Intramitochondrial bodies in sheep adrenal zona glomerulosa cells

Summary Relatively large, mostly rounded, very electron dense intramitochondrial bodies in adrenal zona glomerulosa cells of sheep are described and their nature and connection to protein in the mitochondria discussed. The so called azocarmine granules seen in the light microscope may be identical with the intramitochondrial bodies in the zona glomerulosa cells.     

Electron microscopic morphometry of the zona glomerulosa cells of the Wistar rat.

Results of morphometric measurements were compared on the cells of the zona glomerulosa (ZG) of the adrenal gland of the rat. The mean area covered by the cells, the nuclei, the mitochondria and the lipid droplets, as well as the surface density of the endoplasmic reticulum membrane and of internal mitochondrial membrane indicated an inhomogeneity even in the ZG cells of the same animal, with considerable deviations in the ZG of different animals. The dark and light ZG cells were compared too. The results suggest hormonal-functional differences, both among the normal animals and among the cells of the same animals.     

Adrenal cortical foci and nodules in Sprague-Dawley rats treated with N-nitrosomorpholine. Light and electron microscopic findings

Treatment of male Sprague-Dawley rats with N-nitrosomorpholine (NNM) in the drinking water at a dose of 120 mg/l for 7 weeks resulted in a subsequent enhanced development of focal and nodular lesions in the adrenal cortex. Sequential observation revealed that focal lesions in the zona reticularis/fasciculata or the zona glomerulosa developed both earlier and at a significantly higher incidence in animals treated with carcinogen than in untreated controls. Foci observed within or adjacent to the zona glomerulosa were all of pale cell appearance and contained large numbers of electron-dense cytoplasmic granules similar to those observed in normal granulosa cells. The foci and nodules which arose in the zona reticularis/fasciculata were, in contrast, characterized by a reduction or loss of the dense osmiophilic droplets normally seen in the cells of this region of the adrenal cortex, a pronounced increase in pleomorphic mitochondria of atypical appearance and the development of vacuoles.     

Investigations on the turnover of adrenocortical mitochondria

The effects of chronic administration of ACTH and dexamethasone on the morphology of mitochondria in zona glomerulosa cells of the rat adrenal cortex were investigated by stereological techniques. It was found that the volume of the mitochondrial compartment as well as the surface of the outer and inner mitochondrial membranes were significantly increased or decreased in relation to the number of days of ACTH- or dexamethasone-treatment. In ACTH-administered rats, the average volume of individual mitochondria decreased significantly up to the 6th day of treatment and then showed a conspicuous increase from the 6th to the 15th day, whereas in dexamethasone administered animals this parameter, after a small increase during the first 6 days of treatment, displayed a significant decrease. The number of mitochondria per cell showed a dramatic increase during the first 6 days of treatment with ACTH and continued to increase, but only slightly, with the subsequent treatment. In contrast, this parameter showed a parabolic decrease as a function of the duration of treatment in animals receiving dexamethasone. In the light of evidence showing that dexamethasone blocks ACTH-release, these findings are discussed and interpreted to indicate that ACTH is involved in the maintenance and stimulation of the growth and proliferative activity of mitochondria in rat adrenal zona glomerulosa.     

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.     

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.     

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.     

Leukotrienes as common intermediates in the cyclic AMP dependent and independent pathways in adrenal steroidogenesis

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and adrenocorticotropin (ACTH). Since the mitochondria can recognize factors generated by AII (cyclic-AMP-independent) and ACTH (cyclic AMP dependent), it is reasonable to postulate the existence of a common intermediate in spite of a different signal transduction mechanism. We have evaluated this hypothesis by stimulation of mitochondria from glomerulosa gland with fractions isolated from glomerulosa gland stimulated with AII or from fasciculata gland stimulated with ACTH; the same fractions were tested using mitochondria from fasciculata cells. Postmitochondrial fractions (PMTS) obtained after incubation of adrenal zona glomerulosa with or without AII (10(-7) M) or ACTH (10(-10) M), were able to increase net progesterone synthesis 5-fold in mitochondria isolated from non-stimulated rat zona glomerulosa. In addition, AII in zona glomerulosa produced in vitro steroidogenic fractions that were able to stimulate mitochondria from zona fasciculata cells. Inhibitors of arachidonic acid release and metabolism blocked corticosterone production in fasciculata cells stimulated with ACTH. This concept is supported by the experiment in which bromophenacylbromide and nordihydroguaiaretic acid also blocked the formation of an activated PMTS. In fact, non-activated PMTS, in the presence of exogenous arachidonic acid AA, behaved as an activated PMTS from ACTH stimulated cells. We suggest that the mechanisms of action of ACTH and AII involve an increase in the release of AA and an activation of the enzyme system which converts AA in leukotriene products.     

Gestational changes in hamster adrenal cortex: Morphometric and ultrastructural stereologic studies

Summary In the hamster, the weight of the adrenal glands increases during the course of gestation, with the highest value at day 5. In comparison to non-pregnant control animals, there were no changes in the volume of the zona glomerulosa (ZG) and zona fasciculata (ZF), while the volume of the zona reticularis (ZR) increased notably. The average volume of ZG-cells rose at day 5 of pregnancy and thereafter gradually decreased to that of control hamsters. A marked drop in the volume of ZF-cells was seen at days 5 and 10 of pregnancy, whereas at day 15 the cells were larger than in controls. At day 5 of pregnancy, a conspicuous increase in the cell volume was found in ZR, followed by lower values at day 10 and again higher than in control hamsters at day 15. The total number of parenchymal cells in hamster adrenal cortex increased at day 10 of gestation, then underwent a marked decrease, reaching the control value at the final day of pregnancy; this drop was mainly due to a reduction in the number of ZF-cells. The changes in the cell volume were paralleled by rather proportional changes in the volume of the mitochondrial compartment and in the quantity of smooth endoplasmic reticulum. The volume of the lipid-droplet compartment significantly rose in the course of gestation in both ZF- and ZR-cells. The cortisol output by adrenal homogenates gradually decreased during pregnancy.Study performed in partial fulfillment of the Ph.D. Thesis of Magdalena Nowak     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.     

