Mineralocorticoid Receptor-Like Aldosterone-Binding Protein in Cell Culture*

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Abstract
Receptors for mineralocorticoids have been characterized in kidney and to a lesser extent in certain extrarenal tissues but these receptors have not yet been identified in cells in culture. In the current studies we report the presence of mineralocorticoid receptor-like aldosterone-binding proteins in cloned lines of cultured rat pituitary tumor cells (GC, GH3 sublines) that synthesize and secrete GH and, in some cases, PRL. The apparent Kd of these sites for aldosterone determined from a Scatchard analysis of the binding by intact cells at 37 C is 1 nM, and about 8000 sites per cell were detected. The affinity of the receptors for several steroids is in the order of 9a-fluorocortisol > aldosterone > deoxycorticosterone > cortisol > prednisolone » dexamethasone ≍β-methasone > testosterone > triamcinolone ≍ estradiol. In intact cells and under cell-free conditions, aldosterone stimulates a temperature-dependent nuclear binding of these binding proteins. Under the cell-free conditions, this is inhibited by 10 mM sodium molybdate. The mineralocorticoid antagonists prorenone, spironolactone, and progesterone also bind to these sites with affinities similar to those with which they bind to the kidney receptors. Furthermore, in contrast to the case with aldosterone, when prorenone binds to these sites, the complexes do not bind to the nucleus, even after its incubation with intact cells at 37 C. These data suggest that mineralocorticoid antagonists may in general exhibit their agonist-blocking actions by failing to promote nuclear binding of the receptor-antagonist complex. Similar receptors were not found in ACTH-producing mouse AtT-20 cells. The finding of mineralocorticoid receptors in cells of pituitary origin also raises the question of whether this class of hormones can have direct actions on the pituitary. This probable mineralocorticoid receptor-containing cultured cell line should be useful for further studies of these receptors.