A stimulatory effect of interleukin-1 on adrenocortical cortisol secretion mediated by prostaglandins.

Studies using cultured bovine adrenocortical cells now demonstrate that the cytokines interleukin-1 (IL-1) alpha and beta, contrary to previous reports, can stimulate cortisol secretion in vitro in a dose- and time-dependent fashion. However detectable levels of IL-1 receptor could not be demonstrated in adrenal cortical, medullary, or capsular cells by membrane displacement of iodinated IL-1 alpha by unlabeled IL-1 beta, a technique that readily demonstrates specific IL-1 alpha-binding sites on 3T3 fibroblasts. The stimulatory effect of IL-1 on cortisol secretion could be totally blocked by indomethacin, an indication that this effect might be mediated indirectly via local release of prostaglandins (PGs). Subsequent investigations have confirmed that IL-1 does enhance the conversion of [3H]arachidonate to PGs by cultured adrenal cells, and that some of these PGs (PGD2, PGF2 alpha, and PGE2), in turn, can stimulate cortisol production. Taken together these observations suggest that IL-1-induced stimulation of cortisol secretion is mediated through local release of PGs by a small subpopulation of cells within the adrenal gland.

Kinetic analysis of adrenal 3 beta-hydroxysteroid dehydrogenase activity during human development.

Kinetic analyses of microsomal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in adrenal glands from 11 individuals, aged 1-60 yr, were carried out to determine whether changes in substrate or cofactor affinity (Km) or cellular content, as reflected in maximal velocity, could explain the changes in adrenal delta 5-3 beta-hydroxysteroid secretion that occur in late childhood and puberty. The Km values for the cofactor NAD+ were similar regardless which substrate, dehydroepiandrosterone (DHA), pregnenolone, or 17-hydroxypregnenolone (17OH-delta 5P), was used. The Km values for DHA (0.3 microM), pregnenolone (0.4 microM), and 17OH-delta 5P (0.3 microM) were similar and within the intraadrenal concentration ranges for DHA and 17OH-delta 5P previously reported. Each substrate was a competitive inhibitor for the others, with close similarity between affinity and inhibition constants. These observations point to the presence of a single 3 beta-HSD, rather than several substrate-specific variants. There was no change in substrate Km with age; the maximal velocity was lower (0.1-0.6 nmol/mg X min) in a single 1-yr-old infant than in later life, but there was no significant change (mean, 2.9-4.6 nmol/mg X min for the three substrates) between values at 12 and 60 yr. This suggests that ACTH-mediated induction of 3 beta-HSD may be low in infancy and higher in adults, while in vivo studies point to a reduction in actual 3 beta-HSD activity during this period. The likely explanation for this paradox between enzyme levels and final activity is that 3 beta-HSD is progressively inhibited during late childhood and puberty by rising intraadrenal concentrations of various delta 4-3-ketosteroids.

Steroid inhibitory effects upon human adrenal 3 beta-hydroxysteroid dehydrogenase activity.

The inhibitory effects of varying concentrations of steroids upon 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) kinetics were studied in human adrenal microsomes. Each enzyme assay was conducted in triplicate at five different concentrations of three substrates (dehydroepiandrosterone, pregnenolone, and 17OH-pregnenolone), using microsomes from at least three donors. Each steroid was screened for possible inhibition at concentrations of 10(-8) and 10(-6) M and then studied in more detail at five different concentrations. The type of inhibition and the inhibition constant (Ki) were determined by analysis of Lineweaver-Burk and Dixon plots, together with replots of the slopes from the Dixon plots. The mean Km (Michaelis-Menten constant) for the three substrates was 0.42 +/- 0.04 (SE) mumol/liter (n = 73). Each steroid tested, including delta 5-3 beta-hydroxysteroids, estrogens, and several delta 4-3-ketosteroids, with the exception of cortisol, caused significant inhibition of 3 beta-HSD activity, and in each case the steroid appeared to behave as a competitive inhibitor. In most cases the Ki value was approximately 10(-7) M. At micromolar concentrations several steroids, notably estrone and estradiol, caused almost total inhibition of adrenal 3 beta-HSD activity. Comparison of the calculated Ki values with available data concerning changes in intra-adrenal steroid concentrations during childhood suggests that these changes would be sufficient to cause a relative decline in 3 beta-HSD activity during adrenarche. Although postnatal circulating steroid concentrations would appear to be insufficient to influence adrenal steroidogenesis, the high serum levels of placental steroids during fetal life would be expected to cause marked 3 beta-HSD inhibition.

