Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

Summary The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-(1–24) ACTH or anti (17–39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold.The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.T. Garcia-Caballero has a Postdoctoral Fellowship from Xunta de Galicia.     

Investigations on the effects of long-term administration of a methionine-enkephalin analogue on the adrenal zona fasciculata of rats treated with dexamethasone or dexamethasone and ACTH

Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose-dependent increase in the volume of zona fasciculata cells of dexamethasone-administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone-suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata.     

Effects of a prolonged treatment with aminoglutethimide on the zona fasciculata of rat adrenal cortex: A morphometric investigation

Summary The effects of a 7-day administration of aminoglutethimide (AG) on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. There was a 70–74% decrease in the concentration of corticosterone in blood, but no conspicuous qualitative changes suggesting cell degeneration occurred. Morphometry showed that AG induced a significant hypertrophy of the zona fasciculata and its parenchymal cells only in normal animals, which was due to an increase in the volume of the mitochondrial compartment and to proliferation of the smooth endoplasmic reticulum. This response to AG was considered to be non-specific and mediated by the enhanced secretion of ACTH following the decrease in the blood level of corticosterone. AG administration significantly increased the volume of the lipid-droplet compartment and the number of intramitochondrial lipid-like inclusions in both groups of animals. These changes were interpreted as the morphological counterpart of the AG-induced block of cholesterol utilization in steroid synthesis.     

Effects of streptozotocin-induced experimental diabetes on the morphology and function of the zona fasciculata of rat adrenal cortex

The effects of a severe streptozotocin (STZ)-induced diabetes on the morphology and function of the adrenal zona fasciculata were examined in rats with intact or pharmacologically interrupted hypothalamic-hypophyseal-adrenal axis. In animals with an intact hypothalamic-hypophyseal axis, STZ-diabetes induced hypertrophy of the cells of the zona fasciculata and a rise in the plasma corticosterone concentration. Conversely, in rats in which the hypothalamic-hypophyseal axis had been interrupted, experimental diabetes provoked atrophy of the zona fasciculata cells, and a lowering in the plasma corticosterone level. The effects of STZ-diabetes were completely reversed by insulin infusion in both groups of rats. The hypothesis is discussed that the chronic lack of insulin may directly inhibit the growth and steroidogenic capacity of the rat zona fasciculata and that this effect of experimental diabetes may be masked in rats with an intact hypothalamic-hypophyseal axis by the concurrent enhancement of ACTH release due to chronic stress resulting from the metabolic consequences of prolonged diabetes.     

Low-density lipoprotein receptor activity in the guinea pig adrenal cortex. II. Zonal response to aminoglutethimide and 17 alpha-ethinylestradiol

Low-density lipoprotein (LDL) receptor activity and the concentration of cholesterol were measured in the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig to examine the relation between cholesterol content and LDL receptor activity. While the concentration of cholesterol was 2-3-times higher in the outer cortical zone, the maximum high-affinity binding capacity for LDL was essentially the same for the two zones, or slightly higher for the inner zone. Adrenocorticotrophic hormone (ACTH) caused a significant increase in LDL receptor activity only in the outer zone, but led to a reduction in the cholesterol content in both adrenocortical zones. The treatment of animals with 17 alpha-ethinyl-estradiol also resulted in a reduction of cholesterol in both adrenocortical zones, but an increase in LDL receptor number only in the outer zone. The latter effect was partially reversed by the administration of dexamethasone. Aminoglutethimide, which was used in a dose that did not block steroidogenesis but did block the hydrolysis of cholesteryl esters in response to ACTH, did not prevent the ACTH-induced increase in LDL receptor number in the outer zone. Thus, the number of LDL receptors was increased in the zona fasciculata by ACTH in the absence of a reduction in cellular cholesterol content, while the number of LDL receptors in the zona reticularis was not increased by ACTH even in the face of a reduction in cellular cholesterol. Exclusive of the experiments employing aminoglutethimide, when the cellular cholesterol content was plotted against LDL binding activity, an excellent inverse correlation was revealed for the zona fasciculata, but essentially no correlation was noted for the zona reticularis. It is concluded that the outer and inner cortical zones of the guinea pig adrenal are quite distinct in the nature of their LDL receptor activity and regulation: the LDL receptor of the outer zone appears to function in a way similar to what has been reported for the whole adrenal cortex of other species in that receptor number correlates with tissue cholesterol content and is primarily regulated by ACTH; the LDL receptor number of the inner zone, however, does not correlate with tissue cholesterol content and is apparently not regulated by ACTH.     

