Potassium-induced aldosterone biosynthesis in cultured rat zona glomerulosa cells.

Rat adrenal zona glomerulosa cells lost their ability to produce aldosterone from either endogenous precursors or added deoxycorticosterone within 2 days of primary monolayer culture in a medium with a potassium concentration of 6.3 mmol/l. The lost corticosterone methyl oxidase I and II activities were totally regenerated when the ambient potassium concentrations was raised to 31 mmol/l. The conversions of deoxycorticosterone to 18-hydroxycorticosterone and aldosterone were completely restored by culture in a high-potassium medium also in zona glomerulosa cells of rats in which aldosterone biosynthesis had been suppressed by potassium restriction and sodium loading. However, these conversions were not induced in zona fasciculata-reticularis cells. The induction of aldosterone biosynthesis was associated with the appearance of a mitochondrial 49,000 protein cross-reacting with an antibody raised against bovine adrenal cytochrome P-450(11) beta. Thus primary cultures of zona glomerulosa cells are promising models for studying in vitro the molecular mechanisms of long-term adaptation of aldosterone biosynthesis to sodium and potassium intake.

Effects of beta-lipotropin, beta-endorphin, gamma 2-melanotropin and corticotropin on steroid production by isolated human adrenocortical cells.

Normal human adrenocortical cells (from multi-organ donors) were incubated with corticotropin (tetracosactide), highly purified human beta-lipotropin, synthetic human beta-lipotropin, gamma 2-melanocyte stimulating hormone and beta-endorphin. Corticotropin stimulated cortisol, aldosterone and 18-hydroxycorticosterone production starting at 10(-12)-10(-11) mol/l in normal adrenocortical cells. Purified human beta-lipotropin also stimulated steroidogenesis but 100-1000-fold higher concentrations of the peptide were needed. In contrast, synthetic human beta-lipotropin was without any effect on steroid production up to concentrations of 10(-7) mol/l. Synthetic beta-lipotropin (5 x 10(-10) mol/l) did not significantly change the dose-response curve for corticotropin (10(-13) mol/l-10(-9) mol/l) versus the three steroids measured. gamma 2-Melanotropin and beta-endorphin (10(-6) mol/l) stimulated the secretion of cortisol, but not of aldosterone. Since synthetic human beta-lipotropin has no effect on human adrenocortical cells, the purified beta-lipotropin must be contaminated with traces of corticotropin. Since pathologically elevated levels of proopiomelanocortin-derived peptides will rarely exceed plasma concentrations of 10(-10) mol/l, our findings in vitro do not support a physiological or pathophysiological role of the peptides examined in the regulation of adrenal steroid secretion.

Effect of atrial natriuretic factor on aldosterone and its precursor steroid production in adrenal zona glomerulosa cells from spontaneously hypertensive rats.

The effect of alpha-human atrial natriuretic factor (alpha-hANP, 10(-6) M- 10(-8) M) on basal, and maximum angiotensin II (AII, 4.8 X 10(-8) M)-, ACTH (4.3 X 10(-9) M)-, and potassium (8mM)-stimulated levels of corticosterone, 18-hydroxycorticosterone (18-OHB) and aldosterone production were studied in adrenal glomerulosa cells from spontaneously hypertensive rats (SHR) at 14 weeks of age as compared to those in the age-matched Wistar-Kyoto rats (WKY) on a normal sodium diet. Plasma corticosterone, 18-OHB and aldosterone levels and the aldosterone response in vitro to the graded doses of AII were similar in SHR and WKY. Basal, and maximum AII-, ACTH-, and potassium-stimulated levels of corticosterone, 18-OHB and aldosterone also were similar in the cells from SHR and WKY. alpha-hANP similarly inhibited basal and stimulated levels of these corticosteroids in the cells from SHR and WKY. These results indicate that the inhibitory effect of alpha-hANP on aldosteronogenesis is unaltered in SHR at 14 weeks of age on a normal sodium diet.

[The effects of o,p'-DDD on adrenal steroidogenesis and hepatic steroid metabolism].

