Induction of nitric oxide synthase activity in adrenal cells

The induction of nitric oxide synthase (NOS) II by bacterial lypopolysaccharide (LPS) was studied in a steroidogenic mouse tumor adrenal cell line (Y1). Conditioned media from LPS-stimulated peritoneal macrophages induced an increase in NOS II expression as shown by western and northern blot analysis. Accordingly, in the presence of conditioned media an increase in nitrite levels was observed. In addition, steroid production was significantly decreased. In conclusion, NOS II expression could be induced in steroidogenic cells with a concomitant inhibition of steroid production.Fil: Cymeryng, Cora Betriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Lotito, S.P.. Universidad de Buenos Aires; ArgentinaFil: Colonna, C.. Universidad de Buenos Aires; ArgentinaFil: Cornejo Maciel, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Podestá, E. J.. Universidad de Buenos Aires; Argentin

Hypothalamic-pituitary-adrenal axis dysfunction in streptozotocin(STZ)-diabetic rats: effect of lipoic acid and ?-tocopherol treatment

Background and aims: Glucocorticoid release is involved in glucose counterregulation, and a diminished or absent adrenocortical function renders non-diabetic as well as insulin-treated diabetic subjects prone to hypoglycemia. We previously demonstrated that STZ-diabetic rats show increased basal, but markedly reduced ACTH-stimulated corticosterone (CS) levels. Present experiments, performed in STZ-diabetic rats receiving lipoic acid (LA) or ?-tocopherol (aT) treatment, were designed 1) to evaluate the effect of these treatments on oxidative stress parameters, on nitric oxide synthase (NOS) activity, and on steroidogenic function of the adrenal cortex of STZtreated rats, and 2) to test the hypothesis that STZ-induced oxidative stress might modify CS release by affecting pituitary ACTH secretion. Materials and methods: Male Wistar rats (220-240g) were randomly assigned to the following groups: CON (control), STZ (2 injections of 40 mg/ kg STZ separated by 48 h), LA (90 mg/kg ip every 48 h), aT (200 mg/kg/d po), STZ-LA or STZ-aT. LA, aT or vehicle were initiated immediately after the confirmation of hyperglycemia. After 4 weeks, all animals were sacrificed. Oxidative stress parameters and NOS activity, as well as the expression levels of CYP11A1, a key steroidogenic enzyme, and of the ACTH receptor MC2R were evaluated by RT-qPCR in the adrenal cortex. Plasma ACTH and serum CS levels (basal and ACTH-stimulated) were measured by RIA. After normality testing a one-way ANOVA was used for statistical evaluation. Results: STZ rats displayed elevated basal CS levels, and a diminished CS response to ACTH injection. Antioxidants decreased CS levels to those observed in controls (CON: 7.3 ± 2.5*, STZ: 71.7 ± 9.2, STZ aT: 5.8 ± 3.4*, STZ-LA: 19.1 ± 5.5* ng/ml; *p<0.001 vs STZ), and restored the defective response to ACTH. Lower basal ACTH levels were detected in STZ animals; neither LA nor aT had an impact on these changes. At the adrenal level, both drugs precluded the increase in TBARS and carbonyl content, and in the expression levels of catalase and heme oxygenase-1 detected in STZ rats. Both antioxidants also prevented the increase in NOS activity detected in diabetic animals (CON: 88.5 ± 0.5*, STZ: 216.7 ± 9.2, STZ-aT: 108.6 ± 7.4* and STZLA: 117.4 ± 16.8* pmol/min/mg protein; *p<0.01 vs STZ), and corrected the down-regulation of MC2R expression observed in the STZ group. CYP11A1 mRNA levels were not affected by any of these treatments. Conclusion: Our results show that, in STZ-diabetic rats, generation of oxidative stress is associated with an increased activity of the adrenocortical NO generating system, a known negative modulator of CS release. Systemic antioxidant treatment not only normalized oxidative stress parameters and NOS activity, but also corrected the observed effects of STZ-induced diabetes on CS levels. Compared to control animals, STZ-diabetic rats showed lower baseline circulating levels of ACTH, probably resulting from exaggerated basal CS output. Antioxidant therapy restored basal and ACTH-induced glucocorticoid release without modifying plasma ACTH levels, further supporting the role of local regulatory signals, such as NO, in the dysregulation of adrenal steroidogenesis observed in this animal model of diabetes. Supported by: PICT 2008 N103

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E(2)

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nω-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by ≈40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE(2), which in turn releases corticosterone stored in microvesicles and other organelles

Role of CREB on heme oxygenase-1 induction in adrenal cells: Involvement of the PI3K pathway

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe2+. ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at −146 and −37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[−295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[−295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.Fil: Astort, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Repetto, Esteban Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Rocha Viegas, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Mercau, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Sanchez Puch, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Finkielstein, C. V.. Polytechnic Institute and State University; Estados UnidosFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Cymeryng, Cora Betriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Crosstalk between nitric oxide synthases and cyclooxygenase 2 in the adrenal cortex of rats under lipopolysaccharide treatment

The effect of lipopolysaccharide on the modulation of steroid production by adrenal cells has been recently acknowledged. The purpose of this study was to determine the in vivo effects of LPS on adrenal cyclooxygenase 2 (COX-2) expression, analyze its crosstalk with the nitric oxide synthase (NOS) system, and assess its involvement on the modulation of glucocorticoid production. Male Wistar rats were injected with LPS and with specific inhibitors for NOS and COX activities. PGE2 and corticosterone levels were determined by RIA. Protein levels were analyzed by immunoprecipitation and western blotting. Transfection assays were performed in murine adrenocortical Y1 cells. Results show that LPS treatment increases PGE2 production and COX-2 protein levels in the rat adrenal cortex. Systemic inhibition of COX-2 blunted the glucocorticoid response to ACTH, as well as the increase in NOS activity and the NOS-2 expression levels induced by LPS. Conversely, NOS inhibition prevented the LPS-dependent increase in PGE2 production, COX-2 protein levels, and the nitrotyrosine modification of COX-2 protein. Treatment of adrenocortical cells with a NO-donor significantly potentiated the LPS-dependent increase in NFjB activity and COX-2 expression levels. In conclusion, our results show a significant crosstalk between COX-2 and NOS in the adrenal cortex upon LPS stimulation, in which each activity has a positive impact on the other. In particular, as both the activities differently affect adrenal steroid production, we hypothesize that this kind of fine modulation enables the gland to adjust steroidogenesis to prevent either an excessive or an insufficient response to the endotoxin challenge.Fil: Sanchez, Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Mercau, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Repetto, Esteban Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Martinez Calejman, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Astort, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Perez, Matías N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; ArgentinaFil: Arias, Pablo. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cymeryng, Cora Betriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina; Argentin