Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself.

The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10⁻⁹ M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative.

Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, N = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, N = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17-, 21- and 11β-hydroxylases.

The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response (P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%.

Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.     

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration to guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17-hydroxylase and 21-hydroxylase activities were decreased to 20–25% of control values by the higher dose of ABT. Mitochondrial 11β-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3β-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.     

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11β-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C₁₉ steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C₁₉ steroids would be formed. The urinary excretion of the four 11-oxy-C₁₉ steroids as well as many of the major C₂₁ cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C₁₉ steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation.

A secondary goal was to determine to what extent 11-oxy-C₁₉ steroids were metabolites of cortisol or adrenal synthesized 11β-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C₁₉ 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C₁₉ steroids should reflect the absence of 11β-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11β-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11β-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11β-hydroxyandrostenedione.     

Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells

Among the large number of immediate early genes, nuclear proto-oncogenes of the Fos and Jun families, have been postulated to be involved in the long-term effects of several growth factors on cell differentiation and/or multiplication. Since adrenal cell differentiated functions appear to be regulated by specific hormones and growth factors, the effects of these factors on proto-oncogene mRNA levels were analysed in bovine adrenal fasciculata cells (BAC) in culture. Corticotropin (ACTH) and insulin-like growth factor I increased c-fos and jun-B mRNA, but had no effect on c-jun mRNA and these early changes were associated with a later increase in BAC specific function [ACTH receptors, cytochrome P 450 17) and 3β-hydroxysteroid dehydrogenase (3β-HSD)] and an enhanced steroidogenic responsiveness to both ACTH and angiotensin-II (A-II). On the other hand, A-II increased the three proto-oncogene (c-fos, c-jun and jun-B) mRNAs, induced a decreased of P 450 17 and 3β-HSD and caused a marked homologous and heterologous (ACTH) densitization. Transforming growth factor β₁ which only increased jun-B mRNA, markedly reduced BAC differentiated functions and the steroidogenic responsiveness to both ACTH and A-II. Thus, it is postulated that the proto-oncoproteins encoded by the immediate early genes may play a role in the long-term effects of peptide hormones and growth factors on BAC differentiated functions.     

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450_11β and cytochrome P-450_aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Molecular genetic studies on the biosynthesis of aldosterone in humans

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.     

Functional expression of cDNAs for bovine 11 beta-hydroxylase-aldosterone synthases, P450(11 beta)-2 and -3 and their chimeras.

Two molecular species of bovine P450(11β), P450(11β)-2 and P450(11β)-3 have been identified, in which the amino acid differences were found at the 6th, 36th and 82nd positions from the NH₂-termini of the mature proteins. They catalyzed the 11β-, 18- and 19-hydroxylation and aldosterone formation from 11-deoxycorticosterone, and the rate of production of 18-hydroxycorticosterone and aldosterone by P450(11β)-3 was greater than that by P450(11β)-2 [Morohashi et al., J. Biochem. 107 (1990) 635–640].

In this study, chimeric clones were constructed whose 6th, 36th and 82nd amino acid residues were exchanged with each other. Two original clones and six chimeric clones were expressed in COS-7 cells, and their steroidogenic activities studied. The ratio of aldosterone or 18-hydroxycorticosterone production to corticosterone production by one clone was compared with that of the other. The ratios for the four clones having Gly36 [P450(11β)-3 type] were 0.08–0.22, whereas those for the clones having Ser36 [P450(11β)-2 type] were 0.03–0.05, suggesting that the Gly36 structure is important for aldosterone production.     

Influence of 19-nortestosterone and androgens on progesterone biosynthetic enzymes in early human placental explants

We recently showed that the production of progesterone (P4) in human placental explant culture from early gestation is enhanced by treatment with 19-nortestosterone (19-NT) or with certain androgens, namely androsen, namely androstenedione (A-dione), 5-androstane-3, 17β diol (3-diol) and 5-androstane-3β, 17β diol (3β-diol). This stimulation of P4 was explored further in this study. There was little metabolism of radioactive P4 when incubated for 24 h in the presence or absene of these steroids. The role of different steroids in the regulation of P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was evaluated by measuring the conversion of P4 derived from unlabelled 25-hydroxycholesterol and from labelled pregnenolone, respectively. The results showed that 19-NT, A-dione and 3-diol stimulated (P450scc) activity; however, 3β-diol was ineffective. While 19-NT and 3β-diol enhanced the bioconversion of pregnenoloe to P4, A-dione and 3-diol were without effect.

