Glucocorticoid receptor monoclonal antibodies define the biological action of RU 38486 in intact B16 melanoma cells.
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The mechanism of action of the synthetic glucocorticoid antagonist, RU 38486, has yet to be completely elucidated. Although RU 38486 is a potent antiglucocorticoid in vivo, several studies have indicated that it has some agonist activities in vitro, such as high-affinity steroid binding to the receptor, activation, and DNA binding. Nevertheless, these in vitro postbinding events do not lead to any known gene expression. To understand the action of the glucocorticoid antagonist RU 38486, we studied glucocorticoid receptor localization on a mouse melanoma cell line (B16C3) by indirect immunofluorescent staining techniques, using monoclonal antibodies to the glucocorticoid receptor. Our data in intact cells suggest that, unlike glucocorticoid agonists such as triamcinolone acetonide, and similar to the glucocorticoid antagonist cortexolone, RU 38486-bound receptors do not translocate to the nucleus and hence do not allow for transcription of glucocorticoid-regulated genes to occur. Passage through the nuclear membrane may be a rate-limiting step in the action of glucocorticoid antagonists, and translocation may in itself be an important regulatory mechanism of steroid hormone action. (+info)
Effects of gonadotropin on ovarian intrafollicular processes during the development of oocyte maturational competence in a teleost, the Atlantic croaker: evidence for two distinct stages of gonadotropin control of final oocyte maturation.
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Full-grown oocytes of Atlantic croaker are insensitive to maturation-inducing steroid (MIS) unless they are primed with gonadotropin (GtH). The objective of this study was to examine the mechanism of GtH-induced maturational competence in croaker oocytes. Specifically, we determined the in vitro secretion of steroids by intact ovarian follicles of unprimed or hCG-primed fish, the direct effects of steroids on maturational competence, and the effects of steroid (cyanoketone), protein (cycloheximide), and RNA (actinomycin D) synthesis inhibitors on hCG-induced maturational competence and steroidogenesis in vitro. The steroid content of the incubation medium after hCG treatment was measured by RIA. The effects of hCG or exogenous steroid treatment on maturational competence were determined by recording the incidence of germinal vesicle breakdown (GVBD) after MIS-induced GVBD in a standard bioassay. Our major findings were: (1) induction of maturational competence occurred after exposure of ovarian follicles to hCG either in vivo or in vitro; (2) MIS secretion was detected in follicles of hCG-primed fish but not unprimed fish, and no MIS secretion was observed during hCG induction of maturational competence in vitro; (3) treatment with cyanoketone blocked the hCG-dependent secretion of testosterone and estradiol but not the development of maturational competence; (4) treatment with MIS or various other exogenous steroids in the absence of hCG did not induce maturational competence; and (5) hCG-induced maturational competence was inhibited by cycloheximide and actinomycin D. Therefore, the mechanisms of GtH induction of oocyte maturation in Atlantic croaker can be described in two distinct stages: a delta-4 steroid-(including MIS) and estrogen-independent priming stage followed by a MIS-mediated GVBD stage. The priming stage may involve mechanisms requiring RNA as well as protein synthesis. (+info)
Fluconazole inhibits human adrenocortical steroidogenesis in vitro.
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Evaluation of a radioimmunoassay of urinary cortisol without extraction.
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We evaluated the Kallestad "Quanticoat" cortisol RIA for direct (no extraction) measurement of urinary free cortisol, which requires no solvent extraction. An analytical-recovery study showed a linear regression of y (measured) = 0.65x (added) + 37.5 micrograms/L (Sy.x = 21.4 micrograms/L, r = 0.978, n = 48). Recovery appeared to vary with the urine used and with the concentrations of cortisol added. Within- and between-run CVs were less than or equal to 4.1% and less than or equal to 3.8%, respectively. Cross reactivities were low, except for prednisolone (20.5%). This no-extraction method gave higher values for urinary free cortisol than did either an RIA method involving extraction or an HPLC method. A comparison study with the HPLC (x) and with the method involving extraction (x') gave the following Deming-debiased regression equations: y = 1.60x + 68.8 (Sy.x = 34.4, n = 29) and y = 1.33x' + 0.69 (Sy.x = 40.3, n = 66), respectively. We conclude that the no-extraction method may give misleading results for patients' diagnosis or management if this cross reactivity is not taken into account. (+info)
Partial deficiency of adrenal 11-hydroxylase. A possible cause of primary hypertension.
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Results of supraphysiological adrenocorticotropic hormone (ACTH) stimulation of biosynthetic pathways of adrenal zona fasciculata indicate that a deficiency of 11-hydroxylase exists in patients with essential hypertension. The deficiency is suggested by the much greater stimulus of synthesis of deoxycorticosterone (DOC) and deoxycortisol in hypertensive subjects than in controls (p less than 0.001). No significant difference in the synthesis of cortisol, corticosterone, progesterone, 17-hydroxyprogesterone (17-OHP), and delta-4-androstenedione (D4) was observed between the two groups. The ratios for synthesis of DOC and corticosterone and for deoxycortisol and cortisol found in hypertensive patients were significantly higher than those found in controls (p less than 0.001); no significant difference was observed in the synthesis of 17-OHP and progesterone. The synthesis of DOC and deoxycortisol was not significantly correlated with either blood pressure or plasma renin activity. Plasma renin activity was significantly lower in hypertensive subjects than in normotensive subjects (p less than 0.0001), while no difference was found in aldosterone secretion between the two groups. The 11-hydroxylase deficiency in the adrenal zona fasciculata may be one of the genetic factors causing hypertension together with environmental factors (particularly salt intake and work-related stress). The investigation performed in our study may be useful for the evaluation of adrenal zona fasciculata enzymatic activities during the study of hypertensive patients. (+info)
Successful control of Cushing's disease in the elderly with long term metyrapone.
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A 77-year old woman with pituitary-driven Cushing's disease is described. The condition was completely controlled on long-term treatment with metyrapone. (+info)
Direct solid-phase radioimmunoassay for screening 17 alpha-hydroxyprogesterone in whole-blood samples from newborns.
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We describe a direct, solid-phase RIA for 17 alpha-hydroxyprogesterone (17-OH-P) that we are using to screen neonates for congenital adrenal hyperplasia. Phosphate buffer containing danazol and anti-17-OH-P is placed in tubes coated with antibody to IgG. The tubes also contain standards, controls, or blood samples on filter paper discs 3 mm in diameter. 125I-labeled 17-OH-P is added to each tube. The mixture is vortex-mixed and incubated overnight. The fluid and disc are removed, the radioactivity remaining in the tubes is counted, and the amount of 17-OH-P per disc is calculated by using a log-logit transformation of the standard curve. Results compare favorably with those by two extraction assays. Inter- and intra-assay CVs were less than 11% and less than 9%, respectively. Sensitivity was 2 pg per assay tube. There is no significant cross reactivity with structurally related steroids at their physiological concentrations. Analytical recovery of added 17-OH-P averaged 104%. 17-OH-P in whole blood spotted on filter paper is stable for at least six months. (+info)