O,p'-DDD is used for the treatment of adrenocortical carcinoma and Cushing's disease. The inhibitory effect of this drug on the adrenal steroid biosynthesis has been described by many authors, but there are very few reports about the sites of action of this drug on adrenal steroid synthesis. This paper presents in vitro studies on adrenal steroidogenesis and hepatic steroid metabolism. The effects of o,p'-DDD on adrenal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 11 beta-hydroxylase (11 beta-OHlase) and 18-hydroxylase (18-OHlase) were examined in vitro using mitochondrial and microsomal fractions prepared by standard centrifugation procedures from the homogenate of bovine adrenal cortices. The concentrations of o,p'-DDD inducing 50% inhibition of 3 beta-HSD, 11 beta-OHlase and 18-OHlase were 8 X 10(-6) M, 1 X 10(-5) M and 3 X 10(-6) M, respectively. This study clearly demonstrates the marked inhibitory effects of o,p'-DDD on 3 beta-HSD in vitro, which was not described previously. The inhibitory effects of o,p'-DDD on these 3 enzymes were diminished by an addition of 0.05 approximately 0.5 mM of cofactor (NADPH or NAD). The results indicate that o,p'-DDD may reduce NADPH or NAD utilization, resulting in the inhibition of steroidogenesis. The effects of o,p'-DDD on hepatic 5 beta-reductase were examined in vitro using rat liver homogenate. O,p'-DDD inhibits 5 beta-reductase, resulting in the decrease of conversion of cortisol to dihydrocortisol and tetrahydrocortisol at the concentration of 10(-3) M.

[The effect of ketoconazole and some other inhibitors of steroidogenesis on P-450 11 beta-catalyzed reactions].

Ketoconazole (Nizoral), an orally active antimycotic agent with a broad spectrum, has been reported to interfere with steroidogenesis both in patient and in vivo rat studies. It has also been shown that the same drug inhibits some P-450--catalyzed reactions in adrenal cortex mitochondria. In the present work, we studied the inhibitory effect of Ketoconazole, along with some other known inhibitors of steroidogenesis, on the reconstituted steroid monooxygenase system, which consists of adrenodoxin, its reductase and P-450 11 beta as the protein components being purified from bovine adrenal cortex mitochondria. The results indicated that; Ketoconazole completely inhibited hydroxylation of deoxycorticosterone at the 11 beta-position to form corticosterone and at the 18-position to form 18-hydroxydeoxycorticosterone. The Ki value for Ketoconazole, calculated either from the 11 beta-hydroxylase reaction or the 18-hydroxylase reaction, was 0.56 microM, which was comparable to the value obtained for metyrapone in the same system. Ketoconazole also inhibited 18-hydroxylation of corticosterone to form 18-hydroxycorticosterone, with 50% inhibitory concentration of less than 0.03 microgram/ml. The corresponding value for this inhibitor in the deoxycorticosterone 18-hydroxylase reaction was found to be 0.3 microgram/ml. The contrast between these values for the two substrates is striking. Thus, in a series of reaction steps, the inhibitory effect of corticosterone to 18-hydroxycorticosterone was more potent than deoxycorticosterone to 18-hydroxycorticosterone reaction. Both trilostane and o, p'-DDD over the wide concentration range failed to inhibit any of the reconstituted P-450 11 beta system similar to those applied to the Ketoconazole study.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the renin-angiotensin system in the regulation of late steps in aldosterone biosynthesis by sodium intake of potassium-deficient rats.

The role of the renin-angiotensin system in the adaptation of late steps in aldosterone biosynthesis to sodium intake was studied in potassium-deficient rats. Capsular portions of adrenal glands were incubated with [3H]corticosterone and conversion to aldosterone and 18-hydroxycorticosterone was measured by double isotope dilution and multiple paper chromatography. Sodium loading of sodium- and potassium-depleted rats resulted in a rapid and extensive fall in PRA but only in a delayed and gradual suppression of aldosterone biosynthesis. Treatment with the converting enzyme inhibitor, captopril, did not affect aldosterone biosynthesis in rats with established sodium and potassium deficiency, but blocked the stimulation of the conversion of corticosterone to aldosterone and 18-hydroxycorticosterone by sodium restriction of potassium-depleted rats. Infusion of a high dose of angiotensin II into potassium-deficient rats stimulated aldosterone biosynthesis depending upon the concurrent sodium intake. Accordingly, the renin-angiotensin system plays an important but limited role in the control of late steps of aldosterone biosynthesis by sodium intake. Angiotensin II seems to be essential for the induction but not for the maintenance of a high activity of the enzyme(s) involved in the conversion of corticosterone to aldosterone during combined sodium and potassium restriction. The sensitivity of the zona glomerulosa to the long term stimulatory action of angiotensin II varies with the sodium intake and appears to be regulated by the plasma potassium concentration and unknown other mediators.

