Sodium depletion of rats induces a mitochondrial protein in the zona glomerulosa of the adrenal cortex

Sodium restriction of rats resulted in a marked increase in the concentration of a mitochondrial protein with a mol. wt of 49000 in the zona glomerulosa of the adrenal cortex. The same protein had been previously induced in potassium deficient rats by potassium repletion. Since both sodium depletion and potassium repletion are known to stimulate the activity of the mitochondrial mixed function oxidase involved in the conversion of corticosterone to aldosterone, the present observation supports the hypothesis that the potassium-induced protein might represent cytochrome P-450CMO (corticosterone methyl oxidase).     

Adaptation of aldosterone biosynthesis to sodium and potassium intake in the rat

The steroidogenic response of rat adrenal zona glomerulosa to stimulators is variable and depends on the activity of biosynthetic steps involved in the conversion of deoxycorticosterone (DOC) to aldosterone (Aldo). Corticosterone methyl oxidations (CMO) 1 and 2 are stimulated by sodium restriction and suppressed by potassium restriction. These slow alterations are accompanied by the appearance or disappearance of a specific zona glomerulosa mitochondrial protein with a molecular weight of 49,000. Induction of CMO 1 and 2 activities and the appearance of the 49 K protein can also be elicited in vitro by culture of rat zone glomerulosa cells in a medium with a high potassium concentration. The 49 K protein crossreacts with a monoclonal antibody raised against purified bovine adrenal cytochrome P-450(11 beta). The same antibody stains a protein with a molecular weight of 51,000 in rat zona fasciculata mitochondria and in zone glomerulosa mitochondria of rats in which CMO 1 and 2 activities have been suppressed by potassium restriction and sodium loading. The 51 K crossreactive protein was purified to electrophoretic homogeneity by chromatography on octyl-sepharose. In a reconstituted enzyme system, it converted DOC to corticosterone (B) and to 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) but not to 18-hydroxycorticosterone (18-OH-B) or Aldo. A partially purified 49 K protein preparation from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen converted DOC to B, 18-OH-DOC, 18-OH-B and Aldo. According to these results, rat adrenal cytochrome P-450(11 beta) exists in two different forms, with both of them capable of hydroxylating DOC in either the 11 beta- of the 18-position, but with only the 49 K form capable of catalyzing CMO 1 and 2. The adaptation of aldosterone biosynthesis to sodium deficiency or potassium intake in rats is due to the appearance of the 49 K form of the enzyme in zona glomerulosa mitochondria.     

An ultrastructural, morphometric and autoradiographic study of the effects of 7,12-dimethylbenzanthracene on the rat adrenal cortex.

The effects of chronic treatment (up to 9 consecutive days) with 7,12-dimethylbenzathracene (DMBA) on the adrenal glands of adult male Wistar rats were investigated. Morphometry showed that DMBA provokes atrophy of the zona reticularis which was due to the decrease in both cell volume and number. The zona fasciculata showed only a decrease in the cell volume, whereas the zona glomerulosa did not display any significant changes. Autoradiography demonstrated that DMBA induces a significant increase in the number of mitoses and "S" phase cells in the zona glomerulosa and outer zona fasciculata, which may be interpreted as a repair mechanism of the DMBA-provoked slight necrosis in the inner adrenocortical layers. The mechanism(s) underlying the cytotoxic effect of DMBA is discussed in the light of our ultrastructural observations showing that the chemical causes a decrease in the volume of the mitochondrial and nuclear compartments and in the surface of smooth endoplasmic reticulum as well as an increase in the volume of the lipid compartment.     

Zona glomerulosa of the adrenal gland in a transgenic strain of rat: a morphologic and functional study

Transgenic rats for the murine Ren-2 gene display high blood pressure, low circulating levels of angiotensin II, and high renin content in the adrenal glands. Moreover, transgenic rats possess and increased aldosterone secretion (maximal from 6 to 18 weeks of age), paralleling the development of hypertension. To investigate further the cytophysiology of the adrenal glands of this strain of rats, we performed a combined morphometric and functional study of the zona glomerulosa of 10-week-old female transgenic rats. Morphometry did not reveal notable differences between zona glomerulosa cells of transgenic and age- and sex-matched Sprague-Dawley rats, with the exception of a marked accumulation of lipid droplets, in which cholesterol and cholesterol esters are stored. The volume of the lipid-droplet compartment underwent a significant decrease when transgenic rats were previously injected with angiotensin II or ACTH. Dispersed zona glomerulosa cells of transgenic rats showed a significantly higher basal aldosterone secretion, but their response to angiotensin II and ACTH was similar to that of Sprague-Dawley animals. Angiotensin II-receptor number and affinity were not dissimilar in zona glomerulosa cells of transgenic and Sprague-Dawley rats. These data suggest that the sustained stimulation of the adrenal renin-angiotensin system in transgenic animals causes an increase in the accumulation in zona glomerulosa cells of cholesterol available for steroidogenesis, as indicated by the expanded volume of the lipid-droplet compartment and the elevated basal steroidogenesis. However, the basal hyperfunction of the zona glomerulosa in transgenic animals does not appear to be coupled with an enhanced responsivity to its main secretagogues, at least in terms of aldosterone secretion.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.     

