Extracellular Matrix Protein Signatures of the Outer and Inner Zones of the Rat Adrenal Cortex.

This study analyzes the extracellular matrix (ECM) signatures of the outer (OF = capsule + subcapsular + zona glomerulosa cells) and inner fractions (IF = zona fasciculata cells) of the rat adrenal cortex, which comprise two distinct microenvironment niches. Proteomic profiles of decellularized OF and IF samples, male and female rats, identified 252 proteins, with 32 classified as ECM-component and ECM-related. Among these, 25 proteins were differentially regulated: 17 more abundant in OF, including Col1a1, Col1a2, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, Lama5, Lamb2, Lamc1, Eln, Emilin, Fbln5, Fbn1, Fbn2, Nid1, and Ltbp4, and eight more abundant in IF, including Col4a1, Col4a2, Lama2, Lama4, Lamb1, Fn1, Hspg2, and Ecm1. Eln, Tnc, and Nid2 were abundant in the female OF, while Lama2, Lama5, Lamb2, and Lamc1 were more abundant in the male IF. The complex protein signature of the OF suggests areas of tissue stress, stiffness, and regulatory proteins for growth factor signaling. The higher concentrations of Col4a1 and Col4a2 and their role in steroidogenesis should be further investigated in IF. These findings could significantly enhance our understanding of adrenal cortex functionality and its implications for human health and disease. Key findings were validated, and data are available in ProteomeXchange (PXD046828).

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex.

Steroid hormones are synthesized through enzymatic reactions using cholesterol as the substrate. In steroidogenic cells, the required cholesterol for steroidogenesis can be obtained from blood circulation or synthesized de novo from acetate. One of the key enzymes that control cholesterol synthesis is 24-dehydrocholesterol reductase (encoded by DHCR24). In humans and rats, DHCR24 is highly expressed in the adrenal gland, especially in the zona fasciculata. We recently reported that DHCR24 was expressed in the mouse adrenal gland's inner cortex and also found that thyroid hormone treatment significantly upregulated the expression of Dhcr24 in the mouse adrenal gland. In the present study, we showed the cellular expression of DHCR24 in mouse adrenal glands in early postnatal stages. We found that the expression pattern of DHCR24 was similar to the X-zone marker gene 20αHSD in most developmental stages. This finding indicates that most steroidogenic adrenocortical cells in the mouse adrenal gland do not synthesize cholesterol locally. Unlike the 20αHSD-positive X-zone regresses during pregnancy, some DHCR24-positive cells remain present in parous females. Conditional knockout mice showed that the removal of Dhcr24 in steroidogenic cells did not affect the overall development of the adrenal gland or the secretion of corticosterone under acute stress. Whether DHCR24 plays a role in conditions where a continuous high amount of corticosterone production is needed requires further investigation.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

A Rat Model of Post-Traumatic Stress Syndrome Causes Phenotype-Associated Morphological Changes and Hypofunction of the Adrenal Gland.

BACKGROUND: Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production. METHODS: Rats were exposed to PSS for ten days. Thirty days later, the rats' anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex. RESULTS: Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxiety phenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats. CONCLUSION: Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Changes in Transcriptional Regulation of Postnatal Morphogenesis of the Adrenal Zona Fasciculata Caused by Endocrine Disruptor Dichlorodiphenyltrichloroethane.

We studied the expression of transcriptional factors regulating postnatal morphogenesis of the adrenal zona fasciculata in rats after developmental exposure to endocrine disruptor DDT. It was found that tissue reparation after trophic disorders and cell death triggered by prenatal and postnatal exposure to DDT was accompanied by an increase in the number of Oct4- and Shh-expressing cells forming a pool located outside the regeneration zones and involved in the maintenance of tissue homeostasis in the zona fasciculata. DDT exposure also disrupted the expression of antiproliferative factor Hhex. The data showed that proliferation of fasciculata cells after termination of adrenal cortex growth was downregulated by inhibition of the expression of Oct4 and Shh and suppression of canonical Wnt signaling, i.e. due to a decrease in the reserve cell pool essential for physiological regeneration, which can reduce the reactive potential of the zona fasciculata.