Plasma 18-hydroxycorticosterone during continuous ambulatory peritoneal dialysis.

Continuous ambulatory peritoneal dialysis (CAPD) entails the continuous presence of hypertonic dialysate in the peritoneal cavity. We postulated that the continuous, gradual ultrafiltration produces chronic activation of the renin-angiotensin system and the adrenal zona glomerulosa. To explore this hypothesis, we measured plasma levels of PRA, active renin (AR), total renin (TR), inactive renin (IR), 18-hydroxycorticosterone (18-OH-B), and aldosterone (PAC) under basal and stimulated conditions. At 0800 and 1200 hr after overnight recumbency, plasma levels of PRA, AR, TR, IR, and 18-OH-B were elevated above the range for sodium-replete recumbent normal subjects. PAC, however, was normal. The increase in TR was due predominantly to an increase in AR. After the combined stimulus of 4 hr ambulation and the ultrafiltration induced by a 2 L exchange, plasma levels of PRA, AR, TR, IR, and PAC were within the range for sodium-replete upright normal subjects. Plasma 18-OH-B levels, however, remained markedly elevated. Graded intravenous infusion of ACTH at rates of 0.03 to 10 ng/min demonstrated that the threshold for an ACTH-stimulated rise in plasma 18-OH-B and PAC is at least as low as that for cortisol and corticosterone. We conclude that CAPD produces activation of the renin-angiotensin system. The high circulating levels of PRA, AR, and, presumably, angiotensin II result in increased secretion of 18-OH-B by the adrenal zona glomerulosa.

Corticoidogenesis in Bufo arenarum H. I. In vitro biosynthesis of [3H]pregnenolone and [3H]corticosterone metabolites and of endogenous 3-oxo-4-ene intermediates at 28 degrees C and 37 degrees C.

Sliced interrenal tissue from Bufo arenarum H. was incubated without exogenous substrate (to determine endogenous corticosterone (B) and progesterone), as well as in the presence of [3H]pregnenolone ([3H]Pre) and [3H]corticosterone, at 28 and at 37 degrees C. When yields of radiometabolites were analyzed as such, [3H]Pre at 28 degrees C was found to be a good precursor for 18-hydroxycorticosterone (18-HO-B), aldosterone (aldo), and an unknown N very similar but not equal to 19-hydroxycorticosterone. However, [3H]Pre at 28 degrees C was not a good precursor for corticosterone. When the same tritiated substrate was incubated at 37 degrees C, the yields of 18-HO-B and N diminished significantly, aldo remained as it was at 28 degrees C, and B increased. [3H]B was a poor precursor to aldo, 18-HO-B, and N, irrespective of incubation temperatures, but a good precursor to material with the mobility of 11-dehydro-B and an aldo-like compound X. Measurement of endogenous B and progesterone pools followed by calculations of specific activities lead to the conclusion that these differences should not be ascribed to artifactual competitions between precursor pools for enzyme systems. This species is thus characteristic in its capacity to bypass, at least at 28 degrees C and under the in vitro conditions employed, corticosterone during aldo biosynthesis. Furthermore, the vast ambient temperature ranges to which this and many other amphibian species are exposed and the temperature dependence observed in corticoidogenesis of B. arenarum suggest that corticoidogenic studies in these species should be carried out at more than one temperature.

Regulation of 18-hydroxycorticosterone formation in bovine adrenocortical mitochondria.

The conversion of deoxycorticosterone to 18-hydroxycorticosterone was examined by using sonicated mitochondrial suspension of bovine adrenocortex. The KM's of deoxycorticosterone for the 11 beta- and 18-hydroxylations were in the same range (1 microM), while the turnover number for the 11 beta-hydroxylation (50 nmol/min/nmol cytochrome P-450) was 6 times as great as that for the 18-hydroxylation (7.3). The KM and turnover number for the 18-hydroxylation of corticosterone were 6 microM and 0.4, respectively. Those for the 11 beta-hydroxylation of 18-hydroxy-11-deoxycorticosterone were 120 microM and 5. When products were analysed in the incubation of deoxycorticosterone with the mitochondrial suspension containing a larger amount of cytochrome P-450, the formation of 18-hydroxycorticosterone was observed in addition to the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone. The kinetic interrelation between the two pathways was further examined together with consideration of the cytochrome P-450-linked hydroxylation system. The result suggests that the pathway via 18-hydroxy-11-deoxycorticosterone substantially participates in the formation of 18-hydroxycorticosterone from deoxycorticosterone. The perturbation of this network by an artificial means, such as the addition of Triton X-100, revealed that the detergent (0.02%) facilitated the production of 18-hydroxycorticosterone from deoxycorticosterone, regardless of its inhibitory effect on the production of corticosterone and 18-hydroxy-11-deoxycorticosterone from deoxycorticosterone. These studies provide an important insight into the regulation mechanism of 18-hydroxycorticosterone formation from the precursors on the mitochondrial level.