Ultrastructural localization of mercury in adrenals from rats exposed to methyl mercury

Accumulations of mercury have been demonstrated in adrenal glands by light and electron microscopy with a highly sensitive histochemical technique. Rats were exposed to methyl mercury in drinking water (20 mg/l) for 7-180 days, or were given intraperitoneal injections of methyl mercury (daily dose 100 or 200 micrograms). The amount and location of the mercury deposits were dependent upon the exposure time, the method of administration and the amount administered. In rats exposed to methyl mercury in drinking water, accumulations were often observed in both the zona glomerulosa and reticularis. They appeared first in the zona glomerulosa of animals treated for 1 week. In the zona fasciculata, deposits were observed only in the animals treated for 50 to 180 days. In animals treated for 180 days the cytoplasm of the cells in the zona fasciculata was heavily vacuolated and distinct necrotic cells were observed in other cortical zones. In the chromaffin cells, a slight increase in the amount of deposits was observed with increasing exposure time. Both epinephrenic and norepinephrenic cells contained deposits. Only a few deposits were observed in the cortical and chromaffin cells of animals treated with intraperitoneal injections. Ultrastructural deposits were observed in the lysosomes of cortical cells and in both lysosomes and secretory granules of chromaffin cells.     

Effect of acute heat stress on rat adrenal cortex — a morphological and ultrastructural study

The stereological structure of rat adrenal gland was analysed by light and electron microscopy after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions a significant increase in plasma corticotrophin (ACTH), serum corticosterone and aldosterone levels were observed. Histological and stereological investigation at light microscopy showed significant decrease in volume density of capsule and zona glomerulosa, increase in volume of fasciculata cells, and decrease of numerical density of zona fasciculata cells and mean diameter of blood vessels. At the ultrastructural level, volume density of nuclei and mitochondria of zona glomerulosa cells were significantly increased and that of lipid droplets decreased. Volume density of mitochondria of fasciculata cells was significantly increased, while number of lipid droplets per μm² of cell was reduced. In the cells of zona reticularis significant increase in the number of lipid droplets was found. The response of zona glomerulosa may be interpreted as immediate reaction to dehydration, while alterations detected in zona fasciculata, which were less extensive, were related to purely stressogenic effects of high ambiental temperature.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.     

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.     

Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.     

Effect of long-term inhibition of hydroxy-methylglutaryl coenzyme A reductase by mevinolin on the zona fasciculata of rat adrenal cortex. A combined morphometric and biochemical study

The effects of a 7-day infusion with mevinolin, a potent competitive inhibitor of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase, on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. In both groups of animals, the drug caused a lowering in plasma and intra-adrenal cholesterol concentrations, as well as a slight decrease in the blood level of corticosterone. Morphometry of zona fasciculata cells showed that specific mevinolin-induced changes (i.e. those occurring in both groups of rats and therefore not due to enhanced release of ACTH following decrease in circulating corticosterone) are severe lipid-droplet depletion and a conspicuous increase in smooth endoplasmic reticulum (SER) and peroxisomes. The hypothesis is discussed that these morphological changes express a compensatory response of zona fasciculata cells to counteract the mevinolin-induced inhibition of cholesterol synthesis in both liver and adrenal cortex.     

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.     

