Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.

RNA-Seq Reveals Sub-Zones in Mouse Adrenal Zona Fasciculata and the Sexually Dimorphic Responses to Thyroid Hormone.

The sex-specific prevalence of adrenal diseases has been known for a long time. However, the reason for the high prevalence of these diseases in females is not completely understood. Mouse studies have shown that the adult adrenal gland is sexually dimorphic at different levels such as transcriptome, histology, and cell renewal. Here we used RNA-seq to show that in prepubertal mice, male and female adrenal glands were not only sexually dimorphic but also responded differently to the same external stimulus. We previously reported that thyroid hormone receptor ß1 (TRß1) in the adrenal gland is mainly expressed in the inner cortex and the fate of this TRß1-expressing cell population can be changed by thyroid hormone (triiodothyronine; T3) treatment. In the present study, we found that adrenal glands in prepubertal mice were sexually dimorphic at the level of the transcriptome. Under T3 treatment, prepubertal females had 1162 genes differentially expressed between the saline and T3 groups, whereas in males of the same age, only 512 genes were T3-responsive. Immunostaining demonstrated that several top sexually dimorphic T3-responsive genes, including Cyp2f2 and Dhcr24, were specifically expressed in the adrenal inner cortex, precisely in an area partially overlapping with the X-zone. Under T3 treatment, a unique cortical layer that surrounds the adrenal X-zone expanded significantly, forming a distinct layer peculiar to females. Our findings identified novel marker genes for the inner adrenal cortex, indicating there are different sub-zones in the zona fasciculata. The results also highlight the sex-specific response to thyroid hormone in the mouse adrenal gland.

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.

Atypical antipsychotic drug modulates early life infection induced impairment of hypothalamic-pituitary-adrenal axis: An age related study in mice.

Evidences from human and animal studies indicate that exposure to infection during early life act as a stressor to impair the hypothalamic-pituitary-adrenal (HPA) axis and may be one of the contributing factors of mental illness of later life. Several atypical antipsychotic drugs (AAPDs) proved to be effective in alleviating psychiatric illness through normalization of HPA axis. However, AAPD are least tried to evaluate their efficacy in modulation of HPA axis impaired under infection. The present study elucidated that the treatment with AAPD paliperidone (PAL: 0.025 mg/kg/bw and 0.05 mg/kg/bw) during periadolescence period (postnatal day 35- postnatal day 56) dose-dependently normalized the HPA axis of the female mice who were gestationally (gestational day 15 and 17) exposed to bacterial endotoxin lipopolysaccharide (LPS: 800 µg/kg/bw; intraperitoneally). The effectiveness of PAL treatment in counteracting the LPS induced hyperactivity of HPA axis was age-related, better observed at postnatal day 120 than at postnatal day 200. The PAL modulation of HPA axis reflected at different levels: inhibition of hypothalamic CRF expression and reduction in plasma levels of adrenocorticotropin and corticosterone. Histopathological alterations such as hypertrophy and/or hyperplasia in cortical zona fasciculata as well as medullary chromaffin cells of adrenal also normalized on PAL treatment. The comparatively long wash out period after drug treatment (postnatal day 57- postnatal day 200) along with age related hormonal imbalance could be correlated to less effectiveness of PAL on HPA axis at postnatal day 200. PAL modulation of HPA axis might be through maintenance of cytokines and reproductive axis homeostasis.

Effects of Adipocyte-derived Factors on the Adrenal Cortex.

