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When colonic crypt cells isolated from intact rats are incubated with [3H]corticosterone specific nuclear binding is displaced by neither aldosterone nor the antiglucocorticoid RU38486, suggesting that [3H]corticosterone is binding to a site distinct from classical mineralocorticoid and glucocorticoid receptors. TLC revealed that the predominant nuclear [3H]steroid in the nucleus of [3H]corticosterone-incubated colonic crypt cells is [3H]11-dehydrocorticosterone. Where the enzyme 11 beta-hydroxysteroid dehydrogenase converting corticosterone to 11-dehydrocorticosterone is absent (cytosol preparations), [3H]corticosterone binds to classical glucocorticoid and mineralocorticoid receptors; in whole cells when 11 beta-hydroxysteroid dehydrogenase is blocked by carbenoxolone, cytoplasmic and nuclear binding of authentic [3H]corticosterone rises. Saturation and Scatchard analyses of nuclear [3H]11-dehydrocorticosterone binding demonstrate a single saturable binding site with a dissociation constant of < or = 10 nM at 22 C. We interpret these studies as evidence for a novel 11-dehydrocorticosterone-preferring receptor that may mediate glucocorticoid effects in tissues with high level of 11 beta-hydroxysteroid dehydrogenase activity.

The human progesterone receptor (hPR) exists as two isoforms, hPR-A and hPR-B, which differ only in that hPR-A lacks 164 amino acids present at the amino-terminus of hPR-B. In this study we have separately expressed hPR-A and hPR-B and asked whether the progesterone-binding mechanisms are the same or different for the two forms of hPR and for their mixture. We investigated 1) the cooperativity of binding [3H]progesterone to the receptor, as measured by the Hill coefficient (nH); and 2) the dissociation rate of [3H]progesterone from the receptor. To compare the effects of dimerization, these ligand-binding properties were measured over a range of receptor concentrations. Binding of [3H]progesterone to hPR-A was positively cooperative at all concentrations used; the limiting value for the Hill coefficient was 1.47 +/- 0.11 at high receptor concentrations (5-19 nM) and 1.31 +/- 0.06 at low receptor concentrations (1-4 nM). Similarly, little change was observed in the dissociation rate constant over the same concentration range; the values at high and low concentrations were 4.59 +/- 0.15 and 3.03 +/- 0.25 x 10(-3) min-1, respectively. By contrast, the hPR-B concentration had a marked effect on positive cooperative binding and the dissociation rate of progesterone. At high hPR-B concentrations (3-5 nM), the limiting Hill coefficient was 1.49 +/- 0.11, which is indicative of moderately strong positive cooperativity, whereas at lower hPR-B concentrations (1-3 nM), the Hill coefficient was reduced to 1.1, which is essentially noncooperative. The [3H]progesterone dissociation rate was 4.52 +/- 0.44 x 10(-3) min-1 at the higher concentrations of hPR-B and was increased to 1.6 +/- 0.11 x 10(-3) min-1 at the lower concentrations. Thus, over the same concentration range where hPR-A exhibited no significant change in positive cooperativity or the dissociation rate, these progesterone-binding properties were highly dependent on the concentration of hPR-B. When hPR-A and hPR-B were mixed, positive cooperative binding and the dissociation rate were more similar to hPR-B than to hPR-A, in that both binding parameters were dependent on the concentration of receptor. However, the hPR-AB mixture differed from hPR-B alone in that the mixture required a greater receptor concentration (7-10 vs. 3-5 nM) to exhibit positive cooperativity and the increased dissociation rate. These results show, first, that each hPR isoform displays different [3H]progesterone-binding properties, which are most prominent at low concentrations of receptor, and second, that one isoform can influence the other. As the two receptor forms differ only at the N-terminus, yet positive cooperativity and changes in the dissociation rate constant are indicative of conformational changes affecting hormone binding, these results also strongly suggest that the N-terminus may directly or indirectly interact with the C-terminal ligand-binding domain.

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.

Org 31710 and Org 31806 are new antiprogestagens. These compounds were tested for their binding affinity to various steroid receptors and for their bio-activity in various animal models and the results were compared with those of RU38486 and ZK 98299. Both Org compounds are strong antiprogestagens with little antiglucocortocoid activity and are devoid of other hormonal activities except for some weak androgenic and anti-androgenic activity. The two compounds are more potent than RU38486 and ZK 98299 with respect to their anti-progestational activity and are more selective. The Org compounds are effective in inhibiting the development of tumours in the 7,12-dimethylbenz(a)anthracene (DMBA) rat model. Org 31710 and Org 31806 may be applied for both the treatment of breast tumours and the improvement of fertility control.

To determine whether the steroid antagonist RU486 mediates its antiglucocorticoid and antiprogestin activities by the same or different receptor mechanisms, a direct comparison of RU486 interaction with glucocorticoid (GR) and progesterone (PR) receptors was made. The effects of RU486 on transformation of GR and PR 8-10S complexes in the intact cell and in vitro were analyzed by sucrose density gradient centrifugation, and the in vitro stability of receptor-heat shock protein-90 interactions was analyzed by coimmunoprecipitation. Compared to agonist, RU486 binding produced a reduction in the amount of GR converted from 8S to 4S and stabilized the GR-heat shock protein-90 complex. By contrast, PR-RU486 complexes were transformed both in vitro and in the intact cell to the same extent as receptor-agonist complexes. PR-RU486 complexes sedimented at 5-6S, whereas PR-R5020, GR-RU486, and GR-agonist complexes sedimented at 4S. The portion of GR that undergoes nuclear transformation when bound to RU486 was examined for binding to the glucocorticoid-progesterone response element of the mouse mammary tumor virus by an immunoprecipitation assay. The nuclear-transformed GR-RU486 complex bound the glucocorticoid-progesterone response element with the same affinity as the nuclear-transformed GR-triamcinolone acetonide complex. The electrophoretic mobilities of GR-RU486 complexes and GR-agonist complexes were the same, as determined by gel retardation assay. These results suggest that RU486 exerts its antiglucocorticoid activity at two levels of receptor action: prevention of complete GR transformation and alteration of a step subsequent to GR-DNA binding. As an antiprogestin, RU486 action is exerted predominantly at a post-DNA-binding step.