Stress-induced changes in the aged-rat adrenal cortex. Histological and histomorphometric study.

BACKGROUND: Stress exposure exerts direct effects on the morphology and functionality of the adrenal cortex. In addition, ageing effects growth, differentiation, apoptosis and cellularity of the cortex. The missing data is the combined effect of stress and ageing on the adrenal cortex. The aim of this study was to demonstrate the structural changes in the adrenal cortex following the exposure to stress in the adult and aged albino rats. MATERIALS AND METHODS: Forty rats were divided into groups I and II (adult and senile). Each group was further subdivided into subgroups a and b (control and stressed). Light and electron microscopic studies were done. Area per cent of collagen fibres (Masson's trichrome-stained sections), number of proliferating cells (optical density immunoreactivity in the Ki67 stained sections) and thickness of the three adrenal zones were also measured. RESULTS: Lamellar separation of the capsule with subcapsular spindle cell hyperplasia and areas of ghost cells were observed in zona glomerulosa (ZG) and zona fasciculata (ZF) in group I-b. Separation and indentation of the capsule with its lamellar separation were observed in group II-a with the existence of multiple scattered degenerative foci in ZF and zona reticularis (ZR). Similar and aggressive was the architectural pattern of ZF in group II-b with the presence of areas of homogenous degeneration. The nuclei of ZG had marginated chromatin in group I-b and were pyknotic with deformed irregular outlines in group II-b. Multiple lysosomes and vacuolar degeneration mitochondria were also seen in group I-b. The nuclei of ZF were irregular with condensed marginated heterochromatin in group I-b, irregular with scattered chromatin in group II-a and indented with areas of chromatin destruction in group II-b. Mitochondria with disrupted cristae and cristolysis were also detected in group I-b. Numerous lipofuscin granules and dilated smooth endoplasmic reticulum were revealed in group II-b. The mean collagen fibre area per cent and the mean number of the proliferating cells in group II-b were significantly higher by 39% and 23%. The thickness of ZG decreased significantly by 20% in group I-b. Contrary, the thickness of both ZF and ZR increased significantly by 10% in group I-b. CONCLUSIONS: Histological alterations occurred in the adrenal cortex in response to stress, especially when coupled with the advance of age. This was accompanied by increase in the area per cent of collagen fibres and increase in the mean number of the proliferating cells in the adrenal cortex.

Decrease in Lipid Droplets in Adrenal Cortex of Male Wistar Rats after Chronic Exposure to Energy Drinks.

Background and objectives: Energy drinks are popular non-alcoholic beverages. They are consumed in large amounts, mainly by active, young people. Although they are easily accessible and marketed as safe, numerous cases of adverse effects have been published, including cardiac arrest, arrythmias, acute hepatitis, and renal failure. The aim of the current study is the assessment of energy drink influence on the histological structure of adrenal cortex in rats. Material and Methods: 15 male young Wistar rats were equally divided into three groups: control (C), experimental (E) and reversibility control (RC). C group received water and standard rodent food ad libitum while both E and RC groups had additionally unlimited access to energy drinks. C and E groups were decapitated after 8 weeks and RC was given another 8 weeks without energy drinks. Adrenal glands were embedded in paraffin blocks and 5 µm slides were prepared and stained according to standard H&E and Masson's trichrome protocols. Additionally, immunohistochemical stainings against Ki-67, p53, CTGF and caspase-3 were prepared. Results: Decreased vacuolization and numerous pyknotic nuclei were noted in E and RC groups. Overexpression of caspase-3 was noted both subcapsular in zona glomerulosa and along sinusoids in zona fasciculata. Increased collagen deposition in zona glomerulosa and zona fasciculata of E and RC was observed. Insular and irregular overexpression of CTGF was noted. The overall picture of CTGF expression matched the Masson's trichrome. No significant difference was observed in Ki-67 expression. Conclusions: The results of the current study suggest that the stimulation is so intense that it causes significant damage to adrenal cortical cells, resulting in their apoptosis. It seems, however, that the observed effects are at least partially reversible.