BACKGROUND AND OBJECTIVE: Obesity is highly complicated by hypertension and hyperglycemia. In particular, it has been proposed that obesity-related hypertension is caused by adipocyte-derived factors that are recognized as undetermined proteins secreted from adipocytes. Adipocyte-derived factors have been known to be related to aldosterone secretion in the adrenal gland. So far, Wnt proteins, CTRP-1, VLDL, LDL, HDL and leptin have been demonstrated to stimulate aldosterone secretion. In contrast, it has not yet been clarified whether adipocyte-derived factors also affect adrenal cortisol secretion. METHODS AND RESULTS: In the present study, we investigated the effect of adipocyte-derived factors on cortisol synthase gene CYP11B1 mRNA expression in vitro study using adrenocortical carcinoma H295R cells and mouse fibroblast 3T3-L1cells. Interestingly, adipocyte-derived factors were demonstrated to have the ability to stimulate CYP11B1 mRNA expression. CONCLUSION: Since CYP11B1 is well known as a limiting enzyme of cortisol synthesis, our study suggests that adipocyte-derived factors may stimulate cortisol secretion, as well as aldosterone secretion. Taken together, adipocyte-derived factors may be the cause of metabolic syndrome due to their stimulating effects on aldosterone/cortisol secretion. Therefore, the innovation of novel drugs against them may possibly be a new approach against metabolic syndrome.

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 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.

Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats.

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

Effects of acrolein on the production of corticosterone in male rats.

Acrolein, an α, ß-unsaturated aldehyde, exists in a wide range of sources. Acrolein can be not only generated from all types of smoke but also produced endogenously from the metabolism by lipid peroxidation. The cellular influence of acrolein is due to its electrophilic character via binding to and depleting cellular nucleophiles. Although the toxicity of acrolein has been extensively studied, there is relatively little information about its impact on hormone release. This study aimed at the effect of acrolein on hypothalamic-pituitary-adrenal (H-P-A) axis. In an in vivo study, male rats were administrated with acrolein for 1 or 3days. The plasma corticosterone in response to a single injection of adrenocorticotropic hormone (ACTH) increased slowly in acrolein-pretreated rats than in control rats. Further investigating the steroidogenic pathway, the protein expressions of steroidogenic acute regulatory protein (StAR) and the upper receptor-melanocortin 2 receptor (MC2R) were attenuated in acrolein-treated groups. Another experiment using trilostane showed less activity of P450scc in zona fasciculata-reticularis (ZFR) cells in acrolein-treated groups. In addition to the suppressed ability of corticosterone production in ZFR cells, acrolein even had extended influence at higher concentrations. The lower ACTH was observed in the plasma from acrolein-pretreated rats. In an in vitro study, ZFR cells were incubated with acrolein and the results showed that corticosterone concentrations in media were decreased in a dose-dependent manner. Acrolein also desensitized the response of the ZFR cells to ACTH. These results suggested that acrolein decreased the releasing ability of corticosterone via an inhibition on the response of ZFR cells to ACTH and the reduction of protein expressions of StAR and MC2R as well as the activity of P450scc in rat ZFR cells. The present evidences showed that the H-P-A axis was affected by the administration of acrolein.

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.

Ca2+ and K+ channels of normal human adrenal zona fasciculata cells: properties and modulation by ACTH and AngII.

In whole cell patch clamp recordings, we found that normal human adrenal zona fasciculata (AZF) cells express voltage-gated, rapidly inactivating Ca(2+) and K(+) currents and a noninactivating, leak-type K(+) current. Characterization of these currents with respect to voltage-dependent gating and kinetic properties, pharmacology, and modulation by the peptide hormones adrenocorticotropic hormone (ACTH) and AngII, in conjunction with Northern blot analysis, identified these channels as Cav3.2 (encoded by CACNA1H), Kv1.4 (KCNA4), and TREK-1 (KCNK2). In particular, the low voltage-activated, rapidly inactivating and slowly deactivating Ca(2+) current (Cav3.2) was potently blocked by Ni(2+) with an IC50 of 3 µM. The voltage-gated, rapidly inactivating K(+) current (Kv1.4) was robustly expressed in nearly every cell, with a current density of 95.0 ± 7.2 pA/pF (n = 64). The noninactivating, outwardly rectifying K(+) current (TREK-1) grew to a stable maximum over a period of minutes when recording at a holding potential of -80 mV. This noninactivating K(+) current was markedly activated by cinnamyl 1-3,4-dihydroxy-α-cyanocinnamate (CDC) and arachidonic acid (AA) and inhibited almost completely by forskolin, properties which are specific to TREK-1 among the K2P family of K(+) channels. The activation of TREK-1 by AA and inhibition by forskolin were closely linked to membrane hyperpolarization and depolarization, respectively. ACTH and AngII selectively inhibited the noninactivating K(+) current in human AZF cells at concentrations that stimulated cortisol secretion. Accordingly, mibefradil and CDC at concentrations that, respectively, blocked Cav3.2 and activated TREK-1, each inhibited both ACTH- and AngII-stimulated cortisol secretion. These results characterize the major Ca(2+) and K(+) channels expressed by normal human AZF cells and identify TREK-1 as the primary leak-type channel involved in establishing the membrane potential. These findings also suggest a model for cortisol secretion in human AZF cells wherein ACTH and AngII receptor activation is coupled to membrane depolarization and the activation of Cav3.2 channels through inhibition of hTREK-1.

Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTH-stimulated bovine adrenal fasciculata cells.

AIMS: We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. MAIN METHODS: The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography-mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. KEY FINDINGS: An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. SIGNIFICANCE: Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.

Adrenocorticotropin hormone (ACTH) effects on MAPK phosphorylation in human fasciculata cells and in embryonic kidney 293 cells expressing human melanocortin 2 receptor (MC2R) and MC2R accessory protein (MRAP)ß.

Adrenocorticotropin hormone (ACTH) exerts trophic effects on adrenocortical cells. We studied the phosphorylation of mitogen-activated proteins kinases (MAPKs) in human embryonic kidney cells stably expressing the ACTH receptor, MC2R, and its accessory protein MRAPß and in primary cultures of human adrenal fasciculata cells. ACTH induced a maximal increase in p44/p42(mapk) and of p38 MAPK phosphorylation after 5min. Neither the overexpression of wild-type arrestin2, arrestin3 or their respective dominant negative forms affected p44/p42(mapk) phosphorylation. However, preincubation with the recycling inhibitors brefeldin A and monensin attenuated both cAMP accumulation and p44/p42(mapk) phosphorylation proportionally. Cyclic AMP-related PKA inhibitors (H89, KI(6-22)) and Rp-cAMPS decreased p44/p42(mapk) phosphorylation but not ACTH-mediated cAMP production. The selective Epac1/2 activator, 8-pCPT-2'-O-MecAMP, did not modify the effect of ACTH. Thus, cAMP/PKA, but not cAMP/Epac1/2 pathways, or arrestin-coupled internalization of MC2R is involved in ACTH-induced p44/p42(mapk) phosphorylation by human MC2R. Together, ACTH binding to MC2R stimulates PKA-dependent p44/p42(mapk) phosphorylation.

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.

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: a possible role for cyclin E.

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex.

Effects and mechanisms of nonylphenol on corticosterone release in rat zona fasciculata-reticularis cells.

Alkylphenol ethoxylate, consisting of ∼80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactant. The primary degradation product of NPEO, nonylphenol (NP), interferes with reproduction, induces cell death in gonads, and leads to changes in other reproductive parameters. With such apparent stress, NP is believed to induce stress response mechanism, i.e., adrenal cortical hormone. However, the effects and action mechanisms of NP on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of NP on corticosterone release. ZFR cells were incubated with NP in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxyl cholesterol (25-OH-cholesterol), pregnenolone, progesterone, or deoxycorticosterone at 37°C for 1 h. The concentrations of corticosterone or pregnenolone in the spent media were measured by radioimmunoassay. The expressions of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc) protein, and 11ß-hydroxylase in the cells were measured by Western blot. The data demonstrated that (1) NP stimulated corticosterone release induced by ACTH, 8-Br-cAMP, FSK, 25-OH-cholesterol, pregnenolone, progesterone, or deoxycorticosterone; (2) NP significantly increased pregnenolone release in the control, 25-OH-cholesterol, trilostane, and 25-OH-cholesterol + trilostane groups; (3) NP-stimulated corticosterone release was estrogen receptor dependent, but mediated by nitric oxide and p38 mitogen-activated protein kinase pathway independent; and (4) NP did not affect StAR, 11ß-hydroxylase, or P450scc protein expression. These results suggest that NP acts directly on rat ZFR cells to stimulate corticosterone release and that the stimulation mechanism of NP mediates through post-cAMP corticosterone manufacture enzymes, i.e., P450scc and 11ß-hydroxylase.