Stereological analysis of the guinea pig adrenal: effects of dexamethasone and ACTH treatment with emphasis on the inner cortex

This paper describes the morphological responses of adult male guinea pig adrenals to dexamethasone (DEX) and adrenocorticotrophic hormone (ACTH), in both qualitative and quantitative terms. Most organelles and inclusions are affected, but their responses often vary in the different cell types examined: zona fasciculata externa and interna, and zona reticularis. Following DEX the volume of lipid droplets increases in cells of zona fasciculata externa but decreases in zona reticularis; smooth endoplasmic reticulum decreases in fasciculata externa but increases in reticularis. Following ACTH, exactly the opposite occurs. This strongly suggests differing functions for these subcellular entities in each cell type, particularly for the smooth reticulum, as well as for the cells themselves. The volume of the Golgi complex markedly decreases following DEX in all cells but increases only in zona fasciculata interna and zona reticularis following ACTH. These deeper cortical cells are known to secrete at least one sulfated steroid, dehydroepiandrosterone sulfate, and these changes in the Golgi complex strengthen the suggestion that the Golgi plays a role in sulfation of steroids. Mitochondrial volume and number decrease in all cells following DEX, supporting their role in steroidogenesis. Further decreases in their volume, accompanied by increases in their number following ACTH, may be related to a proliferation of mitochondria in response to ACTH. Changes in peroxisome volume and number, following DEX and ACTH, suggest a possible role for these organelles in steroid cell metabolism. Lysosomes decrease in volume in all cells following ACTH. This does not support the recently suggested concept that they play a role in steroid secretion.     

Ultrastructural Characteristics of Adult Rat Adrenocortical Cells Maintained in vitro With and Without ACTH

Bhattacharyya, T. K., Butler, D. G., Price, C. S. 1980. Ultrastructural characteristics of adult rat adrenocortical cells maintained in vitro with and without ACTH. (Ramsay Wright Zoological Laboratories, Department of Zoology, University of Toronto, and Division of Endocrinology, Toronto Western Hospital, and Department of Medicine, University of Toronto, Canada.) — Acta zool. (Stockh.) 61(1): 9–21. The ultrastructural morphology of adrenocortical cells of adult rats maintained in culture for four days with and without corticotropin (ACTH) was studied in comparison to freshly dissociated cells. Identification of cells belonging to zona glomerulosa, zona fasciculata, and zona reticularis was made on the characteristics of the mitochondria and smooth endoplasmic reticulum (SER), and was confirmed by comparison with cellular layers from intact adrenals. Without ACTH, glomerulosa and fasciculata cells showed disappearance of SER, atrophied Golgi apparatus (GA), and a striking proliferation of granular reticulum. Fascicular cells had a loss in mitochondrial matrix density and the mitochondrial cristae showed a tendency to convert to lamellar glomerulosa-type cristae. Zona reticularis cells were not strikingly altered. Maintenance with ACTH led to increased cell size and islet formation of cortical cells. Glomerulosa cells had normal appearance and fasciculata cells manifested a pronounced development of GA and SER, and a normal configuration of mitochondria. The effects of ACTH on glomerulosa cells suggest a trophic influence of ACTH on these cells in vitro. The alterations observed in fascicular cell mitochondria and SER can be explained in terms of known concepts of steroid biosynthesis and basically agree with the patterns of steroid synthesis observed in these cells previously (Price et al. 1975).     

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

Fine structure of the rabbit adrenal cortex and the effects of short-term ACTH administration

Summary The fine structure of the rabbit adrenal cortex was investigated. The parenchymal cells display the ultrastructural features of steroid-producing cells, and also contain numerous electron-dense bodies frequently located near intercellular canaliculi, which open into the subendothelial space. Short-term ACTH-administration induced a noticeable decrease in the volume of the lipid compartment in the cells of all three cortical zones and a significant increase in the volume of dense bodies in the cells of zona fasciculata and zona reticularis. The hypothesis that these dense bodies are secretory granules is discussed in the light of biochemical evidence showing that ACTH increases the concentration of both corticosterone and cortisol in the decapsulated-enucleated adrenal homogenate and does not affect the activity of two lysosome-marker enzymes (i.e., acid phosphatase and -glucuronidase).     

Studies on cytogenesis in adult rat adrenal cortex: circadian and zonal variations and their modulation by adrenocorticotropic hormone

Circadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4 h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6 h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.