Chromatin Fractal Organization, Textural Patterns, and Circularity of Nuclear Envelope in Adrenal Zona Fasciculata Cells.

Despite previous research efforts in the fields of histology and cell physiology, the relationship between chromatin structural organization and nuclear shape remains unclear. The aim of this research was to test the existence and strength of correlations between mathematical parameters of chromatin microarchitecture and roundness of the nuclear envelope. On a sample of 240 nuclei of adrenal zona fasciculata cells stained using the DNA-specific Feulgen method, we quantified fractal parameters such as fractal dimension and lacunarity, as well as textural parameters such as angular second moment (ASM), entropy, inverse difference moment, contrast, and variance. Circularity of the nuclear envelope was determined from the nuclear area and perimeter. The results indicate that there is a statistically significant negative correlation between chromatin ASM and circularity. Moreover, there was a statistically significant positive correlation between chromatin fractal dimension and envelope circularity. This is the first study to demonstrate these relationships in adrenal tissue, and also one of the first studies to test the connection between circularity and fractal and gray-level co-occurrence matrix parameters in DNA-specific Feulgen stain. The results could be useful both as an addition to the current knowledge on chromatin/nuclear envelope interactions, and for design of future computer-assisted research software for evaluation of nuclear morphology.

Zona fasciculata 21-hydroxysteroids and precursor-to-product ratios in 21-hydroxylase deficiency: further characterization of classic and non-classic patients and heterozygote carriers.

INTRODUCTION: Although much is known about the increased levels of the 21-hydroxylase substrates 17-hydroxyprogesterone (17OHP) and 21-deoxycortisol (21DF) - the biochemical markers of all forms of 21-hydroxylase deficiency (21OHD), only limited information is available on the zona fasciculata (ZF) products distal to the enzymatic block: 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), and corticosterone (B). OBJECTIVE: To investigate whether basal and post-ACTH levels of S, DOC, and B and the 21-hydroxylase precursor-to-product ratios determined by tandem mass spectrometry preceded by high-performance liquid chromatography separation (liquid chromatography-tandem mass spectrometry) could disclose distinct profiles in genotypically confirmed classic (no.=14) and non-classic (NC) (no.=18) patients, heterozygote carriers (no.=61) and wildtypes (WT) (no.=27) for 21OHD. RESULTS: Salt wasting (SW) and simple virilizing (SV) had higher basal levels of DOC with no further increase in response to ACTH. Stimulated DOC was similar in 21OHD patients and carriers but was reduced as compared to WT. ACTH-stimulated B increased gradually from SW and SV through WT. The post-ACTH 21DF/B ratio was able to detect 92% of the carriers among WT. All NC patients could be detected by post-ACTH 17OHP/DOC and 21DF/B, with no overlap with 21OHD carriers. CONCLUSION: Although 21-hydroxylase is a key enzymatic step in both 17-hydroxy and 17-deoxy pathways of ZF, the reaction is mostly affected in the latter pathway, leading to a significant impairment of B production, which may further characterize the 21OHD subtypes. Also, the precursor-to-product ratios, particularly 21DF/B, can demonstrate the distinctive outline of 21OHD subtypes, including carriers and normal subjects.

Adrenal glands of slaughtered bulls, heifers and cows: a histological study.

The study involved histological and immunohistochemical examinations of the adrenal glands of healthy slaughtered cattle. Glands of 13 bulls, 10 heifers and 10 cows were examined. The following histological findings were observed: Unequal thickness of connective capsule and nodular formations of the zona glomerulosa (ZG), eosinophilic granules in cells of the ZG, globoid arrangement of the zona fasciculata, nodules or pegs of cortical tissue in the medulla, mutual interlacing of superficial and deep zones of the medulla, proliferation of cortical or medullary cells into the blood vessels wall situated in the medulla and focal inflammatory infiltrates. Cortical cells and noradrenalin-secreting (N) cells in the medulla expressed cytoplasmic positivity of S100 protein. Both adrenalin (A) cells and N cells were positive in synaptophysin. The majority of the cells in the cortex and in the medulla displayed were positive for chromogranin A. Electron microscopy showed structureless, electrondense particles of varying size and shape, mostly displaying the having mostly character of secretory granules.

Simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by semi-micro high-performance liquid chromatography with an immobilized cholesterol oxidase as a pre-column reactor: application to bovine adrenal fasciculata cells.

A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 µg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 µg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.

Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs.

