Aldosterone-binding sites in pregnant and lactating rat mammary glands

We have identified and characterized aldosterone-binding sites in the mammary gland of pregnant and lactating rats by using the highly specific glucocorticoid RU26988 (11 beta, 17 beta-dihydroxy-17 alpha-propynyl-androsta-1,4,6-triene-3-one) to eliminate aldosterone binding to classical glucocorticoid (type II)-binding sites. The hierarchy of affinity of steroids competing for aldosterone in the presence of RU26988 was similar in mammary glands of pregnant and lactating rats and kidneys of lactating rats: deoxycorticosterone greater than aldosterone greater than or equal to corticosterone greater than dexamethasone; this pattern is similar but not identical to that for the mineralocorticoid (type I) receptor of a classical mineralocorticoid target organ, the kidney of the virgin female rat, where aldosterone has a higher affinity than corticosterone. This pattern (aldosterone greater than corticosterone) was also found in renal cytosols of pregnant rats. To define these aldosterone-binding sites in terms of dissociation constant and number of sites, it was necessary to pretreat cytosols with charcoal. Without this treatment, curvilinear Scatchard plots were obtained. After pretreatment with charcoal, subsequent Scatchard analysis indicated a single class of site with low capacity (3-33 fmol/mg protein) and dissociation constants of about 1-3 nM in kidneys and mammary glands of pregnant and lactating rats. The occupancy of this site in vivo may depend on the relative tissue concentrations of aldosterone or corticosterone, which, in turn, are modulated by the levels of extravascular transcortin.