Changes in Secretory Activity of Adrenal Zona Fasciculata Cells in Pubertal Rats Exposed to Low Doses of DDT in Different Periods of Ontogeny.

We studied secretory activity of adrenal zona fasciculata cells in pubertal rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal periods and only during postnatal period. In exposed animals, circulatory disturbances leading to degeneration and necrosis of cells as well as regeneration foci were revealed in the outer zona fasciculata. In rats exposed to DDT during the prenatal and postnatal periods, compensatory increase in secretory activity of cells due to increase in mitochondria content was noted in the inner zona fasciculata, which determined elevated serum concentration of corticosterone. In rats exposed to DDT only during the postnatal development, functional activity of zona fasciculata cells was suppressed, which attested to delayed upregulation of secretion.

ACTH Modulates PTP-PEST Activity and Promotes Its Interaction With Paxillin.

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased KM and VM . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. J. Cell. Biochem. 117: 2170-2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Guanosine triphosphate can directly regulate cortisol production by activating Ca(2+)-messenger systems in bovine adrenal fasciculata cells.

Adenosine triphosphate (ATP) is known to stimulate cortisol production in vitro, however, the effect of guanosine triphosphate (GTP) on cortisol production is not known. We studied the effect of GTP on cortisol production and investigated the regulation of intracellular signal transduction systems, including the cyclic AMP-dependent and Ca(2+)-messenger systems, in bovine adrenal fasciculata cells. GTP clearly induced cortisol biosynthesis but only to a level less than half the adrenocorticotropic hormone (ACTH)-induced maximum. The binding site for [γ-(35)S]-GTPγS was shown to differ completely from that for ATP and also from those for Gs and Gi, as indicated by the fact that binding was not influenced by pretreatment with cholera toxin and pertussis toxin. GTP significantly increased cytosolic calcium ([Ca(2+)]i) and inositol 1, 4, 5-triphosphate without affecting cyclic AMP formation. GTP-induced cortisol production was suppressed by H-9 and Calphostin C (specific protein kinase C inhibitors) but not by H-8 and KT5720 (specific inhibitors of cyclic AMP-dependent protein kinase), suggesting that GTP activates cortisol biosynthesis possibly via a protein kinase C-dependent pathway. Extracellular calcium may be essential for GTP activity since GTP-induced cortisol production was almost completely suppressed in its absence. In conclusion, it can be postulated that GTP-induced steroid secretion in bovine adrenal fasciculata cells is under paracrine or autocrine control.

Adrenal fasciculata cells express T-type and rapidly and slowly activating L-type Ca2+ channels that regulate cortisol secretion.

In whole cell patch-clamp recordings, we characterized the L-type Ca(2+) currents in bovine adrenal zona fasciculata (AZF) cells and explored their role, along with the role of T-type channels, in ACTH- and angiotensin II (ANG II)-stimulated cortisol secretion. Two distinct dihydropyridine-sensitive L-type currents were identified, both of which were activated at relatively hyperpolarized potentials. One activated with rapid kinetics and, in conjunction with Northern blotting and PCR, was determined to be Cav1.3. The other, expressed in approximately one-half of AZF cells, activated with extremely slow voltage-dependent kinetics and combined properties not previously reported for an L-type Ca(2+) channel. The T-type Ca(2+) channel antagonist 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2) inhibited Cav3.2 current in these cells, as well as ACTH- and ANG II-stimulated cortisol secretion, at concentrations that did not affect L-type currents. In contrast, nifedipine specifically inhibited L-type currents and cortisol secretion, but less effectively than TTA-P2. Diphenylbutylpiperidine Ca(2+) antagonists, including pimozide, penfluridol, and fluspirilene, and the dihydropyridine niguldipine blocked Cav3.2 and L-type currents and inhibited ACTH-stimulated cortisol secretion with similar potency. This study shows that bovine AZF cells express three Ca(2+) channels, the voltage-dependent gating and kinetics of which could orchestrate complex mechanisms linking peptide hormone receptors to cortisol secretion through action potentials or sustained depolarization. The function of the novel, slowly activating L-type channel is of particular interest in this respect. Regardless, the well-correlated selective inhibition of T- and L-type currents and ACTH- and ANG II-stimulated cortisol secretion by TTA-P2 and nifedipine establish the critical importance of these channels in AZF cell physiology.