Multiple treatments with SRIH-14 or octreotide affect adrenal zona glomerulosa in adult male rats

Somatostatin analogues are currently used to treat various disorders such as hypersecretion and different neuroendocrine tumors. In this study we examined the effects on the adrenal cortex of somatostatin (SRIH-14) and octreotide administered subcutaneously twice daily for 5 days to adult male rats. Control rats received saline under the same regime. After sacrifice, the adrenal glands were removed and examined morphometrically using the M(42) multipurpose test system. Blood samples were prepared for biochemical tests. Both SRIH-14 and octreotide induced morphofunctional changes in adrenal zona glomerulosa. We found significant decreases (p < 0.05) in the absolute cell and nuclear volumes of zona glomerulosa in both experimental groups in comparison to the control. The serum aldosterone level was 11% lower (p < 0.05) in the SRIH-14 and 13% (p < 0.05) lower in the octreotide-treated group in comparison with the control group. Morphometric parameters of zona fasciculata and zona reticulata and corticosterone levels were not altered significantly (p > 0.05) in either treated group. It may therefore be concluded that both SRIH-14 and octreotide affected zona glomerulosa in the same manner by decreasing morphofunctional characteristics.     

Autometallographic demonstration of gold in the adrenal gland of rats exposed to sodium aurothiomalate

The presence of gold was investigated in sections of the adrenal glands from rats which had been exposed to intraperitoneal sodium aurothiomalate (32 to 120 mg). Gold was histochemically detected in cortical endocrine cells, chromaffin cells and in fibroblasts and macrophages of both the cortex and medulla. Invisible traces of gold were silver enhanced by autometallography making them readily visible at both the light and electron microscopic levels. The intracellular staining intensity was dose-dependent. In general, the number as well as the staining intensity of individual cells, were highest in the zona glomerulosa and zona reticularis. In gold-containing cells the silver-amplified deposits were present in lysosomes.     

Structural and functional studies of the adrenal zona glomerulosa in sodium-depleted and sodium-loaded sheep

Cell and Tissue Research - The effects of alterations in sodium status upon the morphology of the adrenal zona glomerulosa in sheep have been examined qualitatively and quantitatively, using...     

Effects of ACTH-(11-24) on the corticosteroid production of isolated adrenocortical cells

The steroidogenic action of ACTH-(11-24) was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(11-24) stimulated the corticosterone production of zona fasciculata cells and the aldosterone production of zona glomerulosa cells; in addition, it potentiated the effects of ACTH-(1-39) on both cell systems. It is suggested that the ACTH molecule contains more active sites for steroidogenesis than usually acknowledged, as has been found for lipolysis and behavior.     

Interaction of ACTH, beta-endorphin and alpha-melanocyte stimulating hormone in relation to the corticosteroid production of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells.

The combined effects of ACTH, beta-endorphin (beta-EP) and alpha-MSH were studied on the corticosteroidogenesis of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells. beta-EP potentiated the effects of ACTH and alpha-MSH on the zona fasciculata corticosterone production but inhibited those on the zona glomerulosa aldosterone production. beta-EP did not affect the combined action of 4 x 10(-11) M ACTH and 5 x 10(-9) M alpha-MSH on the zona fasciculata or the zona glomerulosa cells, but it inhibited the stimulatory action of the combination of 1.6 x 10(-10) M ACTH and 10(-9) M alpha-MSH on the zona glomerulosa aldosterone production. An interaction of ACTH, beta-EP and alpha-MSH in relation to the zona fasciculata and zona glomerulosa corticosteroid production was found.     

Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.     

Effect of pituitary intermediate lobe extract on steroid production by the isolated zona glomerulosa and fasciculata cells

The effect of intermediate lobe extract (ILE) on aldosterone and corticosterone production of the zona glomerulosa cells and on corticosterone production of the zona fasciculata cells was investigated. The slope of the dose-response curve of ILE dilution was steeper than that of alpha h 1-39 ACTH measured on zona glomerulosa steroid production. The ED50 of both ILE and ACTH was lower when measured on zona glomerulosa than on zona fasciculata steroid production. It is supposed that a hormone (or some other substance) in ILE alters the sensitivity to ACTH of the zona glomerulosa cells.     

