(+)-ATPase activity. In this laboratory, two soluble subfractions (peaks I and II) were previously separated from rat cerebral cortex, which had opposite effects on Na+, K(+)-ATPase activity. Peak I stimulated and peak II inhibited the enzyme (Rodríguez de Lores Arnaiz and Antonelli de Gómez de Lima, Neurochem Res 11:933-947, 1986). The same effects are now reported for K(+)-p-nitrophenyphosphatase activity. Localization of high-affinity ouabain binding in rat brain was done by quantitative autoradiography using a microcomputer digital imaging system. Peak I did not modify, whereas peak II blocked ouabain binding in areas 3-4 of cerebral cortex, dentate gyrus, stria terminalis, thalamic nuclei, and basal ganglia. Similar results were obtained when ouabain binding was determined in rabbit cerebral cortex and by a conventional filtration assay in nerve ending membranes obtained from rat cerebral cortex. These results favour the idea that the factor present in peak II fraction might behave as an ouabain-like substance.

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

“…[ 3 H]-ouabain binding was carried out by a filtration assay using [ 3 H]ouabain. Binding was performed in triplicate in a medium (0.5 ml final volume) consisting of 3 mM MgCl 2 , 2 mM H 3 PO 4 , 0.25 mM sucrose, 0.25 mM EDTA, 30 mM imidazol-HCl buffer, pH 7.4 [16], 250 μg cerebral cortex membranes protein and 45 nM [ 3 H]-ouabain, unless otherwise stated. When indicated, neurotensin, levocabastine and /or SR 48692 were included in the incubation medium.…”

[3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

“…[3H]-Ouabain autoradiography, along with in situ hybridization histochemistry (Stahl and Baskin, 1990), is a powerful quantitative approach for analyzing changes in catalytic subunit molecules of the Na,K-APase in small, defined anatomic regions of the brain that are otherwise unsuitable for study using biochemical filtration assays. QAR should be useful in analyzing the response of the Na,K-ATPase system to modulation of function (Antonelli et al, 1991;Swann and Steketee, 1989;Swann, 1987;Swann, 1984a,b;Swann et al, 1982) or to disease states such as epilepsy .…”

Desipramine modulates3H-ouabain binding in rat hypothalamus

“…Ouabain tightly binds to the phosphorylated form of the cardiac glycoside binding site of the catalytic subunit with a 1:1 stoichiometry, thus providing an invaluable tool for determining the number of binding sites. 3 H-ouabain binding was assessed by quantitative autoradiography (QAR), a technique that proved useful for studying receptor binding affinity and site number in neural tissue (Antonelli et al, 1989), as well as for Na 1 ,K 1 -ATPase modulation in discrete brain areas (Antonelli et al, 1991).…”