Renin knockout rat: control of adrenal aldosterone and corticosterone synthesis in vitro and adrenal gene expression.

The classic renin-angiotensin system is partly responsible for controlling aldosterone secretion from the adrenal cortex via the peptide angiotensin II (ANG II). In addition, there is a local adrenocortical renin-angiotensin system that may be involved in the control of aldosterone synthesis in the zona glomerulosa (ZG). To characterize the long-term control of adrenal steroidogenesis, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro and steroidogenic enzyme expression to wild-type (WT) controls (Dahl S rat). Adrenal capsules (ZG; aldosterone production) and subcapsules [zona reticularis/fasciculata (ZFR); corticosterone production] were separately dispersed and studied in vitro. Plasma renin activity and ANG II concentrations were extremely low in the KO rats. Basal and cAMP-stimulated aldosterone production was significantly reduced in renin KO ZG cells, whereas corticosterone production was not different between WT and KO ZFR cells. As expected, adrenal renin mRNA expression was lower in the renin KO compared with the WT rat. Real-time PCR and immunohistochemical analysis showed a significant decrease in P450aldo (Cyp11b2) mRNA and protein expression in the ZG from the renin KO rat. The reduction in aldosterone synthesis in the ZG of the renin KO adrenal seems to be accounted for by a specific decrease in P450aldo and may be due to the absence of chronic stimulation of the ZG by circulating ANG II or to a reduction in locally released ANG II within the adrenal gland.

Female baboon adrenal zona fasciculata and zona reticularis regulatory and functional proteins decrease across the life course.

Debate exists on life-course adrenocortical zonal function trajectories. Rapid, phasic blood steroid concentration changes, such as circadian rhythms and acute stress responses, complicate quantification. To avoid pitfalls and account for life-stage changes in adrenocortical activity indices, we quantified zonae fasciculata (ZF) and reticularis (ZR) across the life-course, by immunohistochemistry of key regulatory and functional proteins. In 28 female baboon adrenals (7.5-22.1 years), we quantified 12 key proteins involved in cell metabolism, division, proliferation, steroidogenesis (including steroid acute regulatory protein, StAR), oxidative stress, and glucocorticoid and mitochondrial function. Life-course abundance of ten ZF proteins decreased with age. Cell cycle inhibitor and oxidative stress markers increased. Seven of the 12 proteins changed in the same direction for ZR and ZF. Importantly, ZF StAR decreased, while ZR StAR was unchanged. Findings indicate ZF function decreased, and less markedly ZR function, with age. Causes and aging consequences of these changes remain to be determined.

Znrf3 exon 2 deletion mice do not recapitulate congenital adrenal hypoplasia.

Wnt/ß-catenin signaling is essential for adrenocortical development. Zinc and ring finger 3 (ZNRF3), an E3 ubiquitin ligase that attenuates Wnt/ß-catenin signaling, is negatively regulated by R-spondin via an extracellular domain that is partially encoded by exon 2 of ZNRF3. We recently identified ZNRF3 exon 2 deletions in three individuals with congenital adrenal hypoplasia. ZNRF3 exon 2 deletion impairs R-spondin binding, thereby attenuating ß-catenin expression and eventually leading to the development of congenital adrenal hypoplasia. To elucidate the influence of ZNRF3/Znrf3 exon 2 deletion on adrenocortical development, we generated homozygous Znrf3 exon 2 deletion (Znrf3Δ2/Δ2) mice. Whereas the adrenal glands of Znrf3Δ2/Δ2 mice did not show gross morphological changes at birth, moderate hyperplasia of the zona fasciculata (ZF), dispersed medulla arrangement, and a radially spreading zone with macrophage infiltration between the ZF and medulla were observed at 6 weeks of age. 20α-hydroxysteroid dehydrogenase, a marker of the adrenal X-zone, was hardly detected by immunostaining, and gene expression was significantly downregulated. The number of activated ß-catenin-positive cells decreased in the zona glomerulosa, consistent with the results of in situ hybridization for Axin2, a Wnt/ß-catenin target gene. Plasma ACTH and serum corticosterone levels in Znrf3Δ2/Δ2 mice did not differ significantly from those in wild-type mice. These results show a species-specific difference in the effects of ZNRF3/Znrf3 exon 2 deletions in humans and mice; Znrf3Δ2/Δ2 mice do not develop congenital adrenal hypoplasia but instead exhibit moderate ZF hyperplasia, dispersed medulla arrangement, X-zone dysplasia, and macrophage infiltration occurred in the inner cortex.