Hypercortisolism caused by an adrenocortical tumor (AT) results from adrenocorticotropic hormone (ACTH)-independent hypersecretion of glucocorticoids. Studies in humans demonstrate that steroidogenesis in ATs may be stimulated by ectopic or overexpressed eutopic G protein-coupled receptors. We report on a screening of 23 surgically removed, cortisol-secreting ATs for the expression of receptors for luteinizing hormone (LH), gastric-inhibitory polypeptide (GIP), and vasopressin (V(1a), V(1b), and V(2)). Normal adrenal glands served as control tissues. Abundance of mRNA for these receptors was quantified using quantitative polymerase chain reaction (QPCR), and the presence and localization of these receptors were determined by immunohistochemistry. In both normal adrenal glands and ATs, mRNA encoding for all receptors was present, although the expression abundance of the V(1b) receptor was very low. The mRNA expression abundance for GIP and V(2) receptors in ATs were significantly lower (0.03 and 0.01, respectively) than in normal adrenal glands. The zona fasciculata of normal adrenal glands stained immunonegative for the GIP receptor. In contrast, islands of GIP receptor-immunopositive cells were detected in about half of the ATs. The zona fasciculata of both normal adrenal glands and AT tissue were immunopositive for LH receptor; in ATs in a homogenous or heterogenous pattern. In normal adrenal glands, no immunolabeling for V(1b)R and V(2) receptor was present, but in ATs, V(2) receptor-immunopositive cells were detected. In conclusion, QPCR analysis did not reveal overexpression of LH, GIP, V(1a), V(1b), or V(2) receptors in the ATs. However, the ectopic expression of GIP and V(2) receptor proteins in tumorous zona fasciculata tissue may play a role in the pathogenesis of canine cortisol-secreting ATs.

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.

Coregulatory protein-orphan nuclear receptor interactions in the human adrenal cortex.

The capacity of the adrenal to produce steroids is controlled in part through the transcriptional regulation of steroid enzymes. The orphan nuclear receptor steroidogenic factor 1 (SF-1) is central to the transcriptional regulation of all steroid hydroxylase enzymes, whereas nur77 can preferentially regulate steroid enzyme genes relevant to cortisol production. We hypothesised that, in the presence of secretagogues, SF-1 and nur77 may differentially interact with coregulatory proteins in the human adrenal cortex. Both coregulatory proteins, steroid receptor coactivator (SRC-1) and silencing mediator for retinoid and thyroid hormones (SMRT), were found to be expressed in the zona fasciculata and reticularis in the human adrenal cortex, but were largely absent from the zona glomerulosa. Both coregulatory proteins were colocalised with SF-1 and nur77. In the H295R adrenal tumour cell line, SF-1 and nur77 transcripts were increased in cells in the presence of forskolin, whereas nur77 mRNA was also induced with angiotensin II (AII). The coactivator SRC-1 mRNA was increased in the presence of both forskolin and AII. Forskolin induced recruitment of SRC-1 to the SF-1 response element and induced SRC-1-SF-1 interactions, whereas AII increased recruitment of SRC-1 to the nur77 response element and induced SRC-1-nur77 interactions. The corepressor SMRT interacted with SF-1 in the presence of AII and with nur77 in cells treated with forskolin. Orphan nuclear receptor-coregulatory protein interactions may have consequences for the regulation of key steroidogenic enzymes in the human adrenal cortex.

Laminin isoforms in fetal and adult human adrenal cortex.

Laminin has been proposed to influence the function of human adrenal cortex. We have studied the distribution of laminin (Ln) chains using immunofluorescence in human fetal and adult adrenal cortex. In the fetal gland Ln alpha2- and alpha5-chains were weakly expressed in the definitive zone, whereas Ln alpha4-, beta1-, and gamma1-chains occurred around vessels. In the adult gland, Ln alpha2-, alpha5-, and gamma1-chains were found in epithelial basement membranes (BM) in all cortical zones, Ln alpha4-chain in vessels, Ln beta1-chain in outer zone, and Ln beta2-chain in the two inner zones of the cortex, respectively. Among the integrins in adult gland, integrin alpha(3)-subunit was confined to basal surfaces of cortical cells, alpha(6) to vessels, alpha(1) to the stroma, and alpha(2) diffusely to epithelial cells. Lutheran glycoprotein and dystroglycan occurred in the fetal gland diffusely in the definitive zone and throughout the epithelium in the adult. The isoform composition of BM of the adult adrenal gland is distinct, with Ln-2 and -10 in BM of the outer zone and Ln-4 and -11 in BM of the two inner zones. The results suggest that integrin alpha(3)beta(1) and Lutheran are candidate receptors for Ln-10 and -11, whereas dystroglycan probably binds Ln-2 and -4.

Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis. Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17alpha-hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis. In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.

Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor.

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.

Stimulation by interleukin-6 and inhibition by tumor necrosis factor of cortisol release from bovine adrenal zona fasciculata cells through their receptors.

Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are synthesized and released from adrenal cells. Therefore, the effects of TNF-alpha and IL-6 on cortisol release from bovine zona fasciculata (ZF) cells were investigated. IL-6 (10-1000 pg/mL) significantly increased basal and adrenocorticotropic hormone (ACTH)-stimulated cortisol release in a concentration-dependent manner. This stimulatory effect of IL-6 became apparent at intervals as short as 4 h and continued through 24 h. IL-6 also potentiated the cortisol release stimulated by the adenylyl cyclase activator forskolin. By contrast, TNF-alpha (0.1-10 ng) inhibited basal and ACTH-stimulated cortisol release in a concentration-dependent manner. The inhibitory effects of TNF-alpha on cortisol release were significant at time intervals as short as 4 h and continued through 24 h. TNF-alpha inhibited forskolin-stimulated cortisol release. Binding studies demonstrated that ZF cells have IL-6 receptors (100 receptors/cell, Kd of 7.5 x 10(-11)) and TNF receptors (200 receptors/cell, Kd of 2.4 x 10(-9) M). Immunohistochemical analysis provided evidence that the majority of ZF cells have IL-6 receptors, TNF type 1 receptors, and TNF type 2 receptors. Because IL-6 and TNF-alpha are released from the adrenal cortex and these cytokines modify the release of cortisol from the ZF, IL-6 and TNF-alpha may play a paracrine or autocrine role in the regulation of adrenal function.

The in vivo effects of adrenocorticotropin and sodium restriction on the formation of the different species of steroidogenic acute regulatory protein in rat adrenal.

We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.

An adrenocorticotropin-regulated phosphoprotein intermediary in steroid synthesis is similar to an acyl-CoA thioesterase enzyme.

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur J. Biochem. 224, 709-716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues.

Immunohistochemical analysis of AT1 receptor versus P450c17 and 3 beta HSD expression in ovine adrenals.

Previous studies in ovine adrenocortical cells in vitro have shown angiotensin II (AII) receptors are expressed on the zona fasciculata (ZF) cells and are functionally coupled to phosphoinositidase C and increased [Ca2+]i, but AII stimulation does not cause an acute change in cortisol biosynthesis. AII can, however, chronically regulate differential expression of P450c17 and 3 beta HSD in ovine adrenocortical cells in vitro. We have stained ovine adrenal sections with specific antisera to the angiotensin II Type-1 receptor (AT1-R), as well as P450c17 and 3 beta HSD in order to further test the hypothesis that changes in AT1-R expression underlie changes in zonal expression of P450c17 and 3 beta HSD in vivo. AT1-R expression was found to be highest in the outermost layer of cells (zona glomerulosa, ZG) which stained negatively for P450c17 and only faintly positive for 3 beta HSD, as expected. The adjacent layer of cells (ZF) stained much less strongly for AT1-R but stronger for P450c17 and 3 beta HSD. These findings are consistent with our previously reported in vitro expression data, and suggest that the transition from ZG to ZF phenotype, i.e. increased P450c17 and 3 beta HSD expression, may require reduced expression of AT1-R, but maintenance of reduced levels of AT1-R expression in the ovine ZF still allows for differential control of the P450c17: 3 beta HSD ratio. Thus, even though there is no acute cortisol response to AII alone in these cells, AII stimulation can oppose C19 steroid production in the face of cortisol biosynthesis by the ZF in response to agonists such as ACTH.

Characterization of the cDNA corresponding to a phosphoprotein (p43) intermediary in the action of ACTH.

We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.

Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones.

Previously we demonstrated the presence of InsP3R-I, -II and -III subtypes in the zona glomerulosa. Now we have examined the expression of different subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) in the inner zones of rat adrenal cortex. RNA extracted from decapsulated adrenal tissue (zonae fasciculata-reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequent polymerase chain reaction revealed the presence of InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to demonstrate the expression of any known subtypes of InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are of medullary origin.

Regulation of ACTH receptor mRNA and binding sites by ACTH and angiotensin II in cultured human and bovine adrenal fasciculata cells.