Absent aldosterone response to metoclopramide in patients with high spinal cord transection: evidence that metoclopramide stimulates aldosterone secretion through central pathways

This study evaluates dopaminergic regulation of aldosterone secretion in 6 patients with high spinal cord transections. Administration of the dopamine antagonist metoclopramide resulted in a marked rise in plasma aldosterone and 18-hydroxycorticosterone levels in 12 normal individuals, but no change in plasma levels of these zona glomerulosa corticosteroid products in spinal cord patients. Spinal cord transected patients also did not have the rise in plasma renin activity that was observed in normals following metoclopramide administration. Basal levels of aldosterone, 18 hydroxycorticosterone, corticosterone and renin activity as well as the aldosterone responses to graded dose infusion of adrenocorticotropin were similar in the spinal cord patients and the normals. These data suggest that dopaminergic regulation of adrenal zona glomerulosa corticosteroid and renal renin secretion is absent in patients with high spinal cord transections, suggesting that intact neural pathways from the central nervous system are necessary for metoclopramide stimulation of aldosterone and renin secretion in men. Since basal plasma aldosterone levels were normal in spinal cord transected patients, it appears that the absence of dopaminergic control does not result in elevated secretion.     

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca²⁺ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration took place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the glomerulosa are also described.     

Ontogeny of angiotensin II type 1 receptor and cytochrome P450(c11) in the sheep adrenal gland

In the present study we investigated the ontogeny of the expression of the type 1 angiotensin receptor (AT(1)R mRNA) and the zonal localization of AT(1)R immunoreactivity (AT(1)R-ir) and cytochrome P450(c11) (CYP11B-ir) in the sheep adrenal gland. In the adult sheep and in the fetus from as early as 90 days gestation, intense AT(1)R-ir was observed predominantly in the zona glomerulosa and to a lesser extent in the zona fasciculata, and it was not detectable in the adrenal medulla. AT(1)R mRNA decreased 4-fold between 105 days and 120 days, whereas AT(1)R mRNA levels remained relatively constant between 120 days and the newborn period. In contrast, both in the adult sheep and in the fetal sheep from as early as 90 days gestation, intense CYP11B-ir was consistently detected throughout the adrenal cortex and in steroidogenic cells that surround the central adrenal vein. In conclusion, we speculate that the presence of AT(1)R in the zona fasciculata, and the higher levels of expression of AT(1)R at around 100 days gestation, may suggest that suppression of CYP17 is mediated via AT(1)R at this time. The abundant expression of AT(1)R-ir and CYP11B-ir in the zona glomerulosa of the fetal sheep adrenal gland would also suggest that lack of angiotensin II stimulation of aldosterone secretion is not due to an absence of AT(1)R or CYP11B in the zona glomerulosa.     

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.     

Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.     

Effects of the hypocholesterolemic drug, 4-aminopyrazolo (3,4-d) pyrimidine (4-APP), on the hamster adrenal cortex. An ultrastructural and functional study.

The aim of this study was to gain insight into the effects of 4-aminopyrazolo(3,4-d)pyrimidine (4-APP), a hypocholesterolemic drug, on the adrenal cortex of the hamster, representing an animal species in which steroidogenesis primarily relies on utilization of cholesterol synthesized de novo in the gland. 4-APP administration (1.5 mg/animal day for 3 days) to intact or dexamethasone-suppressed hamsters resulted in a marked proliferation of adrenocortical cells. However, the volume of parenchymal cells was unchanged in intact animals and lowered in the zona glomerulosa (ZG) and zona reticularis (ZR) of dexamethasone-administered hamsters. In both groups of animals, 4-APP strikingly increased the volume of the lipid-droplet compartment and markedly reduced the surface area of smooth endoplasmic reticulum in ZF cells, without significantly affecting the volume of the mitochondrial compartment and the surface area of mitochondrial cristae. These morphologic changes displayed no evident correlation with adrenal cortisol content and secretion. Since most of the 4-APP-induced changes were not prevented by dexamethasone, it seems legitimate to suggest that they could mainly depend on a direct effect of 4-APP on the hamster adrenocortical cells.     

CYTOLOGICAL ALTERATIONS RELATED TO STIMULATION OF THE ZONA GLOMERULOSA OF THE ADRENAL GLAND

The structure of the zona glomerulosa of the rat adrenal gland stimulated by sodium restriction has been studied by light and electron microscopy. The major changes observed during the course of the experiment in stimulated glands involve cytoplasmic droplets, mitochondria, and the endoplasmic reticulum. There is a progressive decrease in the number of cytoplasmic droplets of low electron opacity. Numerous, greatly elongated mitochondria containing parallel arrays of tubules are noted. These tubules extend from within the mitochondria through gaps in the mitochondrial-limiting membranes into the cytoplasm. In addition, amorphous intramitochondrial deposits, possibly aldosterone precursors, are seen. Increased amounts of smooth-surfaced endoplasmic reticulum, often showing complex arrangements, are another feature of the stimulated zona glomerulosa. Other alterations include the presence of large numbers of dense bodies as well as cytoplasmic droplets of high electron opacity. These observations are discussed in relation to the biosynthesis of aldosterone.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.