Gap junction proteins and cell-cell communication in the three functional zones of the adrenal gland

Mouse and monkey adrenal glands were used to study the relationships between gap junction protein expression, intercellular communication and adrenal zonation. Dye communication patterns were determined by incubating freshly excised and hemisected adrenal glands in Lucifer yellow, a gap junction permeable fluorescent dye. Immunohistochemical techniques were used to localize adrenal gap junction proteins. The combination of these two techniques permitted the correlation of gap junction proteins with dye transfer and hormone responses in specialized regions of the adrenal cortex. Lucifer yellow dye communication was most pronounced in the inner glucocorticoid/androgen-producing regions (zona fasciculata/zona reticularis), but was virtually absent in the outer mainly mineralocorticoid-producing region (zona glomerulosa). This pattern of dye communication was coincident with immunohistochemical localization of the gap junction protein, alpha(1)Cx43. The variations in communication and alpha(1)Cx43 expression within the adrenal cortex are thought to be relevant to normal physiological regulation of the adrenal gland.     

Bimodal inhibition of connexin 43 gap junctions decreases ACTH-induced steroidogenesis and increases bovine adrenal cell population growth

In order to elucidate the role of gap junctions in adrenal cell responses, we measured the effect of inhibiting gap junctions with 18-alpha glycerrhetinic acid (GA; a potent inhibitor of cell-cell communication) and connexin antisense transfection on cell proliferation and adrenocorticotropin (ACTH)-stimulated steroidogenesis. In these experiments we utilized a bovine adrenocortical cell (SBAC) population, which responds to ACTH treatment with a dose-dependent increase in steroid production, an increase in connexin 43 (alpha(1)-Cx43) gap junction protein concentrations, and a decrease in cell population growth. SBAC cell populations treated with GA had increased growth rates, decreased ACTH-stimulated steroidogenesis, but no reduction in alpha(1)-Cx43 gap junction protein contents. In contrast, when SBAC cells were transfected with alpha(1)-Cx43 antisense cDNA, gap junction protein concentration was dramatically reduced as expected, unlike the GA-treated cell populations. Cell populations transfected with alpha(1)-Cx43-antisense also exhibited increased growth rates and a decreased steroidogenic response to ACTH treatment as compared with control or vector-only transfected cell populations. The decreased responsiveness and increased number of cells in the population after gap junction function was decreased by either GA treatment or antisense transfection, suggests that gap junctions may be necessary factors in ACTH-stimulated responsiveness and growth control in the adrenal gland.     

Alpha1 connexin 43 gap junctions are decreased in human adrenocortical tumors

Gap junctional communication disorders have been implicated in the etiology of benign and malignant tumors. Understanding the type, distribution, and frequency of gap junctions in adrenal disorders should provide insight into the role of gap junctions in adrenal carcinogenesis as well as information that may be useful in developing improved diagnosis and treatment of adrenal diseases. Using immunocytochemical techniques, we have characterized and compared alpha1 connexins 43 gap junction protein levels in normal adrenal glands to those in benign and malignant adrenocortical human tumors. In addition, gap junction protein levels were studied in a human adrenal cancer cell line (H295). In both normal and neoplastic adrenal tissues, only alpha1 connexin 43 could be detected, whereas beta1 connexin 32 and beta2 connexin 26 were not found. In the normal adrenal gland, the zona fasciculata was demonstrated to have the highest number of gap junctions per cell (mean +/- SEM, 13.78 +/- 1.93). In contrast, in benign adrenocortical adenomas, the number of gap junctions per cell compared to that detected in normal adrenal glands was significantly reduced (mean +/- SEM, 4.6 +/- 1.17; P < or = 0.05), and the lowest number was found in malignant adrenocortical tumors (1.42 +/- 0.58; P < or = 0.05). Similarly, there were few or no alpha1 connexin 43 gap junctions in the H295 population. There was a progressive decrease in gap junction plaques in adrenocortical cancer cell populations compared to those in normal cell populations. Therefore, analysis of gap junction protein may be helpful for the differential diagnosis of benign and malignant adrenal tumors. The induction of gap junctions in malignant cells may provide a novel therapeutic strategy for adrenal cancer.     

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.     

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

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

Adrenocorticotropin increases interleukin-6 release from rat adrenal zona glomerulosa cells.

