Functional zonation of the guinea pig adrenal cortex: differences in mitochondrial steroid metabolism between the inner and outer zones

Previous studies established that cells isolated from the chromatically distinct inner (primarily zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex had vastly different steroidogenic capabilities; the outer zone produced far more cortisol than the inner zone. The mechanism(s) responsible for those differences were investigated by comparing mitochondrial steroid metabolism in the inner and outer zones. Cytochrome P-450 concentrations were similar in the two zones, but 11 beta-hydroxylase activity was approximately twice as great in the outer zone. More importantly, cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis, was nearly 10 times greater in outer than inner zone mitochondria. Free cholesterol concentrations were also far higher in outer zone mitochondria. The results suggest that the relatively low level of steroid secretion by cells of the zona reticularis is attributable, at least in part, to deficiencies in mitochondrial cholesterol content and/or metabolism.     

Zonal expression of endothelial nitric oxide synthase in sheep and rhesus adrenal cortex.

There is mounting evidence that nitric oxide (NO) may inhibit adrenal steroidogenesis by binding to the heme group of P450 enzymes, particularly the rate-limiting steps cholesterol side-change cleavage P450, aldosterone synthase P450, and 17 alpha-hydroxylase/C(17/20)-lyase P450. Using immunohistochemistry, nitrotyrosine was detectable throughout the ovine adrenal cortex, and endothelial NO synthase (eNOS) was further identified in zona glomerulosa (ZG) and at a higher level throughout the zona fasciculata, increasing toward the medulla. Caveolin-1, 90-kDa heat shock protein, ERK-1/2, and Akt, all known and proposed regulators of eNOS activity, were detected throughout the ovine adrenal cortex. Western immunoblotting confirmed the identity of these proteins as well as the absence of neuronal NOS, inducible NOS, caveolin-2, and caveolin-3. Through dual immunostaining we further identified for the first time a zona intermedia without strong staining for 17 alpha-hydroxylase/C(17/20)-lyase P450 or angiotensin II type 1 receptor, but positive for eNOS. Rhesus adrenals also stained positively for eNOS, but staining was seen only in the ZG and zona reticularis. We conclude that eNOS may play a role in controlling zone-specific aldosterone synthase vs. 11 beta-hydroxylase activities of the single CYP11B gene in sheep. In the rhesus monkey, NO may modulate ZG aldosterone synthase, but it is not needed for control of the distinct 11 beta-hydroxylase in the zona fasciculata. In the zona reticularis, however, eNOS may control C(19) steroid production at the level of 17 alpha-hydroxylase vs. 17,20-lyase activity otherwise unopposed by 3beta-hydroxysteroid dehydrogenase.     

Immunohistochemistry of cytochrome P-450 21-hydroxylase: microscopic examination of the enzyme in the bovine adrenal cortex and kidney

Cytochrome P-450 specific for steroid 21-hydroxylase (P-450C21) localized in bovine adrenal cortex and kidney was immunocytochemically observed by the peroxidase-antiperoxidase method using a specific antibody. P-450C21 was present in all three zones of the adrenal cortex. Immunoreactivity for P-450C21 was intense in the zona glomerulosa and inner reticularis and faint in the area between the zona glomerulosa and outer fasciculata, probably representing the zona intermedia. The positive stain was only observed in parenchymal cells. The immunoreactivity varied within each zone, especially in the zona reticularis. In the kidney, immunoreactivity for P-450C21 was exclusively localized in the distal and cortical and medullary collecting tubules. This corresponds to the site of mineralocorticoid action in the kidney. No immunoreactivity was observed in the liver and aorta.     

Molecular nature of aldosterone synthase, a member of cytochrome P-450(11 beta) family

The molecular nature of the aldosterone synthesizing enzymes of cattle and rat is discussed. In bovine adrenal cortex, one molecular species of cytochrome P-450(11 beta) catalyzes aldosterone synthesis as well as 11 beta-hydroxylation. The intactness of the mitochondrial membrane surrounding P-450(11 beta) in the zonae fasciculata-reticularis is essential to keep the aldosterone synthesizing activity of the cytochrome in these zones latent. In rat adrenal cortex, two distinct molecules belonging to a P-450(11 beta) family exist. One is 11 beta-hydroxylase, and the other aldosterone synthase.     

Immunohistochemical analysis of cytochrome P-450 17 alpha-hydroxylase in pig adrenal cortex, testis and ovary

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-450(17 alpha] was immunohistochemically observed in pig adrenal cortex, testis and ovary by the biotin-streptavidin method using a specific antibody against P-450(17 alpha) purified from neonatal pig testis. In the adrenal cortex, P-450(17 alpha) was present in the zona fasciculata and reticularis while no immunoreactivity was observed in the zona glomerulosa, confirming the absence of 17-hydroxylated steroid synthesis in the zona glomerulosa. In the testis, P-450(17 alpha) was present exclusively in Leydig cells and immunoreactivity was absent in seminiferous tubules. In the ovary, immunoreactivity was observed only in the theca interna but not in the membrana granulosa of follicles. Among the tissues examined, the relative intensity of immunoreactivity was greatest in the Leydig cells, and progressively less in theca interna cells, outer fasciculata cells and inner fasciculata and reticularis cells.     

