Intraadrenal Concentrations Research Articles

21-Hydroxylase Deficiency Transiently Mimicking Combined 21- and 11β-Hydroxylase Deficiency

21-Hydroxylase deficiency (21OHD) is the commonest form of congenital adrenal hyperplasia, while 11betaOHD represents 5% of cases. Although both result from mutations in distinct genes, cases of 'apparent' combined 21OHD and 11betaOHD (AC21,11OHD) have been occasionally reported. A 6 year-old girl, born with ambiguous genitalia and salt-loss, had serum elevations (ng/dl) of androstenedione (>1,000), 17-hydroxyprogesterone (17OHP; 38,483), 21-deoxycortisol (21DF; 23,338), and 11-deoxycortisol (S; 4,928), suggesting AC21,11OHD. CYP21A and CYP11B1 genotyping identified mutations only in the former. On follow-up, serum S became normal but 17OHP and 21DF were still elevated. ACTH stimulation disclosed elevated levels of 17OHP and 21DF, but unresponsive S and undetectable deoxycorticosterone. The hormonal pattern initially suggested AC21,11OHD, but subsequent normalization of S showed transient 11-hydroxylase inhibition. This may have occurred by enzyme or co-enzyme immaturity or functional discrepancy, but also by selective inhibition of 11betaOH by excess intra-adrenal concentration of androgens, acting as pseudo-substrates for this enzyme.

11beta-hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex.

Although oxidation of cortisol or corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11beta-HSD type 2 (11beta-HSD2) gene, and found a marked 11beta-HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11-dehydrocorticosterone (DH-B), the inactive forms to which the former glucocorticoids are converted by 11beta-HSD. Addition of the 11beta-HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH-B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH-B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration-dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11beta-hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11beta-HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra-adrenal concentration of non-11beta-hydroxylated steroid hormones.

The possible involvement of pancreatic polypeptide in the paracrine regulation of human and rat adrenal cortex

Pancreatic polypeptide (PP) is a member of a family of 36-amino acid braingut peptides, including neuropeptide Y (NPY) and polypeptide YY (PYY) and acting through many subtypes of Y receptors belonging to the superfamily of the G protein-coupled receptors. PP was found to increase both glucocorticoid and cyclic-AMP production by dispersed rat and human adrenocortical cells in a concentration-dependent manner. Minimal and maximal effective concentrations were 10-10 and 10-8 M, respectively. The glucocorticoid secretagogue effect of 10-8 M PP was blocked by the protein kinase A (PKA) unhibitor H-89, but not by the ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP) Autoradiography showed the presence of [125I]PP binding sites in the inner zones of rat and human adrenal cortex, which were not displaced by NPY, PYY, ACTH or CIP. Sizable amounts of PP-immunoreactivity were detected in the medulla of both rat and human adrenals (about 50–100 fmol/mg); this content may give rise, upon submaximal stimulation of PP release, to local intraadrenal concentrations of about 10-8/10-7 M. Collectively, these findings allow us to draw the following conclusions: (i) PP stimulates glucocorticoid secretion, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway, and (ii) PP could be included in that group of regulatory peptides, contained in adrenal medulla, which are able to control the secretory function of the cortex acting in a paracrine manner.

Vasopressin: A potent autocrine/paracrine regulator of mammal adrenal functions.

The control of adrenal functions by locally secreted neuropeptides or neutotransmitters is of great physiological importance. Vasopressin (VP) is one of these autocrine/paracrine regulators. We demonstrated by RT-PCR and perifusion experiments that rat and human adrenal medulla expressed and released vasopressin under basal conditions and under stimulation by acetylcholine. Intra-adrenal concentrations of VP may be sufficient to activate adrenal VP receptors. In the cortex, only the V1a receptor subtype has been detected. It triggered both steroid secretion and cortical growth. In the medulla, both V1a and V1b receptor subtypes were expressed. V1b receptors were mainly present on chromaffin cells and stimulated catecholamine secretion. The role of the V1a receptor remains unclear. Pathophysiological studies also revealed that human pheochromocytoma did not overexpress vasopressin receptors but might oversecrete vasopressin causing high plasma VP concentrations and elevated blood pressure.

Effect of Hypophysectomy on Corticotropin-Releasing Hormone and Adrenocorticotropin Immunoreactivities in the Rat Adrenal Gland

It has previously been shown that rat adrenal zona medullaris possesses an interleukin-1β (IL-1β)-responsive peripheral branch of the CRH/ACTH system that duplicates the hypothalamopituitary central one (Mazzocchi et al., Mol Cell. Neurosci. 4:267, 1993). The intraadrenal content of corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) immunoreactivities (ir), as well as IL-1β-stimulated release of CRH-ir and ACTH-ir, increased in relation to the number of days elapsed from hypophysectomy; the effect of hypophysectomy required at least 48 h to become significant and reached its maximum after 72 h. The action of IL-1β on ACTH-ir release was annulled by simultaneous exposure to α-helical-CRH, an antagonist of CRH. ACTH infusion, at a rate restoring a normal blood level of the hormone, prevented the effect of hypophysectomy on intraadrenal concentrations of both CRH-ir and ACTH-ir; similarly, the hypophysectomy-evoked rise in intraadrenal ACTH-ir content was completely annulled by treating hypophysectomized rats with CRH or dexamethasone. Taken together our findings suggest that the elimination of the central branch of CRH/ACTH system induces a marked increase in the activity of the intraadrenal peripheral one. The hypothesis is advanced that the hypophysectomy-induced lowering of circulating ACTH and the consequent drop in the production of adrenal glucocorticoids enhances, via a classic negative feedback mechanism, gene expression of CRH and ACTH in adrenal medullary chromaffin cells.