Central effects of ghrelin on the adrenal cortex: a morphological and hormonal study.

Ghrelin, a growth hormone secretagogue that exerts an important role in appetite and weight regulation, participates in the activation of the hypothalamo-pituitary-adrenal (HPA) axis. Male Wistar rats (5/group) received daily for 5 days, via an ICV (intracerebroventricular) cannula, 5 microl phosphate buffered saline with or without 1 microg of rat ghrelin. Two hours after the last injection, blood and adrenal glands were collected from decapitated rats for blood hormone analyses and histologic and morphometric processing. Ghrelin treatment resulted in increased (p<0.05) body weight (13%), absolute whole adrenal gland weight (18%) and whole adrenal gland volume (20%). The absolute volumes of the entire adrenal cortex, ZG, ZF, and ZR also increased (p<0.05) after ghrelin by 20%, 21%, 21% and 11%, respectively. Ghrelin-treated rats had elevated (p<0.05) blood concentrations of ACTH, aldosterone and corticosterone (68%, 32% and 67%, respectively). The data clearly provide both morphological and hormonal status that ghrelin acts centrally to exert a global stimulatory effect on the adrenal cortex. Clarifying of the ghrelin precise role in the multiple networks affecting the stress hormone release, besides its well known energy and metabolic unbalance effects, remains a very important research goal.

N6-substituted cAMP analogs inhibit bTREK-1 K+ channels and stimulate cortisol secretion by a protein kinase A-independent mechanism.

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels whose inhibition by cAMP is coupled to membrane depolarization and cortisol secretion through complex signaling mechanisms. cAMP analogs with substitutions in the 6 position of the adenine ring selectively activate cAMP-dependent protein kinase (PKA) but not exchange proteins activated by cAMP (Epacs). In whole-cell patch-clamp recordings from AZF cells, we found that 6-benzoyl-cAMP (6-Bnz-cAMP) and 6-monobutyryl-cAMP potently inhibit bTREK-1 K+ channels, even under conditions in which PKA activity was abolished. Specifically, when applied through the patch electrode, 6-Bnz-cAMP inhibited bTREK-1 with an IC(50) of less than 0.2 microM. Inhibition of bTREK-1 by 6-Bnz-cAMP was not diminished by PKA antagonists, including N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), adenosine 3'-5'cyclic monophosphothiate, Rp-isomer, protein kinase inhibitor (PKI) (6-22) amide, and myristoylated PKI (14-22), applied alone or in combination, externally and intracellularly through the patch pipette. Under similar conditions, these same antagonists completely blocked PKA activation by 6-Bnz-cAMP. Inhibition of bTREK-1 by 6-Bnz-cAMP was voltage-independent and eliminated in the absence of ATP in the pipette solution. 6-Bnz-cAMP also produced delayed increases in cortisol synthesis and the expression of CYP11a1 mRNA that were only partially blocked by PKA antagonists. These results indicate that 6-Bnz-cAMP and other 6-substituted cAMP analogs can inhibit bTREK-1 K+ channels and stimulate delayed increases in cortisol synthesis by AZF cells through a PKA- and Epac-independent mechanism. They also suggest that adrenocorticotropin and cAMP function in these cells through a third cAMP-dependent protein. Finally, although 6-modified cAMP analogs exhibit high selectivity in activating PKA over Epac, they also may interact with other unidentified proteins expressed by eukaryotic cells.