Interleukin-6 (IL-6) is produced by adrenal zona glomerulosa cells; its release is stimulated by several secretagogues, including IL-1 alpha, IL-1 beta, and angiotensin II. The present study reports that ACTH (0.1-100 nM) increased the release of IL-6 from primary cultures of rat adrenal cells in a concentration-dependent manner. This increase was accompanied by an increase in cAMP content in cell extracts and in the incubation medium. The dynamics of IL-6 release from the adrenal cells also were investigated using a perifusion system; approximately 50 min were required for the effects of IL-1 alpha, IL-1 beta, and ACTH on IL-6 release to become apparent. Following withdrawal of the secretagogues, IL-6 release returned to basal levels within 90-120 min. In some experiments, the adrenal zona glomerulosa was separated from the zona fasciculata/reticularis to determine the origin of secretagogue-stimulated IL-6 release. PGE2 and forskolin increased IL-6 release from both cell types, but maximal release from zona glomerulosa cells was more than 10-fold greater than that from zona fasciculata/reticularis cells. ACTH (0.1-100 nM) increased intracellular cAMP levels in cells from both cell types in a concentration-dependent manner, but increased IL-6 release only from zona glomerulosa cells. Dexamethasone, an inhibitor of IL-6 production in several tissues, had no effect on either basal or stimulated IL-6 production in the adrenal. Because IL-1 beta is produced primarily by tissues of the immune system, whereas ACTH is a classical endocrine hormone, we investigated the effect of interaction of these proteins on IL-6 release from the adrenal. Together, IL-1 beta and ACTH stimulation of IL-6 release was greater than the sum of the effects of each substance separately; however, IL-1 beta did not potentiate the effect of ACTH on cAMP levels. Similarly, IL-1 beta potentiated IL-6 release stimulated by forskolin and (Bu)2cAMP. Thus, the adrenal may be an important convergence point between the immune and endocrine systems, and because IL-6 release is regulated by IL-1 alpha, IL-1 beta, ACTH, and angiotensin II, and this cytokine stimulates corticosterone release, IL-6 may play an important paracrine role in integrating the signals derived from these systems.     

Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa cell preparations.

The actions of forskolin have been investigated to determine to what extent its effects on steroidogenesis in rat adrenal preparations are dependent on activation of adenylate cyclase. In zona glomerulosa preparations, stimulation of both aldosterone and corticosterone production was obtained at concentrations of forskolin between 1 and 10 mumol/l. The effects of 10 mumol forskolin/l were additive with those of low doses (1 pmol/l) of corticotrophin (ACTH), but not with those of high doses (1 nmol/l) of ACTH. In contrast, in zona fasciculata/reticularis cells, doses of forskolin up to 10 mumol/l produced no significant stimulation of corticosterone production either alone or in the presence of ACTH (1 pmol/l and 1 nmol/l). The response to 1 nmol ACTH/l was attenuated in the presence of forskolin (10 mumol/l) in both zona glomerulosa and zona fasciculata/reticularis cell preparations. Cyclic AMP production increased progressively with dose up to 100 mumol forskolin/l in zona glomerulosa cells, whereas corticosterone production was maximal between 10 and 30 mumol forskolin/l and decreased at 100 mumol forskolin/l. In zona fasciculata/reticularis cells, cyclic AMP production was also increased by forskolin (1 and 10 mumol/l). The stimulation of zona glomerulosa steroidogenesis by forskolin (1-10 mumol/l) and ACTH (1-100 pmol/l) were both reduced by the adenylate cyclase inhibitor, N6-phenylisopropyladenosine (100 mumol/l). The calcium channel inhibitor, nifedipine, only reduced the steroidogenic response to forskolin (3 mumol/l) at doses of 300 mumol/l whereas the response to 8.4 mmol K+/l was inhibited at 10 mumol nifedipine/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine adrenal cells secrete interleukin-6 and tumor necrosis factor in vitro

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted and/or synthesized by the rat and human adrenal cortex. In this study, the release of IL-6 and TNF from bovine adrenal cells was determined. Bovine adrenal glands were collected from an abattoir and dissected into the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR), and medulla. The tissues were enzymatically dispersed to single cells and cultured for 4-6 days. The cells were then exposed (4 h) to angiotensin II (AII), adrenocorticotrophic hormone (ACTH), phorbol dibutyrate (PDB), interleukin-1beta (IL-1beta), interleukin-1alpha (IL-1alpha), and endotoxin (LPS). The IL-6 and TNF content of the incubation medium was determined by bioassays. The release of IL-6 and TNF from the ZG, ZF, ZR, and medulla was increased by PDB, IL-1alpha, IL-1beta, and LPS. In contrast, ACTH and AII increased IL-6 release from the ZG, ZF, and ZR but had no effect on IL-6 release from the medulla. ACTH decreased TNF release from all adrenal cortical zones but had no effect on TNF release from the medulla. Immunohistochemistry utilizing antibodies against TNFalpha demonstrated TNFalpha-containing cells throughout the adrenal gland. The majority of the cells of the ZG, ZF, and ZR contained TNFalpha. However, the cells of the ZG contained more TNFalpha than the cells of the ZR or ZF. Small patches of TNFalpha-containing cells were also found in the adrenal medulla and capsule. These findings support the hypothesis that IL-6 and TNF may have autocrine/paracrine effects on the adrenal gland.     

