Identification of Mineralocorticoid Binding Sites in Rat Brain by Competition Studies and Density Gradient Centrifugation

A search for mineralocorticoid binding sites in neural tissue was carried out on the basis of competition by two mineralocorticoid antagonists (spironolactone and glycyrrhetinic acid), a mineralocorticoid agonist (9α-fluorocortisol) and a pure glucocorticoid (RU 26988). The hippocampus was selected for these studies, in view of its preferential concentration of binding sites for pH]-aldosterone (ALDO) and two glucocorticoids:PH]-corticosterone (CORT) and PH]-dexa-methasone (DEX). Inhibition studies performed with 5 · 10–4–5 · 10–9 M spironolactone and with 5 · 10–4–5 · 10–8 M glycyrrhetinic acid, showed a dose-response reduction of PH]-ALDO, PH]-CORT and PH]-DEX binding, implying that in brain these compounds did not behave as exclusive ALDO antagonists. Two concentrations of 9α-fluorocortisol (5 · 10–9 and 2.5 · 10–8 M) preferentially displaced PH]-ALDO in comparison to PH]-DEX or PH]-CORT. Relative binding affinity (RBA) of 9α-fluorocortisol was also higher for the mineralocorticoid than for the glucocorticoids. RU 26988 (5 · 10-6–2.5 · 10–8 M) gave differential inhibition of the three ligands and its RBA for PH]-DEX site was twice as high as for [3H]- CORT, and three orders of magnitude higher than for PH]-ALDO binding sites, thus clearly separating sites for gluco- and mineralocorticoids. Further evidence for the presence of separate binding molecules for mineralo- and glucocorticoids in hippocampal cytosol was provided by ultracentrifugation on 16–41% glycerol gradients containing molybdate, which yielded sedimentation values of 9.88 ± 0.27 for PH]-DEX (n = 5), 10.48 ± 0.27 for PH]-CORT (n = 9) and 11.3 ± 0.13 for PH]-ALDO (n = 7). Whereas data for PH]-CORT and PH]-DEX were not significantly different among themselves, those for [3H]-ALDO were higher (p < 0.05 and p < 0.01, respectively). Moreover, glycerol gradients of cytosol samples incubated with [3H]-ALDO in the presence of a 100-fold excess RU 26988 (circumstances under which 81% of glucocorticoid sites were blocked), revealed a 23% reduction of PH]-ALDO only. These results, based on competition studies, indicate that distinct populations of mineralocorticoid and glucocorticoid sites coexist in brain tissue, and further suggest the tentative identification of the 11.3 S peak as that of the mineralocorticoid binding site in cytosol from hippocampus.