Role of adrenal renin in the regulation of adrenal steroidogenesis by corticotropin.

The major regulator of mineralocorticoid production in the adrenal is angiotensin II produced by the action of renal renin. The discovery that the rodent adrenal also synthesizes renin and angiotensinogen suggests there is autocrine regulation of mineralocorticoid synthesis. The transgenic rat [TGR(mREN2)27] expresses the Ren-2d gene predominantly in the adrenal. Despite suppressed kidney and plasma renin, these animals develop fulminant hypertension between 5 and 15 weeks of age. Corticosteroid concentrations are significantly elevated during hypertension development. We assessed steroidogenesis in TGR(mREN2)27 rats by analyzing the expression of the mRNAs for three steroidogenic enzymes: P450scc, the rate-limiting step of steroidogenesis; P450c11 beta, which converts deoxycorticosterone to corticosterone in the zona fasciculata/reticularis; and P450c11AS, which converts deoxycorticosterone to aldosterone in the zona glomerulosa. P450c11AS mRNA, but neither P450c11 beta nor P450scc mRNA, was overexpressed in the adrenal gland of TGR(mREN2)27 rats. In situ hybridization with specific probes for P450c11 beta and P450c11AS mRNA localized the former exclusively to the zona fasciculata and the latter to the zona glomerulosa. In TGR(mREN2)27 rats, the size of the adrenal and number of P450c11AS-expressing zona glomerulosa cells were about twice those of a normal Sprague-Dawley rat. Both animals respond to corticotropin similarly; corticotropin had no effect on the expression of P450scc and P45011 beta mRNAs, rendered P450c11AS mRNA undetectable, and simultaneously altered the morphology of the adrenal cortex, resulting in a lack of zona glomerulosa-like cells. Thus, the local renin-angiotensin system has a major effect on the basal expression of P450c11AS mRNA, but little effect on the corticotropin-regulated expression of P450scc, P450c11 beta, and P450c11AS mRNAs.     

Aldosterone secretion a molecular perspective.

The major mineralocorticoid hormone aldosterone is secreted from the zona glomerulosa of the adrenal cortex. Aldosterone is synthesized from cholesterol via a series of hydroxylations and oxidations. The enzymes involved in these reactions are mostly members of the cytochrome P450 superfamily. The final steps of this pathway, the conversion of 11-deoxycorticosterone (DOC) to aldosterone, require conversion via the intermediates 18-hydroxy-DOC or corticosterone and 18-hydroxycorticosterone. There are significant differences between species in the number of genes that encode the P450(11beta)-related enzymes (CYP11B) involved in these steps and the zonal distribution of their expression. One enzyme is capable of 11-hydroxylation, 18-hydroxylation, and 18-oxidation of DOC to aldosterone. The genetic basis of four diseases-congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency, glucocorticoid-remediable aldosteronism, aldosterone synthase deficiency type I and type II-is explicable by mutations in these cytochrome P450(11beta)-related genes. (Trends Endocrinol Metab 1997;8:346-354). (c) 1997, Elsevier Science Inc.     

The impact of polymorphisms in the gene encoding aldosterone synthase (CYP11B2) on steroid synthesis and blood pressure regulation

The terminal stages in the synthesis of aldosterone and cortisol are catalysed by the enzymes aldosterone synthase and 11beta-hydroxylase respectively. We have previously reported that polymorphic variation in the 5' promoter region (-344C/T) of the gene encoding aldosterone synthase (CYP11B2) is associated with increased aldosterone metabolite excretion and with hypertension associated with a raised aldosterone to renin ratio (ARR). Additionally, basal and ACTH-stimulated plasma levels of 11-deoxycortisol, the precursor of cortisol, are higher in subjects carrying the T-allelic variant. We have now identified in a family study (573 individuals from 105 extended families ascertained through a hypertensive proband) that excretion of the main metabolite of this steroid (tetrahydro-11-deoxycortisol, THS) is heritable (19.4%) and that the T-allele of CYP11B2 is more strongly associated with higher THS levels than the C-allele. Raised plasma and urinary levels of 11-deoxycortisol suggest that there is relative inefficiency of 11beta-hydroxylation in the zona fasciculata; the P450 enzyme responsible for this step is encoded by the gene CYP11B1, which is highly homologous with and adjacent to CYP11B2. The association of genetic variation in the promoter of CYP11B2 which, in the adrenal cortex, is only expressed in zona glomerulosa, and zona fasciculata 11beta-hydroxylation function is paradoxical. There may be linkage dys-equilibrium between this polymorphism and a quantitative trait locus (QTL) in CYP11B1. Chronic alteration of 11beta-hydroxylase activity may increase ACTH drive to the adrenal cortex, altering the regulation of aldosterone synthesis. This may explain, at least partly, the association between CYP11B2 polymorphisms and hypertension.     