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

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

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocytosis of connexin protein in adrenal cells

The ability of adrenocorticotropin (ACTH) to affect gap junctions was examined in adrenal cells in vivo and in vitro. Treatment with ACTH increased the size and number of gap junction plaques on the cell membranes in hypophysectomized animals and in adrenal culture. Intracellular (cytoplasmic) annular gap junctions were observed in cells of the inner adrenal cortical zones and in adrenal cell cultures. To investigate the relationship of annular gap junctions to surface junctions, adrenal cells in culture were transfected with cDNA encoding a green fluorescent protein tagged connexin 43 construct (Cx43-GFP), and subsequently studied by time-lapse video microscopy, immunocytochemistry, and transmission electron microscopy (TEM). Internalization of part or all of a surface gap junction plaque resulted in annular gap junction formation. These studies support the hypothesis that cytoplasmic vesicles, initially described with TEM methods, can result from removal of gap junction plaques from the cell surface. It is suggested that hormones can play a regulatory role in cell-cell communication by influencing the availability of gap junction protein at the cell surface and that hormonally-sensitive processes might serve as a means of altering intercellular communication.     

Distribution of extracellular signal-regulated protein kinases 1 and 2 in the rat adrenal and their activation by angiotensin II

The adrenal gland of the rat is continuously regenerated through proliferation of a stem cell population in the outer part of the gland. To clarify the location of proliferative events within the adrenal gland, and the factors that stimulate them, rat adrenal capsule preparations, consisting of capsule, zona glomerulosa (ZG) and the outer zona fasciculata (ZF) were maintained in vitro under different conditions of stimulation, for varying periods. Sites of proliferation were identified by 5-bromo-2'-deoxy-uridine (BrdU) staining, and the distribution of classical MAP kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) 1 and 2, immunoreactivity was determined using immunocytochemistry. BrdU staining was limited to the outer glomerulosa and the capsule, where it was enhanced by angiotensin II, but not by a high potassium ion concentration nor by ACTH. In contrast, ERK1/2 immunoreactivity was distributed throughout the ZG and in the medulla, with none detectable in the ZF and reticularis. Furthermore, angiotensin II, potassium ions and ACTH were all shown to induce ERK1 and ERK2 phosphorylation in the ZG. Treatment of adrenal capsule tissue with the specific MAPK kinase inhibitor PD98059 revealed inhibition of ERK1/2 phosphorylation, but no effect on angiotensin II-induced aldosterone secretion. Although the distribution and activation of the MAPK pathway suggest a link with proliferation, the findings clearly designated only the outer part of the glomerulosa and capsule as a potential stem cell population. Further functions should be sought for the apparently silent major part of the glomerulosa.     

Lipoprotein requirements for secretion of ultraviolet-absorbing corticosteroids by guinea pig adrenocortical cells in vitro: inner versus outer cortices; zona glomerulosa versus zona fasciculata

Most studies of lipoprotein requirements for steroid secretion by the adrenal have examined the mixed cell population of the whole gland; none have examined lipoprotein requirements of guinea pig adrenocortical cells. In this study the effect of exogenous lipoprotein on the ability of cells from each of the different regions of the guinea pig adrenal cortex to synthesize and secrete steroids has been analyzed in vitro, under baseline and ACTH-stimulated conditions. Most studies have assessed the effects of lipoprotein on one or a few selected steroids. In this study the effects of lipoprotein and ACTH were examined both by an assay for fluorogenic steroids and by HPLC analysis of the spectrum of UV-absorbing steroids. Guinea pig outer adrenocortices, containing zona glomerulosa and zona fasciculata, maintained in vitro as fragments or as isolated cells, secreted at least 5 times more steroid than the inner cortex, predominantly zona reticularis, and were dependent upon lipoproteins for their secretion. Corticosterone (B) and cortisol (F) were the predominant products of both zones. Aldosterone (Aldo), 18-hydroxycorticosterone (18-OH B), deoxycorticosterone (DOC), 11 beta-hydroxyandrostenedione (11 beta-OH And), androstenedione (And), and deoxycortisol were less abundant products of the outer cortex, while the inner cortex secreted only very small amounts of these steroids. Each of the outer cortical cell types secreted a distinct spectrum of steroids. Aldo, 18-OH B, and DOC were characteristic of glomerulosa cells, but B was most prominent. Fasciculata cells secreted primarily F, with 11 beta-OH And as their next most prominent product. Low density lipoprotein (LDL) enhanced steroid secretion by glomerulosa cells to a greater extent than that by fasciculata cells, but the stimulation of LDL utilization by ACTH was greatest for fasciculata cells. LDL and ACTH also influenced the pattern of steroids secreted by each cell type. Addition of LDL to glomerulosa cells enhanced secretion of DOC and B, but not that of Aldo or 18-OH B. In fasciculata cell cultures, LDL enhanced secretion of both F and 11 beta-OH And. ACTH, particularly in the presence of LDL, stimulated secretion by glomerulosa cells of Aldo and 18-OH B, as well as that of F, And, and 11 beta-OH And. The combined presence of ACTH and LDL in fasciculata cell cultures preferentially stimulated secretion of F and B.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