[Nature of the products of in vitro interrenal synthesis in adult Bufo bufo formosus]. / Nature des produits de la synthèse interrénalienne in vitro chez Bufo bufo formosus adulte.

Corticosterone (B), aldosterone (Aldo), 11-deoxycorticosterone (DOC), 11-dehydrocorticosterone (A), 18-hydroxycorticosterone (18 OH B) and cortisol (F) are identified after incubation of interrenal of Bufo bufo formosus with radioactive progesterone. Yields of radioactive B and Aldo are larger than those of radioactive DOC, A and 18 OH B; yield of radioactive F is the smallest one.

[Corticosteroid biosynthesis in various water-salt dietary regimens]. / Biosintez kortikosteroidnykh gormonov nadpochechnikami pri razlichnykh vodno-solevykh rezhimakh pitaniia.

The production of desoxycorticosterone, 18-hydroxy-desoxycorticosterone and corticosterone by the mouse adrenals increases in vitro whereas 18-hydroxy-corticosterone and/or aldosterone production falls to an uncertain level when the salt consumption rises from about 0.07 to 50 mekv per animal a day under conditions of water consumption ad libitum. Specific activity values of 3H-corticosteroids, being formed by the adrenal cortex capsule from 3H-progesterone, added to the incubated culture, decrease. Aldosterone production by the adrenals remains unchanged under conditions of sodium load with the limited water consumption. It is suggested that the stimulating effect of endogenous ACTH on the adrenal cortex is potentiated because of the increased salt consumption.

Evidence for an aldosterone biosynthetic defect in congenital adrenal hyperplasia.

A search was made for an abnormality in aldosterone biosynthesis in congenital adrenal hyperplasia due to a cortisol 21-hydroxylation defect. Examination of the urinary metabolites of potential C-18-oxygenated steroid precursors revealed an abnormal pattern; however, the locus of the defect was not at the C-21 hydroxylation step, but consisted of overproduction of glomerulosa 18-hydroxylation step, but consisted of overproduction of glomerulus 18-hydroxycorticosterone relative to aldosterone, as seen in the type II corticosterone methyl oxidase defect. This abnormality, which was seen in all salt losers and most nonsalt losers, provided evidence for diminished aldosterone secretory reserve even when values of the hormone are normal or elevated. These findings support the concept that salt-losing and nonsalt-losing forms of the cortisol 21-hydroxylation defect differ only in degree and are not different genotypes. An implication of these findings is that all patients with congenital adrenal hyperplasia with an elevated 18-hydroxycorticosterone to aldosterone metabolite ratio should be considered for mineralocorticoid replacement therapy even if their absolute aldosterone values appear to be normal or elevated.

Fetal rat adrenal growth and steroidogenesis in vitro after maternal dexamethasone treatment.

Two groups of female rats were treated with dexamethasone phosphate (DEX) for 24 h. One group received DEX in drinking water (10 micrograms/ml) on the 15th day of pregnancy (DEX15--16), and the second group on the 17th day of pregnancy (DEX 17--18). The mothers and fetuses were sacrificed on the 20th and 22nd days of pregnancy. DEX reduced adrenal weights and concentration of corticosterone in plasma of the treated females and their fetuses. Fetal body weight was also reduced. However, the lightest adrenal glands were found in fetuses of the DEX17--18 mothers. Furthermore, the 19-day-old fetuses belonging to the DEX17--18 mothers had smallest amounts of corticosterone in plasma, and in plasma, and their adrenal glands produced the lowest amounts of radioactive corticosterone, 11-doxycorticosterone and 18-hydroxy-11-deoxycorticosterone from 4-14C progesterone after 1 h or incubation in vitro. In the 21-day-old fetuses belonging to both the groups of treated mothers, the production of steroids by the adrenal glands was similar and to the same extent reduced in comparison to the control fetuses.