Effects of mevinolin, an inhibitor of cholesterol synthesis, on the morphological and functional responses of rat adrenal zona fasciculata to a prolonged treatment with 4-aminopyrazolo-pyrimidine.

Rat adrenocortical cells are almost completely dependent upon the continuous supply of cholesterol derived from serum lipoproteins. However, a prolonged (5-day) administration of 4-aminopyrazolo-pyrimidine (4-APP), a potent hypocholesterolaemic drug, though provoking a notable decrease in the intra-adrenal concentration of esterified and free cholesterol, did not significantly affect basal plasma level of corticosterone. Morphometry showed a conspicuous hypertrophy of zona fasciculata cells, coupled with a striking proliferation of smooth endoplasmic reticulum (SER) and peroxisomes and with a profound lipid-droplet depletion. The secretory response of zona fasciculata cells to ACTH was still present, but reduced by half with respect to control rats. The simultaneous administration of mevinolin, an inhibitor of cholesterol synthesis, to 4-APP-treated rats caused an additional drop in the intracellular content of free cholesterol and notably lowered basal plasma corticosterone concentration. Mevinolin magnified the 4-APP-induced zona fasciculata cell hypertrophy, as well as SER and peroxisome proliferation. The secretory response to ACTH was completely suppressed. These data are compatible with the view that the morphological changes, which rat zona fasciculata cells undergo during prolonged hypocholesterolaemia, are the expression of the activation of the endogenous cholesterol synthesis. This compensatory response, enabling zona fasciculata cells to maintain a normal basal rate of hormonal output and to respond (though less efficiently) to their main physiological stimulus, seems to be completely independent of any activation of the hypothalamo-hyphophyseal axis, since dexamethasone/ACTH treated rats were used. The hypothesis is advanced that the mechanism underlying this response may involve the decrease of the intracellular free-cholesterol pool.

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.

THE IMPACT OF AMBIENT STEROID CONCENTRATIONS UPON THE RELATIVE ACTIVITIES ADRENAL MICROSOMAL 3β-HYDROXYSTEROID DEHYDROGENASE AND 17, 20-DESMOLASE

We found that intra-adrenal concentrations of most steroids are 2-10 times higher in adults than in infants, and reach levels (10−7-10−5 M) which might influence steroidogenic efficiency. Since 3β-HSD and 17,20-desmolase act on 17OH-pregnenolone to determine relative DHA and cortisol production during human development, we studied their kinetics in adrenal microsomes from 6 kidney donors (12-40 yr) and the effect of varying steroid concentrations on their activities. There was no age-related change in Km for 3β-HSD (0.1-0.5 μM) or desmolase (0.2-1.7 μM). At all ages Vmax for 3β-HSD (3-8 nmol/mg/min) exceeded that for desmolase (0.3-1.4 nmol/mg/min). Physiological intra-adrenal concentrations (10−6 M) of progesterone, 170H-prog., 11-desoxycortisol, corticosterone, androstenedione and testosterone caused >20% inhibition of 3β-HSD, with no effect on desmolase. Estradiol and estrone caused >95% inhibition of 3β-HSD with only minimal impact on desmolase. Kinetic analysis showed competitive and non-competitive inhibition with Ki values of 10−7-10−5 M. These data demonstrate that ambient steroids can render 3β-HSD rate-limiting and permit desmolase to compete for 170H-pregnenolone substrate. This phenomenon provides a rational explanation both for the relative 3β-HSD deficiency of the human fetus and the increased C-19 steroid production during adrenarche.

Dissociation of cortisol and adrenal androgens (AA) in selected patients with hypopituitarism

Dissociation of secretion of cortisol and AA, observed in several physiologic and pathologic conditions, has led to the hypothesis that secretion of AA may be controlled by another hormone in addition to ACTH. An alternate hypothesis attempts to explain all instances of such dissociation by changes in ACTH alone, wherein ACTH controls synthesis of AA by changing the intraadrenal concentration of cortisol. This hypothesis does not allow for dissociation of cortisol and AA in a subject with normal ACTH secretion and intact adrenals. To test this hypothesis, we studied cortisol secretion in 4 patients (20-26 years) with idiopathic hypopituitarism (who never received cortisol replacement therapy) and deficiency of AA as reflected by lack of adrenarche and low levels of serum DHEAS. Determination of serum cortisol and 17-OH progesterone (17 OHP) at 2 h intervals for 2 days and of cortisol and 17 OHP responses to insulin induced hypoglycemia and glucagon infusion revealed normal cortisol and 17 OHP secretion in 2 of the 4 patients. Although intraadrenal concentrations of cortisol and serum levels of ACTH were not measured, there is no reason to suspect that these levels should be abnormal in the 2 patients who had normal cortisol secretion. Low serum levels of DHEAS in these patients cannot be explained by deficiency of ACTH secretion. Our data provide further evidence that secretion of AA may be controlled by another hormone in addition to ACTH.