The actions of N-terminal fragments of corticotrophin on steroidogenesis in dispersed rat adrenal cells in vitro

The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by alpha-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that alpha-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer teh specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5-24), ACTH(1-12), ACTH(1-14), ACTH(1-15), ACTH(1-16) and ACTH(1-17). Their actions were compared with those of alpha-MSH and ACTH(1-24). All of the ACTH-derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of alpha-MSH; minimum effective concentration was 10 nmol/l. Also, like alpha-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5-24), ACTH(1-16) and ACTH(1-17) stimulated fasciculata/reticularis cells at concentrations up to 1 mumol/1. The actions of all of the shorter peptides were thus unlike those of ACTH(1-24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production. The data suggest that the 18-24 region of the ACTH molecule contains the signal for a fasciculata/reticularis response, and the region 1-13 that for glomerulosa specificity. They confirm the view that, in the rat, alpha-MSH itself may be the specific pituitary glomerulosa-stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1-17) analogues should be used with caution.     

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.     

Biosynthetic ad morphological evidence for inhibition of aldosterone production following administration of ACTH to sheep

The effect of ACTH administration for 1-5 days on the morphology and steroidogenic capability of sheep adrenal tissue has been examined. During this period of treatment there was a gradual decline in the in vitro conversion of 3H-labelled precursors to products of solely zona glomerulosa origin (aldosterone and 18-hydroxycorticosterone) while conversion to products of zona fasciculata origin (17-hydroxyprogesterone, 11-deoxycortisol and cortisol) was stimulated throughout. Conversion to DOC, 18-hydroxydeoxycorticosterone and corticosterone (steroids produced by both the zona glomerulosa and the zona fasciculata) declined after initial stimulation. Within 2--3 days of the commencement of treatment, the zona glomerulosa showed a progressive decrease in cell number associated with disruption of cords and cell separation. Ultrastructurally, it was found that typical zona glomerulosa cells had almost disappeared. The majority of residual cells in this area had a structure intermediate between zona glomerulosa and zona fasciculata cells. The similarity in time-course of the alterations in both the morphological and biosynthetic characteristics suggests that the decline in aldosterone output caused by ACTH administration to sheep results from the loss of adrenal zona glomerulosa cells, predominantly due to selective cellular degeneration.     

Seladin-1 expression in rat adrenal gland: effect of adrenocorticotropic hormone treatment

Seladin-1 (KIAA0018) gene is the seventh most highlyexpressed gene in the adult adrenal gland, along with genes coding for steroidogenic enzymes. The aim of the present study was to investigate the localization of the Seladin-1 protein in control and ACTH-treated rat adrenal glands and to verify whether Seladin-1 is involved in secretion. Immunofluorescence studies revealed that Seladin-1 was localized principally in the zona fasciculata, cytoplasm, and nucleus. Expression of Seladin-1 was increased by ACTH treatment, in vivo and in culture conditions. Subcellular fractionation offasciculata cells showed that Seladin-1 was mainly present in the nucleus, membrane, and cytoskeleton fractions and, to a lesser extent, in the cytosol. ACTH treatment decreased Seladin-1 expression in the cytosol, with a concomitant increase in the nuclear fraction. In the glomerulosa and fasciculata cells in culture, ACTH induced a relocalization of Seladin-1 into specific nuclear regions. This ACTH-induced relocalization was abrogated by the pre-treatment of cells with 75 nM U18666A (an inhibitor of Seladin-1). In addition, fasciculata cells exhibited an increase in the basal level of steroid secretion when cultured in the presence of U18666A (25 and 75 nM), although ACTH-induced secretion was decreased. In summary, the present study demonstrates that the protein expression of Seladin-1 is more abundant in fasciculata cells than in glomerulosa cells and that the ACTH treatment increases both expression and nuclear localization of the protein. Results also suggest that depending on its cellular localization, the Delta24-reductase activity of Seladin-1 may play a major role in steroid secretion in the adrenal gland.     

Properties of rat adrenal zona reticularis cells: production and stimulation of certain steroids.

The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay. Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone : corticosterone ratio when compared with zona fasciculata cells. Adrencorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output. In every cell pool, ACTH stimulated the output corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 beta-hydroxylation. Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available. Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells. The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.