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The cortisol/cortisone-responsive AR (AR(ccr)) has two mutations (L701H and T877A) that were found in the MDA PCa human prostate cancer cell lines established from a castrated patient whose metastatic tumor exhibited androgen-independent growth. Cortisol and cortisone bind to the AR(ccr) with high affinity. In the present study, we characterized the structural determinants for ligand binding to the AR(ccr). Our data revealed that many of the C17, C19, and C21 circulating steroids, at concentrations that are found in vivo, functioned as effective activators of the AR(ccr) but had little or no activity via the wild-type AR or GRalpha. Among the synthetic glucocorticoids tested, dexamethasone activated both GRalpha and AR(ccr), whereas triamcinolone was selective for GRalpha. In MDA PCa 2b cells, growth and prostate-specific antigen production were stimulated by potent AR(ccr) agonists such as cortisol or 9alpha-fluorocortisol but not by triamcinolone (which did not bind to or activate the AR(ccr)). Of the potential antagonists tested, bicalutamide (casodex) and GR antagonist RU38486 showed inhibitory activity. We postulate that corticosteroids provide a growth advantage to prostate cancer cells harboring the promiscuous AR(ccr) in androgen-ablated patients and contribute to their transition to androgen-independence. We predict that triamcinolone, a commonly prescribed glucocorticoid, would be a successful therapeutic agent for men with this form of cancer, perhaps in conjunction with the antagonist casodex. We hypothesize that triamcinolone administration would inhibit the hypothalamic-pituitary-adrenal axis, thus suppressing endogenous corticosteroids, which stimulate tumor growth. Triamcinolone, by itself, would not activate the AR(ccr) or promote tumor growth but would provide glucocorticoid activity essential for survival.

2-Methyl-9-fluorocortisol (2Me-9F-COL) was shown to possess extraordinarily intense mineralocorticoid but comparatively slight glucocorticoid activity, as judged by various tests (1). This compound, like other active mineralocorticoids (e.g., desoxycorticosterone and aldosterone), also exhibits a mutual antagonism with glucocorticoids (e.g., cortisone, cortisol) in the regulation of inflammation, anabolism and lymphatic-tissue development (2). On the other hand, the production by typical mineralocorticoids of renal damage and hypertension is not counteracted but rather enhanced by glucocorticoids (3, 4). It was to be expected, therefore, that a compound such as 2Me-9F-COL would produce marked renal lesions and hypertension by virtue of its strong mineralocorticoid potency, despite the fact that it also possesses some glucocorticoid activity. The experiments to be described here demonstrate that this is actually the case; indeed, 2Me-9F-COL proved to be the most potent nephrotoxic steroid among all those h...

Triamcinolone (9α-fluoro-l1β,16α,17α,21-tetrahydroxy-l,4-pregnadiene-3, 20-dione) was studied in rats to assess its adrenocortical activities using the following assays: electrolyte excretion, liver glycogen deposition, growth-survival and anti-inflammatory activity (asbestos pellet technique). Subcutaneously administered triamcinolone was 3 to 12 times more potent than prednisolone and 10 to 40 times more active than hydrocortisone in liver glycogen deposition tests. On oral assay, the relative potency of triamcinolone was higher. Triamcinolone proved to be 5 to 10 times more active than hydrocortisone acetate in the anti-inflammatory assay. Triamcinolone was more efficacious than hydrocortisone and at least as effective as DOC in maintaining the life of immature, male, adrenalectomized rats. Although survival time was prolonged, there was a suppression of growth when compared with the DOC-treated rats. Triamcinolone was found to be a potent diuretic and natriuretic steroid.

A rapid bio-assay for the concomitant assessment of the antiphlogistic and thymolytic activities of parenterally administered corticoids is described. Corticoids are given subcutaneously (single injection) to the rat; at the same time, a 0.5% croton oil solution is applied to the right ear. Ears and thymi are removed and weighed 6 and 8 hr, respectively, after corticoid treatment. The decreases in the phlogistic effects of croton oil and in thymus weights are related to the amounts of administered corticoid. The precision of the antiphlogistic assay is 0.41, while that for the thymus assay is 0.21. Antiphlogistic and thymolytic potencies of corticoids obtained by the combination assay are in good agreement with those obtained by other bio-assay methods. (Endocrinology 79: 463, 1966)

Aldosterone binds to two renal cytosol receptors, one with high affinity and low capacity (Type I or mineralocorticoid) and another with lower affinity but greater capacity (Type II or glucocorticoid receptor). One of the ways to study Type I receptors isolated from Type II receptors is to block the latter with a steroid which shows high affinity for the glucocorticoid receptor and low affinity for the mineralocorticoid receptor. Dexamethasone has been used widely for this purpose but previous investigations and this study have found that dexamethasone competes with [3H]aldosterone for Type I receptor binding with a relative activity of 26% that of aldosterone and therefore is less than ideal as a glucocorticoid receptor blocker. RU-26988 (11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6-trien-20-yn-21-methyl-3-one) is a recently synthesized glucocorticoid that shows very low affinity for the mineralocorticoid receptor. RU-26988 competes with [3H]aldosterone and [3H]2 alpha-methyl-9 alpha-fluorocortisol, a powerful mineralocorticoid, for the cytosol receptor from renal slices of adrenalectomized rats with a relative potency of less than 0.5% in comparison to unlabeled aldosterone and 2 alpha-methyl-9 alpha-fluorocortisol. RU-26988 has over twice the ability of unlabeled dexamethasone to compete with [3H]dexamethasone for binding to the renal cytosol glucocorticoid receptor. Scatchard analysis of [3H]aldosterone binding to rat renal cytosol showed two receptors, one with a calculated dissociation constant (Kd) of 2.81 X 10(-9) M and a second with a calculated Kd of 3.33 X 10(-8) M. The addition of a 100-fold concentration of RU-26988 produced a single line with a Kd of 5.02 X 10(-10) M indicating that the specific blocking of the glucocorticoid receptors allows an accurate determination of the kinetic parameters of the mineralocorticoid receptor by itself. Scatchard analysis of [3H]2 alpha-methyl-9 alpha-fluorocortisol binding produced a straight line with a Kd of 3.7 X 10(-9) M, such as would be produced if it were binding to only one single class of receptors. However, when an excess of RU-26988 was added to block the glucocorticoid receptor, a different straight line was produced by Scatchard's analysis with a Kd of 3.78 X 10(-10) M. Whereas the explanation for this is not apparent, it may be that the much larger concentration of glucocorticoid receptors, for which 2 alpha-methyl-9 alpha-fluorocortisol also has a very great affinity, masks the binding to the mineralocorticoid receptors.

