Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

Immunohistochemical studies on electron transport proteins associated with cythochromes P-450 in steroidogenic tissues II. Microsomal NADPH-cytochrome c reductase in the rat adrenal

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum procued against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase techniques and an indirect fluorecent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Immunohistochemical studies on electron transport proteins associated with cytochromes P-450 in steroidogenic tissues. II. Microsomal NADPH-cytochrome c reductase in the rat adrenal.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum produced against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase technique and an indirect fluorescent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found to differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Zonal distribution of cytochromes P-450 and related enzymes of bovine adrenal cortex--quantitative assay of concentrations and total contents

The zona glomerulosa, zona fasciculata, zona reticularis, and medulla were separated from bovine adrenal glands and cytochromes P-450 and related enzymes in each zone were investigated immunochemically by Western blotting using antisera from chickens or rabbits against cytochromes P-450scc, P-450(11)beta, P-450s21, and b5, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase, NADPH-adrenodoxin reductase, and adrenodoxin. Concentrations of cytochrome P-450(11)beta, NADPH-cytochrome P-450 reductase, and cytochrome b5 per milligram of protein of homogenate were higher in the zona glomerulosa than in the other zones; the levels of the other components were higher in the zona fasciculata. The total enzyme content of all components was the highest in the zona fasciculata. The amount of adrenodoxin was about 10 times that of NADPH-adrenodoxin reductase in each zone.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

18-Hydroxy-11-deoxycortisol: a new steroid isolated from incubations of the adrenal with 11-deoxycortisol

Cortisol has been shown to be metabolized in the zona glomerulosa of the adrenal gland through the same pathway involving the cytochrome P-450, corticosterone methyl oxidase by which corticosterone is transformed to 18-hydroxycorticosterone and aldosterone. When cortisol is the precursor, 18-hydroxycortisol and 18-oxocortisol are formed. 18-Hydroxycortisol can also be made at a similar rate in the bovine zona fasciculata and reticularis as in the zona glomerulosa. We studied the possibility that the formation of 18-hydroxycortisol in the zona fasciculata and reticularis might be through a different pathway involving initial 18-hydroxylation of 11-deoxycortisol before 11 beta-hydroxylation. Rat adrenal capsules or cores were incubated with 10 micrograms of cortisol or 11-deoxycortisol and the formation of 18-hydroxycortisol was measured by radioimmunoassay. Both capsules and cores transformed 11-deoxycortisol to 18-hydroxycortisol, but cortisol was only transformed in the capsular portion. Sixty-two rat adrenals were incubated with 10 mg of 11-deoxycortisol and the putative steroid, 18-hydroxy-11-deoxycortisol, was purified by TLC and HPLC and subjected to gas chromatography mass spectrometry. The mass spectra indicated that the steroid isolated was indeed 18-hydroxy-11-deoxycortisol. The function of this steroid is still unknown.     

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.     

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.     

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.     

Carbonic anhydrase isoenzymes II and I are present in the zona glomerulosa cells of the human adrenal gland

Human carbonic anhydrase isoenzymes I and II (HCA I and II) were purified from human erythrocytes by inhibitor affinity chromatography and ion-exchange chromatography. These isoenzymes were then located in the human adrenal gland using specific polyclonal antisera raised in rabbits and specific detection by immunohistochemical techniques. Both HCA II and I were located in the zona glomerulosa cells, although the staining for HCA I was faint. The cells of the zona fasciculata and the zona reticularis failed to stain with either antiserum. Control stainings with preimmune or anti-HCA VI sera were negative. The presence of HCA II and I in the zona glomerulosa cells may be linked to regulation of the biosynthesis or secretion of mineralocorticoids.     

Characterization of rat capsular adrenal (zona glomerulosa) (Na,K)-ATPase activity

A higher (Na,K)-ATPase activity was found in rat capsular (zona glomerulosa) than in decapsulated (zona fasciculata) adrenal. No unusual characteristics of this enzyme were found in the simulation with Na+ and K+ and the inhibition with ouabain. Angiotensin II, ACTH and serotonin, all potent stimulators of aldosterone biosynthesis, did not affect the enzyme. In conclusion, the characteristics of rat capsular adrenal (Na,K)-ATPase are identical to the classical enzyme. It is not the direct receptor or effector for stimulators of aldosterone biosynthesis but a supportive role in mediating the regulatory signal cannot be ruled out.     

Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa

Chronic administration of somatostatin induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma aldosterone concentration. Zona fasciculata cells did not display any significant change and corticosterone plasma concentration was not significantly affected. These findings are interpreted to indicate that somatostatin exerts a direct inhibitory effect on the growth and steroidogenic capacity of rat adrenal zona glomerulosa.     