Kinetic analysis of inhibition of human adrenal steroidogenesis by ketoconazole.

The kinetics of the inhibitory effects of the imidazole antimicrobial ketoconazole on the activities of the steroidogenic enzymes distal to cholesterol side-chain cleavage were studied in human adrenal microsomal and mitochondrial suspensions. Although ketoconazole was a competitive inhibitor of all five enzyme reactions, the effects on 17-hydroxylase, 17,20-desmolase, and 11-hydroxylase activities (Ki = 10(-8) M) were considerably greater than those on 21-hydroxylase and 3 beta-hydroxysteroid dehydrogenase/isomerase activities (Ki = 10(-4) M). These findings explain the clinical endocrine effects of ketoconazole in the usual therapeutic doses, which include inhibition of cortisol and androgen secretion, compensatory ACTH-mediated secretion of 17-desoxysteroids such as progesterone and aldosterone, and suppression of PRA.

Combined 17-hydroxylase and 17,20-desmolase deficiencies: evidence for synthesis of a defective cytochrome P450c17.

We studied in vivo and in vitro steroidogenesis in six phenotypic female children with 17-hydroxylase deficiency. The diagnosis was suspected as a likely cause of familial low renin hypertension and was confirmed by findings of reduced basal and ACTH-stimulated serum and urinary levels of cortisol and other 17-hydroxysteroids, together with hypergonadotropic hypogonadism in both 46,XY and 46,XX patients, and abnormally increased secretion of 17-desoxysteroids, such as progesterone, 11-deoxycorticosterone, and corticosterone. ACTH stimulation testing demonstrated a lesser degree of 17-hydroxylase deficiency in the obligate heterozygous parents; one father had increased basal serum 17-hydroxyprogesterone values, unresponsive to ACTH, suggesting partial Leydig cell 17,20-desmolase deficiency. In vitro kinetic analysis of testicular microsomal enzymes in the affected 46,XY male pseudohermaphrodites confirmed that both 17-hydroxylase and 17,20-desmolase activities were less than 2% of those in age-matched normal subjects. However, in spite of this virtual absence of both enzymatic activities of cytochrome P450c17, Northern blot analysis demonstrated abundant amounts of RNA in these tests that hybridized to a cDNA specific for this P450 enzyme. Moreover, immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved testicular microsomes showed an apparently normal content of an immunoreactive protein with a mol wt similar to that of authentic P450c17. These results suggest that these patients have a point mutation in the gene for P450c17; the mutant gene is transcribed, but gives rise to a protein defective in normal 17-hydroxylase and 17,20-desmolase activities.

Allgrove syndrome: an autosomal recessive syndrome of ACTH insensitivity, achalasia and alacrima.

Allgrove syndrome (isolated glucocorticoid deficiency, achalasia and alacrima) was found in eight members of an inbred French Canadian/North American Indian pedigree. The high degree of consanguinity supports an autosomal recessive mode of inheritance for this disorder. Six patients presented with hypoglycaemia and other evidence of cortisol deficiency between 2.5 and 8 years of age; however, two others became cortisol deficient after initial testing showed normal cortisol responses to ACTH, evidence that the glucocorticoid insufficiency of this syndrome may not be congenital, but may develop as late as the third decade. No evidence of mineralocorticoid deficiency has been found during 65 patient-years of follow-up. Alacrima was the earliest and most consistent clinical sign of Allgrove syndrome. Other manifestations of peripheral or autonomic neuropathy were found in four patients. The patients showed similar facial features, and three had significant velo-pharyngeal incompetence. All showed oesophageal dysmotility even in the absence of symptomatic dysphagia. In-vitro studies of lymphocyte ACTH binding showed no differences from normal controls. If such lymphocyte binding, as has been suggested, reflects adrenal ACTH receptor activity, these data would suggest that the glucocorticoid deficiency of Allgrove syndrome is not the result of a defect in that receptor. However, the observation that ACTH does not elicit increased adenylate cyclase activity even in normal lymphocytes casts considerable doubt on the physiological significance of ACTH binding to lymphocytes. It seems likely, therefore, that true ACTH receptors are not expressed on peripheral lymphocytes, and any conclusions regarding a possible receptor defect in Allgrove syndrome must await studies of receptor expression on adrenal cell membranes.