Synthesis of aldosterone by mitochondria and homogeneous 11 beta-hydroxylase from beef and pig

It was found that homogeneous 11 beta-hydroxylase from bovine and porcine adrenals catalyzes the conversion of DOC to aldosterone. Mitochondria from both glomerulosa and fasciculata also convert DOC to aldosterone but glomerulosa is much more active than fasciculata. Cholate extracts of mitochondria from the two zones were equally active in converting DOC to aldosterone. Moreover all the enzyme activities of 11 beta-hydroxylase (including 18-hydroxylation and aldehyde synthetase) were precipitated by a polyclonal antibody raised in rabbit against the pure 11 beta-hydroxylase. It is concluded that in beef and pig a single adrenocortical 11 beta-hydroxylase is responsible for the synthesis of aldosterone. To determine the influence of the mitochondrial membrane from glomerulosa and fasciculata on the activities of 11 beta-hydroxylase we examined the activities of rotenone-insensitive reductase enzymes in mitochondria from the two zones. Semidehydroxyascorbate reductase and NADH-cytochrome C reductase activities are considerably more active in glomerulosa than in fasciculata mitochondria. Moreover ascorbate plus NADH (but not ascorbate alone) greatly increases the ability of malate and NADPH to support synthesis of aldosterone without affecting 11 beta- or 18-hydroxylations in mitochondria. It is proposed that maximal synthesis of aldosterone by adrenocortical mitochondria requires in addition to the usual electron transport system (NADPH- greater than ADR- greater than ADX- 11 beta-OHase) an auxilliary system in the outer mitochondrial membrane: NADH- greater than Fp- greater than cyt b- greater than semidehydroascorbate reductase.     

Steroidogenesis activator polypeptide (SAP) in the guinea pig adrenal cortex

Steroidogenesis activator polypeptide (SAP), a cytosolic stimulator of cholesterol side-chain cleavage (cholesterol SCC) previously characterized in the rat, was isolated from guinea pig adrenal cortex. This factor exhibited behavior on reverse-phase high-performance liquid chromatography (HPLC) that was indistinguishable from authentic SAP and crossreacted fully in a SAP radioimmunoassay. In dexamethasone-suppressed guinea pigs neither the concentrations of immunoreactive adrenal SAP nor the levels of cholesterol SCC activity were significantly different between the outer zones (zonae glomerulosa and fasciculata) and the inner zone (zona reticularis). However, at 10 min after treatment of dexamethasone-suppressed animals with ACTH1-24, the outer zone content of SAP was increased 42-fold over unstimulated controls whereas inner zone SAP was elevated only 4-fold. At the same time, cholesterol SCC activity was increased 2-fold in the outer zones but unchanged in the inner zone. In addition to SAP itself, a crossreacting 82 kDa protein (p82)--similar to the putative SAP precursor identified in the rat--was detected on two-dimensional immunoblots of guinea pig whole adrenal homogenate. There were no significant differences in the protein concentrations of p82 or of cytochrome P-450scc between zones, either with or without ACTH treatment. We conclude that the widely reported contrast in corticosteroidogenic potential between the zona fasciculata and the zona reticularis of the guinea pig may reflect a differential capacity to generate SAP, and thus activate cholesterol SCC, in response to ACTH.     

Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenal cortex: histochemical basis for the functional zonation.

Zonal distribution of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenocortex was investigated immunochemically using specific antibodies to these enzymes. Localization of aldosterone synthase cytochrome P-450 (cytochrome P-450aldo), a recently identified enzyme that converts deoxycorticosterone to aldosterone in rat adrenocortex was strictly confined to two or three outermost cell layers in the zona glomerulosa. In contrast, cytochrome P-45011 beta, which forms corticosterone, but not aldosterone, from deoxycorticosterone, was localized in the zona fasciculata-reticularis and not in the zona glomerulosa. Neither enzyme was detected in the medulla or the capsule. The functional zonation of adrenocortex with respect to aldosterone and corticosterone syntheses is, thus, ascribable to the localization of cytochromes P-450aldo and P-45011 beta in the respective zones. When rats were maintained under Na-depleted conditions for 10 days, the zona glomerulosa cells containing cytochrome P-450aldo proliferated to 10-15 layers, the thickness of which was 5-7-fold that in the nonstimulated rats. Proliferation of the cytochrome P-450aldo-positive cells into the zona fasciculata-reticularis was also observed along with arterial walls. Under these conditions, no significant change in the distribution of cytochrome P-45011 beta was noted. These results indicate that the angiotensin-II stimuli, which had been elicited by the low Na treatment, promoted proliferation of the glomerulosa cells, resulting in increased expression of cytochrome P-450aldo in rat adrenocortex.     