Increased adrenal steroidogenesis and suppressed corticosteroid responsiveness in critical COVID-19.

BACKGROUND: The effect of coronavirus disease 2019 (COVID-19) on adrenal endocrine metabolism in critically ill patients remains unclear. This study aimed to investigate the alterations in adrenal steroidogenic activity, elucidate underlying mechanisms, provide in situ histopathological evidence, and examine the clinical implications. METHODS: The comparative analyses of the adrenal cortices from 24 patients with fatal COVID-19 and 20 matched controls were performed, excluding patients previously treated with glucocorticoids. SARS-CoV-2 and its receptors were identified and pathological alterations were examined. Furthermore, histological examinations, immunohistochemical staining and ultrastructural analyses were performed to assess corticosteroid biosynthesis. The zona glomerulosa (ZG) and zona fasciculata (ZF) were then dissected for proteomic analyses. The biological processes that affected steroidogenesis were analyzed by integrating histological, proteomic, and clinical data. Finally, the immunoreactivity and responsive genes of mineralocorticoid and glucocorticoid receptors in essential tissues were quantitatively measured to evaluate corticosteroid responsiveness. FINDINGS: The demographic characteristics of COVID-19 patients were comparable with those of controls. SARS-CoV-2-like particles were identified in the adrenocortical cells of three patients; however, these particles did not affect cellular morphology or steroid synthesis compared with SARS-CoV-2-negative specimens. Although the adrenals exhibited focal necrosis, vacuolization, microthrombi, and inflammation, widespread degeneration was not evident. Notably, corticosteroid biosynthesis was significantly enhanced in both the ZG and ZF of COVID-19 patients. The increase in the inflammatory response and cellular differentiation in the adrenal cortices of patients with critical COVID-19 was positively correlated with heightened steroidogenic activity. Additionally, the appearance of more dual-ZG/ZF identity cells in COVID-19 adrenals was in accordance with the increased steroidogenic function. However, activated mineralocorticoid and glucocorticoid receptors and their responsive genes in vital tissues were markedly reduced in patients with critical COVID-19. INTERPRETATION: Critical COVID-19 was characterized by potentiated adrenal steroidogenesis, associated with increased inflammation, enhanced differentiation and elevated dual-ZG/ZF identity cells, alongside suppressed corticosteroid responsiveness. These alterations implied the reduced effectiveness of conventional corticosteroid therapy and underscored the need for evaluation of the adrenal axis and corticosteroid sensitivity.

Protective effect of grape seed extract against chronic physical stress-induced zona fasciculata injury in male rats: Functional, immunohistochemical and electron microscopic study.

In the present study, we investigated the antioxidant effect of grape seed extract (GSE) against chronic immobilization stress-induced zona fasciculata injury in Wistar male rats. Thirty male rats were divided into three groups: Non-stress group: rats were not subjected to stress protocol and received distilled water orally for 30 days. Stress group: rats received distilled water orally for 15 consecutive days before the induction of chronic immobilization stress experiment (repeated stress for 15 consecutive days), distilled water was continued along with the constant stress experiment. GSE-stress group: rats treated with oral GSE (300 mg/kg), administered orally for 15 consecutive days before the induction of chronic immobilization stress experiment (repeated stress for 15 consecutive days), GSE was continued along with the stress exposure. Chronic stress was induced by placing each animal in a restrainer for 2 h daily for 15 consecutive days in both Stress and GSE-stress groups. The serum corticosterone and adrenal cortex malondialdehyde (MDA) levels were measured as indices of stress. Immunohistochemistry of the inducible nitric oxide synthase (iNOS) as a nitrosative stress marker beside the adrenal cortex's ultrastructure, particularly zona fasciculata, was assessed. Chronic restraint stress significantly elevated the serum corticosterone and adrenal cortex MDA levels, while oral administration of GSE reduced the serum corticosterone level, adrenal cortex MDA levels, and iNOS immunoreactivity in zona fasciculata. Besides, adrenocortical ultrastructure significantly improved. These results suggested that GSE enhanced the antioxidant defense against reactive oxygen species produced under chronic stress conditions, protecting the adrenal cortex. RESEARCH HIGHLIGHTS: This research highlighted the significant protective effects of grape seed extract administration on the histological findings, both in light and electron microscopic studies, as well as the biochemical and functional parameters in cases of stress-induced adrenal cortex injury in rats.

ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms.

Bovine adrenal zona fasciculata (AZF) cells express Ca(v)3.2 T-type Ca(2+) channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca(v)3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3-6 days markedly enhanced the expression of Ca(v)3.2 current. The increase in Ca(v)3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca(v)3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 mum) and 6-benzoyladenosine cAMP (300 mum) induced CACNA1H mRNA, but not Ca(v)3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10-50 mum) enhanced CACNA1H mRNA and Ca(v)3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca(v)3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca(v)3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2'-O-methyl-cAMP and its metabolites 8CPT-2'-OMe-5'-AMP and 8CPT-2'-O-methyl-adenosine increased CACNA1H mRNA and Ca(v)3.2 current; Sp-8CPT-2'-O-methyl-cAMP increased neither Ca(v)3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca(2+) current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca(v)3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca(2+) current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca(v)3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca(v)3.2 expression by the same, unidentified mechanism.

Endotoxin tolerance of adrenal gland: attenuation of corticosterone production in response to lipopolysaccharide and adrenocorticotropic hormone.

OBJECTIVES: Reversible adrenal insufficiency frequently has been diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotropic hormone (ACTH) stimulation. It is generally accepted that a phenomenon called "endotoxin tolerance" contributes to immunosuppression during sepsis. The present study was to investigate whether endotoxin tolerance occurs in the adrenal gland, leading to hyporesponsiveness of adrenal gland during sepsis. DESIGN: Controlled laboratory experiment. SETTING: University research laboratory. SUBJECTS: Sprague-Dawley male rats 200-250 g and primary isolated adrenal fasciculata-reticularis cells. INTERVENTIONS: Rats received intra-arterial injection of purified lipopolysaccharide (0.5 mg/kg) through indwelling femoral arterial catheters, and 24 hrs later the adrenocortical sensitivity to exogenous ACTH (10 ng/kg) was detected. Primary fasciculata-reticularis cells were pretreated with lipopolysaccharide at 0.1-100 ng/mL or with ACTH at 0.01-10 ng/mL and then challenged, in fresh media, with 1 µg/mL lipopolysaccharide or 10 ng/mL ACTH. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor 4 was expressed in adrenal gland and primary fasciculata-reticularis cells. Plasma corticosterone response to ACTH was decreased in rats receiving preinjection of lipopolysaccharide. Lipopolysaccharide pretreatment caused a significant decrease in corticosterone production in response to subsequent ACTH and lipopolysaccharide stimulation in primary fasciculata-reticularis cells. Lipopolysaccharide pretreatment inhibited ACTH- and lipopolysaccharide-induced expression of steroid metabolizing enzymes. Lipopolysaccharide significantly decreased Toll-like receptor 4 and ACTH receptor expression. CONCLUSIONS: Pre-exposure to lipopolysaccharide resulted in hyporesponsiveness to ACTH stimulation in rats. In vitro, lipopolysaccharide pretreatment impaired corticosterone production of fasciculata-reticularis cells in response to ACTH and lipopolysaccharide, which was associated with decreased expression of synthetic enzymes required for corticosterone production. Our results indicate that endotoxin tolerance of adrenal gland is one of the mechanisms for adrenocortical insufficiency during sepsis.

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.