Toxic effect of heavy metals on cells isolated from the rat adrenal and testis

Summary Heavy metals including mercury, cadmium, cobalt, and copper (100 μM) exerted an adverse effect on the viability of isolated rat adrenal capsular (zona glomerulosa), adrenal decapsular (fasciculata and reticularis), and Leydig cells of the testis with mercury being the most potent. Due to the decreased cell viability there was a parallel reduction in corticotropin-stimulated, corticosterone production by adrenal decapsular cells and luteinizing hormone-stimulated testosterone production by Leydig cells. The results indicated a direct toxic action of these heavy metals on steroid-producing cell in the adrenal gland and the tectis. Other metals tested, including lead, zinc, aluminum, chromium, iron, nickel, and lithium, did not exert any deleterious effect on cell viability or hormone-induced steroidogenesis, in adrenal and Leydig cells when tested up to a concentration of 100 μM.     

Effect of calcium(ion) uptake by rat adrenal mitochondrial on pregnenolone formation and spectral properties of cytochrome P-450

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenalgglands. In the presence of succinate, addition of calcium chloride in the concentration range 20–150 μM caused an inhibition of pregnenolone formation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP⁺. Under these conditions, calcium chloride in the concentration range 120–875 μM caused a 2–4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone.The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium the interaction of mitochondrial cytochrome P-450 with endogenous substrate.     

Differential regulation of apolipoprotein-E messenger RNA in zona fasciculata cells of rat adrenal gland determined by in situ hybridization.

Previous studies showed that apolipoprotein-E (apoE) mRNA is regulated in rat adrenal gland by treatments that alter adrenal gland cholesterol content and steroidogenesis. In the present study cell types expressing apoE mRNA were determined by in situ hybridizations using an [alpha-35S]UTP-labeled RNA probe. Autoradiographic grains were counted to compare apoE expression in adrenal glands from control and experimentally treated animals. In control adrenal gland, zona (z.) fasciculata and z. reticularis exhibited the highest level of apoE mRNA expression, with lower levels in z. glomerulosa and medulla. Dexamethasone (DEX) treatment selectively increased apoE mRNA 3-fold in outer z. fasciculata, but not in other adrenal zones. ApoE mRNA expression appeared to be lower in adrenal glands from 4-aminopyrazolopyrimidine-treated rats, in that differences among adrenal gland zones were abolished. DEX treatment increased adrenal gland cholesteryl ester and oil red O staining in z. fasciculata cells in which the apoE mRNA concentration was increased as well as in other cortical cells in which apoE mRNA was unchanged. Aminoglutethimide administration led to a large increase in oil red O staining throughout the cortex, including z. fasciculata, without affecting apoE mRNA expression. These data suggest that adrenal gland apoE mRNA expression is not closely coupled to cellular cholesterol concentrations. Increased apoE mRNA expression in z. fasciculata of DEX-treated animals suggests an inverse relationship between apoE mRNA concentration and the level of steroidogenesis. This result is consistent with the proposal that apoE may play a role in regulating the utilization of cholesterol for steroid production.     

Age-related differences in skeletal muscle protein synthesis: relation to markers of immune activation

A quantitative analysis of zone-specific proliferation was done to determine the recovery of adrenal cortical zonation during regeneration after enucleation. Adult male rats underwent adrenal enucleation [unilateral enucleation (ULE)] or sham surgery, both accompanied by contralateral adrenalectomy. At 2, 5, 10, and 28 days, blood and adrenals were collected to assess functional recovery. Adrenal sections were immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P-450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P-45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Recovery of fasciculata secretory function occurred by 28 days as reflected by plasma ACTH and corticosterone, whereas glomerulosa function reflected by plasma aldosterone remained low at 28 days. At 5 days, ULE adrenals showed increased Ki67+ cells in the glomerulosa and inner fasciculata, whereas at 10 and 28 days increased proliferation was restricted to the outer fasciculata. These data show that enucleation results in transient elevations in glomerulosa and inner fasciculata cell proliferation followed by a delayed increase in the outer fasciculata. To assess adrenal growth in enucleated adrenals previously suppressed by the presence of an intact adrenal, rats underwent ULE and sham surgery; after 4 wk, the intact adrenal was removed and enucleated adrenals were collected at 2, 5, and 10 days. Overall, proliferation was delayed in this model, but at 5 days, Ki67+ cells increased in the outer fasciculata, whereas by 10 days, increased proliferation occurred in the outer and inner fasciculata. The key novel finding of increased proliferation in the inner fasciculata suggests that the delayed growth of the enucleated adrenal results in part from a regenerative response.     

Autoradiographic localization of vitamin A in the adrenal gland of rats.

Cellular distribution of vitamin A in the rat adrenal was evaluated by autoradiography. Vitamin A was concentrated in the lipid droplets of epithelial cells of the zona fasciculata and zona reticularis. A small amount of vitamin A was also present in the cytoplasm and nucleus of these cells. The zona glomerulosa contained very little vitamin A either in lipid droplets or in the remainder of the cell. The medulla had essentially no vitamin A. Such strong cellular specificity for uptake of vitamin A by the cells of the zona fasciculata and zona reticularis supports the hypothesis that vitamin A is involved in synthesis of glucocorticoid hormones.