Differential suppression of the outer and inner zones of the adrenal cortex of the guinea pig

To examine the regulation of the zona fasciculata and the zona reticularis of the guinea pig adrenal cortex, animals were placed on a chronic regimen of dexamethasone. Changes in adrenal zonal weight, cholesterol side-chain cleavage activity, and tissue and serum steroid concentrations were measured after 1 month of dexamethasone administration. With dexamethasone treatment, the weight of the outer zone (glomerulosa/fasciculata) decreased significantly, while the weight of the inner zone (reticularis) did not change. Cholesterol side-chain cleavage activity in mitochondria isolated from the outer zone also declined significantly, while a similar activity in the inner zone did not change. The serum cortisol concentration in response to dexamethasone administration decreased by 85%, as did the concentrations of cortisol and progesterone in the outer zone; the concentration of cortisol in the inner zone decreased by only one third, while the concentration of progesterone did not change. These results in association with previous reports indicate that the zona reticularis of the guinea pig adrenal cortex (in contrast to the zona fasciculata) is not regulated by ACTH in terms of either steroidogenesis or maintenance of cell growth.     

Disorders of steroid 11beta-hydroxylase isozymes.

Steroid 11 beta-hydroxylase activity in the adrenal cortex is required for the synthesis of the major glucocorticoids and mineralocorticoids, but different isozymes mediate this conversion in the zona fasciculata, where cortisol is produced, and the zona glomerulosa, the site of aldosterone synthesis. The isozyme in the latter zone also has 18-hydroxylase and 18-oxidase activities that are required for aldosterone synthesis. Mutations in the genes encoding these isozymes respectively result in defective synthesis of cortisol and aldosterone. Recombinations between the two genes that alter the regulation of the isozyme responsible for aldosterone synthesis cause an inherited form of hypertension, glucocorticoid-suppressible hyperaldosteronism.     

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.     

Zone-specific regulation of two messenger RNAs for P450c11 in the adrenals of pregnant and nonpregnant rats.

Adrenal mitochondria possess two steroidogenic cytochrome P450s. P450c11 converts deoxycorticosterone to corticosterone and aldosterone, and P450scc converts cholesterol to pregnenolone. These P450s receive electrons from NADPH via adrenodoxin reductase and adrenodoxin. A single bovine P450c11 protein has 11-hydroxylase, 18-hydroxylase, and 18-oxidase activities, but this series of enzymatic steps may be mediated by more than one enzyme in rats. Enzymatic assays of purified rat mitochondrial proteins have suggested that one enzyme found in all zones of the adrenal cortex has both 11- and 18-hydroxylase activities, whereas another enzyme, found exclusively in the zona glomerulosa, catalyzes 18-hydroxylation and 18-oxidation of corticosterone. We studied the number and zonal distribution of P450c11 mRNA species in the rat adrenal and how these mRNAs are regulated in the adrenals of normal and pregnant rats. Rats synthesize two similar, but distinct, P450c11 mRNAs. One, P450c11A, is found in both the zona glomerulosa and fasciculata/reticularis, whereas the second, P450c11B, is found only in the zona glomerulosa. The abundance of neither P450c11A mRNA nor P450c11B mRNA is affected by a high-salt diet. However, when rats receive a low-salt diet, P450c11A mRNA decreases and P450c11B mRNA increases. Dexamethasone decreases the amount of P450c11A mRNA without affecting P450c11B mRNA. The combination of a high-salt diet and dexamethasone decreases the amount of both mRNAs further to almost undetectable amounts. Rats given a low-salt diet and dexamethasone have a dramatic increase in the abundance of P450c11B mRNA. Thus both forms of P450c11 mRNA are regulated independently in the rat adrenal cortex. In situ hybridization studies show that only the P450c11 found in the zona glomerulosa is regulated by salt treatment in vivo, whereas glucocorticoid treatment in vivo regulates P450c11 in all zones. In the adrenals of pregnant rats, P450c11B is regulated in a similar fashion to its regulation in the nonpregnant rat adrenal, despite major differences in sodium retention and intravascular volume in pregnant and nonpregnant rats. In the pregnant rat, a low-salt diet increases the abundance of P450c11B to a greater degree than in the nonpregnant rat. By contrast, dexamethasone does not diminish the abundance of P450c11A mRNA in the pregnant rat but reduces it to an almost undetectable amount in the nonpregnant rat. Thus, the regulation of glucocorticoid and mineralocorticoid production in the pregnant and nonpregnant rat occurs by different mechanisms.