The two [3H]aldosterone-binding proteins of rat brain cytosol were characterized by a dextran-coated charcoal method. With molybdate present to stabilize receptors, the affinities of the two sites for [3H]aldosterone in adrenalectomized perfused rat brain cytosols were 0.28 and 18.0 nM at 4 C. High affinity sites comprised 15% of the total receptor number. A small contamination of perfused brain cytosol preparations with corticosteroid-binding globulin (CBG) was found. However, due to the very high affinity of CBG for corticosterone at 4 C, this slight contamination resulted in significant alterations in the apparent affinity of steroids competing for aldosterone-binding sites. Selective precipitation of cytosol receptors with 36% (NH4)2SO4 reduced CBG concentrations to negligible levels. After blockade of low affinity sites with a highly selective glucocorticoid (RU 26988), the order of steroids in competing for the high affinity receptor was desoxycorticosterone greater than fludrocortisone greater than corticosterone greater than aldosterone greater than progesterone greater than dexamethasone. Readdition of a small quantity of dialyzed serum to cytosol preparations yielded a profile of steroid binding similar to that of the kidney mineralocorticoid receptor (aldosterone greater than desoxycorticosterone greater than corticosterone). The distribution of both receptors in brain regions of adrenalectomized rats was determined. Both receptors were at greatest density in the hippocampus and lowest density in the hypothalamus. The high affinity site was at greatest density in limbic regions, whereas the low affinity receptor, apparently identical to the glucocorticoid type II receptor, was at greatest density in cortex and cerebellum. It is concluded that the high affinity aldosterone receptor of rat brain, which had been identified in preliminary studies as a mineralocorticoid receptor, may bind either corticosterone or aldosterone in vivo.

To examine the direct effects of steroids on vascular smooth muscle, we have incubated rat aortic vascular smooth muscle cells in culture either steroid-free, or with natural and synthetic corticosteroids (RU26988, dexamethasone, corticosterone, 9 alpha-fluorocortisol, aldosterone, deoxycorticosterone) or sex steroids (estradiol, 5 alpha-dihydrotestosterone). At the end of 24 h, cultures were pulsed with [35S]methionine for 2 h, the cells lysed, and patterns of incorporation of isotope into protein determined by two-dimensional gel electrophoresis and autoradiography. Neither estradiol nor 5 alpha-dihydrotestosterone altered protein synthetic profiles compared with control (steroid-free) incubations. In contrast, cultures exposed to the six corticosteroids at 10(-7) M showed an identical pattern of response (6 proteins increased, 6 proteins decreased). This response appears to be glucocorticoid specific, since the mineralocorticoids (9 alpha-fluorocortisol, aldosterone, and deoxycorticosterone) did not have any effects over and above those seen with the pure glucocorticoid RU26988. We interpret these data as evidence for a putative glucocorticoid domain of at least 12 proteins in rat vascular smooth muscle cells. In contrast, there appear to be no comparable estrogen-, androgen-, or mineralocorticoid-specific changes in these cells.

Intact and hypophysectomized rats were treated with DCA, cortisone, 9-alpha-fluorohydrocortisone, ACTH and Pitressin both singly and in various combinations. A histologic study was made of changes in the lipid content of the zones of the adrenal cortex in response to specific steroid treatment. DCA and cortisone caused lipid depletion from the zona glomerulosa and zona fasciculata, respectively, while 9-alpha-fluorohydrocortisone caused lipid depletion from both these zones. Pitressin, like DCA, caused lipid depletion from the zona glomerulosa of intact rats, but lipid storage in hypophysectomized rats. As treatment was extended (3–7 weeks), the hormonal effects on adrenal morphology appeared to be directly correlated with time. The zonal response to treatment was also related to the presence or the absence of the pituitary gland. Furthermore, these studies showed that the dose of ACTH is of particular importance in affecting good adrenal zonal maintenance and in offsetting the disuse atrophy induced by s...

MEIER, et at. (1), have demonstrated that foreign body granulomas are provoked in rats by subcutaneous implantation of cotton pellets and that administration of cortisone results in a diminution of granuloma formation. Shipley and Meyer (2) and others (3, 4) have developed assays for anti-inflammatory activity of systemically administered steroids which are based on these principles. Cotton granulomas also have been used to estimate circulating levels of endogenous anti-inflammatory substances during pregnancy and lactation (5, 6). Desaulles, et at. (7) and Stebbins and Stoerk (8) have studied the local effects of several substances in cotton granulomas by impregnating the cotton pellet with the test material before implantation. This cotton granuloma technique lends itself to the study of local steroid action because the same animal can bear both untreated and treated pellets, permitting a comparison.