Trophic and steroidogenic effects of water deprivation on the adrenal gland of the adult female rat

The effects of a 3-day water deprivation were studied in adult female rats in order to know what are the different zones of the adrenal gland and the hormonal factors involved in the growth and the activity of the adrenal gland. Water deprivation significantly increased plasma renin activity (PRA), plasma Angiotensin II (AII), vasopressin (AVP), epinephrine, aldosterone and corticosterone concentrations but did not modify the plasma adrenocorticotropin hormone (ACTH) level. Water deprivation significantly increased the absolute weight of the adrenal capsule containing the zona glomerulosa without modification of the density of cells per area unit suggesting that the growth of the adrenal capsule was due to a cell hyperplasia of the zona glomerulosa. Water deprivation significantly increased the density of AII type 1 (AT₁) receptors in the adrenal capsule but did not modify the density of AII type 2 (AT₂) receptors in the adrenal capsule and core containing the zona fasciculata, the zona reticularis and the medulla. The treatment of dehydrated female rats with captopril, which inhibits the angiotensin converting enzyme (ACE) in order to block the production of AII, significantly decreased the absolute weight of the adrenal capsule, plasma aldosterone and the density of AT₁ receptors in the adrenal capsule. The concentration of corticosterone in the plasma, the density of AT₂ receptors and the density of cells per unit area in the zona glomerulosa of the adrenal capsule were not affected by captopril-treatment. In conclusion, these results suggest that AII seems to be the main factor involved in the stimulation of the growth and the secretion of aldosterone by the adrenal capsule containing the zona glomerulosa during water deprivation. The low level of plasma ACTH is not involved in the growth of the adrenal gland but is probably responsible for the secretion of corticosterone by the zona fasciculata.     

Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF (20 micrograms/kg/h for 7 days) induced atrophy of zona glomerulosa cells and lowering of basal plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system had been interrupted by the simultaneous administration of dexamethasone/captopril and maintenance doses of ACTH/angiotensin II. Chronic ANF treatment also caused comparable reductions in the aldosterone response of zona glomerulosa cells to the acute stimulation with angiotensin II, potassium and ACTH. These data are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa, and that the mechanism underlying this action of ANF does not involve blockade of renin release or ACTH secretion.     

The binding of cortisol to adrenal mitochondria

Binding of tritiated cortisol to adrenal zona glomerulosa mitochondria was studied and compared with that of corticosterone. Cortisol was shown to bind specifically to the inner membrane of zona glomerulosa mitochondria. Corticosterone and cortisol had similar apparent association constants (Ka) and concentrations of binding sites. The methodology was validated by obtaining similar Ka from both binding plots and kinetic data. Cortisol binding was inhibited by pretreatment with sodium dithionite, and displaced by deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone, 11 beta-hydroxy-18-ethynyl-progesterone and metyrapone, but not by cholesterol. These results suggest that cortisol and corticosterone bind to the same cytochrome P-450.     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

Sex differences in adrenocortical structure and function. XI

Summary Adrenal glands from orchectomized and ovariectomized rats, with and without replacement therapy, and also from intact controls of both sexes, were examined by autoradiography with ³H-thymidine. The labelling index after 1 or 2 nucleoside injections was higher in the zona glomerulosa of females than in male rats, while no differences were found in the fascicular and reticular zones. Orchiectomy increased the labelling index in the fascicular and reticular zones, an effect prevented by testosterone. Ovariectomy did not change the labelling index, while estradiol lowered it in the zona glomerulosa. Duration of the S phase was longer in the zona fasciculata cells of males than in females. Both orchiectomy and testosterone shortened this phase in cells of the zona fasciculata and zona reticularis. Ovariectomy prolonged the S phase in the zona fasciculata and shortened this time in the reticular zone, an effect reversed by estradiol.In the glomerular and fascicular zones, cell cycle time was longer in males than in females. Orchiectomy shortened this time in all adrenocortical zones, an effect reversed by testosterone. Ovariectomy shortened cell cycle time in the glomerular and reticular zones and prolonged it in the zona fasciculata; these effects were reversed by estradiol. Turnover rate in adrenocortical cells was markedly higher in females than in males, a difference due to testosterone which markedly decreased turnover rate.     

A study of cell migration in the adrenal cortex of the rat using bromodeoxyuridine

Abstract Bromodeoxyuridine (BrdU) was administered by a single intraperitoneal injection to immature (14 days) male and female and adult male Sprague-Dawley rats. Animals were killed at intervals from 2 hr to 28 days following injection. Labelled cells in the adrenal cortex were identified by an indirect immunoperoxidase technique using a monoclonal antibody to BrdU. At 2 hr, labelling was maximal in the outer zona fasciculata and zona glomerulosa in both prepubertal and adult rats. The numbers of immunopositive cells were greater in the 14 day rats. In both groups, the front of immunopositive cells moved deeper into the cortex with time. These results support the centripetal migration theory of adrenal growth.