The initial rapid stimulation of P4 by 19-NT within 2 h of incubation was not blocked by concurrent treatment with cycloheximide (CH). However, after incubation for 24 h, 70% of the 19-NT-stimulated P4 was abolished by CH. During the same incubation period,] P4 stimulation by A-dione, 3- and 3β-diol were completely blocked by treatment with CH. Thus our observations suggest that 19-NT-stimulated P4 accumulation is due to the combined effects on P450scc adn 3β-HSD enzyme activities. A-dioneand 3-diol increase biosynthesis of P4 by acting selectively on P450scc enzyme. However, the stimulatory action of 3β-diol on P4 is only at the level of 3β-HSD. Since CH blocks the stimulatory actions, the mechanism(s) by which androgens (A-dione, 3-diol and 3β-diol) and norandrogen (19-NT) augment the biosynthetic enzyme activities appears to be mediated by a process inhibited by CH. Since CH interference was absent during the initial rapid P4-stimulation by 19-NT, there may be a direct action of this steroid at the cellular level which is not dependent on new protein synthesis.     

Steroid biosynthesis by zona glomerulosa-fasciculata cells in primary culture of guinea-pig adrenals

Steroidogenesis was studied in guinea-pig glomerulosa-fasciculata cells maintained in primary culture for up to 7 days. The basal secretion which remained stable for the first 2 days in culture rapidly rose to reach a plateau on day 4 at levels 6-7-fold higher than those observed during the first 2 days of culture while the maximal response to ACTH in terms of cortisol and androstenedione secretion was fairly stable throughout the 7-day period. Exposure of glomerulosa-fasciculata cells to ACTH caused a stimulation of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxy-corticosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, 11 beta-hydroxyandrostenedione and aldosterone while, after 48 h of incubation, a marked accumulation of end-products, namely cortisol and 11 beta-hydroxyandrostenedione, was observed. The half-maximal steroidogenic response to ACTH occurred at concentrations varying between 1.7 x 10(-11) and 1.1 x 10(-10) mol/l for the 12 steroids examined. Addition of 8-bromoadenosine 3', 5'-cyclic monophosphate stimulated steroid secretion in a dose-dependent manner. Maximal response to 8-bromoadenosine 3', 5'-cyclic monophosphate was obtained at 1 mmol/l, and no further rise of steroid secretion was observed after addition of ACTH. Incubation of glomerulosa-fasciculata cells with labeled corticosterone, cortisol and androstenedione indicates that only androstenedione can be converted into 11 beta-hydroxyandrostenedione, thus suggesting that this end-product is a good parameter of the C-19 steroid production by guinea-pig glomerulosa-fasciculata cells in primary culture. The present data confirm that guinea-pig glomerulosa-fasciculata cells in primary culture provide an interesting model for the study of the regulation of C-19 steroid formation by the adrenals.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4–5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011 from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M_r 60 kD) and a NADPH-cytochrome P450 reductase (M_r 79 kD).     

Cytochrome P450(11β): Structure-function relationship of the enzyme and its involvement in blood pressure regulation

Cytochrome P450(11β) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11β) family enzymes suggested that the enzymes having both 11β-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11β-hydroxylases of these animals. (2) The molecular nature of the P450(11β) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11β) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11β-, 18- and 19-Hydroxylation activities of adrenal mitochondria prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

18-Ethynyl-deoxycorticosterone inhibition of steroid production is different in freshly isolated compared to cultured calf zona glomerulosa cells

The inhibiting effects of 18-ethynyl-deoxycorticosterone (18-E-DOC) as a mechanism-based inhibitor on the late-steps of the aldosterone biosynthetic pathway were examined in calf adrenal zona glomerulosa cells in primary culture and in freshly isolated calf zona glomerulosa cells. 18-E-DOC inhibited the stimulated secretion of aldosterone and 18-hydroxycorticosterone in a similar dose-response and time fashion. No significant differences were found between the inhibition in cultured and freshly isolated cells (K_i of 0.25 vs 0.26 μM) Corticosterone secretion stimulated by ACTH or angiotensin II was also cultured in freshly isolated zona glomerulosa and fasciculata cells, but was not inhibited in cultured calf adrenal cells. Cortisol secretion stimulated by ACTH was not inhibited by 18-E-DOC in cultured zona fasciculata adrenal cells, but was inhibited in freshly isolated zona fasciculata cells with a K_i of 48 μM. The secretion of 18-hydroxyDOC or 19-hydroxyDOC stimulated by ACTH was not inhibited by 18-E-DOC. The bovine adrenal has been reported to have cytochrome P-450 11β-hydroxylases that can perform the various hydroxylations required for the synthesis of cortisol and aldosterone in the different areas of the adrenal. In other species a distinct 11β-hydroxylase which participates in the biosynthesis of aldosterone and is located in the zona glomerulosa has been described. These studies with the mechanism-based inhibitor, 18-E-DOC, suggest that the bovine adrenal functions in a manner very similar to that of other species and raises the possibility that a distinct 11β-hydroxylase with aldosterone synthase activity might be present, but has not been cloned as yet.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.