Human (HAC) and bovine (BAC) adrenal fasciculata cells express ACTH and angiotensin-II (A-II) receptors. In the present work, we have studied the effects of both hormones on ACTH receptor (ACTH-R) mRNA and binding sites. Both HAC and BAC expressed several ACTH-R transcripts. Although in both cell types, ACTH and A-II increased ACTH-R transcripts in a time- and dose-dependent manner, the maximal effects were different. Thus, ACTH at 10(-9) M enhanced 21- and 5-fold the level of ACTH-R mRNA and binding sites in HAC, whereas in BAC both parameters were enhanced only 3-fold. A-II at 10(-7) M increased 17- and 3.5-fold ACTH-R mRNA and binding sites in HAC, whereas in BAC, it caused only a 2-fold increase in ACTH-R mRNA and a small decrease in receptor number. In HAC, the stimulatory effects of both hormones on ACTH-R mRNA are mainly transcriptional, whereas in BAC they are mainly post-transcriptional, by decreasing the rate of degradation of ACTH-R mRNA. The stimulatory effects of ACTH on ACTH-R in both HAC and BAC were associated with an enhanced steroidogenic response to further hormonal stimulation. In contrast, specific species differences were observed with A-II. Thus, in HAC A-II increased ACTH-R mRNA and binding sites and the ACTH-induced cortisol production, whereas in BAC, A-II caused a slight decrease of ACTH binding sites and steroidogenic desensitization.

Mixed parenchymal-stromal populations of rat adrenocortical cells support the proliferation and differentiation of steroidogenic cells.

The mechanisms which regulate adrenocortical steroidogenesis in differentiated parenchymal cells have been studied in great detail. However, the stem cells that are responsible for regeneration of the adult cortex have never been identified or isolated, and their characteristics are unknown. We have developed a tissue culture system that supports the simultaneous proliferation and differentiation of steroidogenic adrenocortical cells. Utilizing density gradient separation, a cell population composed of a mixture of stromal, endothelial and parenchymal cells (MIX) was isolated from the adult rat adrenal cortex. In primary culture, MIX populations formed high saturation density multilayers from which rounded cells emerged. These cells proliferated, contained lipid, and expressed the steroidogenic enzymes delta 5,3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochrome P-450scc. After selective passaging, rounded MIX-derived cells retained their steroidogenic potential, even in the absence of trophic hormone treatment. In contrast, parenchymal cells obtained from the zonae fasciculata (FASC) and glomerulosa (GLOM) respectively, formed homogeneous monolayers in primary culture, gradually de-differentiated, and no longer responded to trophic hormone treatment after passaging. Thus, primary MIX cultures provided a microenvironment that resulted in the production of adrenocortical cells with stem cell-like qualities. These cultures provide for the first time, a system for the identification of specific inducers that are responsible for both adrenocortical cytogenesis and its associated proliferation and steroidogenic differentiation.

Low sodium intake enhances sensitivity of 11-deoxycortisol and deoxycorticosterone to ACTH in ACTH-suppressed normal subjects.

Continued administration of ACTH to patients with hypopituitarism produced normal increases in steroids dependent on microsomal cytochrome P450(21) and P450(17 alpha) but reduced responses of steroids dependent on mitochondrial cytochrome P450(11 beta-18). To explore possible mechanisms and to determine whether this dissociation occurs with short-term ACTH suppression, we have examined the steroid responses to ACTH after 1 h in 12 normal subjects after equilibration on sodium intakes of 124 mmol/d [normal sodium diet (NSD)], 22 mmol/d [low sodium diet (LSD)], and 240 mmol/d [high sodium diet (HSD)] before and during continued ACTH suppression with dexamethasone (DEX). Two distinct patterns of steroid responses were observed. Deoxycorticosterone (DOC) responses were initially reduced during LSD-DEX but eventually returned to the NSD-control (NSD-CONT) values; in contrast 18-hydroxydeoxycorticosterone and corticosterone remained suppressed. 11-Deoxycortisol and 21-deoxycortisol showed patterns similar to DOC, with a return to normal ACTH responses on LSD-DEX. Basal cortisol levels were reduced and the ACTH response was unchanged by LSD. HSD-DEX reduced basal levels of all steroids as well as their ACTH responses. LSD and/or increased activity of the renin-angiotensin system have a significant impact on 17 alpha- and 21-hydroxylation functions in the zona fasciculata to maintain a normal ACTH response of microsomally dependent steroids under these conditions. In contrast, on HSD-DEX with the renin-angiotensin system suppressed, there is generalized impairment of steroid responses to ACTH.

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.