RESUMO
Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.
Assuntos
Folículo Ovariano , Fator A de Crescimento do Endotélio Vascular , Feminino , Bovinos , Animais , Folículo Ovariano/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células da Granulosa , Células Tecais , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Hormônio Adrenocorticotrópico/metabolismoRESUMO
Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C-C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary.
Assuntos
Hormônio Adrenocorticotrópico , Ovário , Feminino , Bovinos , Animais , Hormônio Adrenocorticotrópico/farmacologia , Interleucina-17 , Ligantes , LeucócitosRESUMO
This research paper addresses the hypothesis that stress, induced by ACTH administration and cortisol release increases somatic cell count (SCC) in mammary secretion, and improves the effectiveness of dry off in goats. We report indicators of milk synthesis and mammary gland involution during dry off. Thirty Saanen goats were subjected to abrupt dry off and treatments: (1) ACTH administration (ACTH) or (2) placebo (Control) on days 1, 3, 6, 9, 12, 15, 30, and 60 of dry off. The expression of target genes in mammary tissue that are related to milk synthesis and cell survival such as insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositol-3-kinase (PIK3CA), protein kinase B (AKT1) and mechanistic target of rapamycin (MTOR), casein (CSN2), lactalbumin (LALBA) and lactoferrin (LF) were evaluated, and plasma cortisol concentration, SCC, leucocyte count, and microbiological analyses in milk and mammary secretions were assessed. ACTH significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue, increased lactoferrin concentration and SCC, and changed immune cell levels in mammary secretions compared to Control. Furthermore, ACTH administration increased the percentage of dry goats compared to the Control (73 vs. 46%, respectively). We conclude that the effect of stress via ACTH administration and cortisol release accelerated mammary involution during the early dry-off period.
Assuntos
Lactação , Leite , Feminino , Animais , Leite/metabolismo , Lactação/fisiologia , Hidrocortisona , Lactoferrina/genética , Glândulas Mamárias Animais/metabolismo , Apoptose , Cabras/fisiologia , Hormônio Adrenocorticotrópico/farmacologiaRESUMO
PURPOSE: To investigate the effects of Periplaneta americana L. on ulcerative colitis (UC) induced by a combination of chronic stress (CS) and 2,4,6-trinitrobenzene sulfonic acid enema (TNBS) in rats. METHODS: The experiment UC model with CS was established in rats by a combination of chronic restraint stress, excess failure, improper, and TNBS. The body weight, disease activity index (DAI), colonic mucosal injury index (CMDI), histopathological score (HS) and pro-inflammatory mediators were measured. The content of corticotropin-releasing hormone (CRH) in hypothalamus or adrenocorticotropic hormone (ACTH) and corticosteroids (CORT) in plasma were evaluated by enzyme-linked immunosorbent assay. The proportion of T lymphocyte subsets was detected by flow cytometry, and gut microbiota was detected by 16S rDNA amplicon sequencing. RESULTS: Weight loss, DAI, CMDI, HS and proinflammatory mediators were reversed in rats by P. americana L. treatment after UC with CS. Increased epidermal growth factor (EGF) was observed in P. americana L. groups. In addition, P. americana L. could reduce the content of CRH and ACTH and regulate the ratio of CD3+, CD3+CD8+ and CD3+CD4+CD25+/CD4+ in spleen. Comparably, P. americana L. changes composition of gut microbiota. CONCLUSIONS: The ethanol extract of Periplaneta Americana L. improves UC induced by a combination of CS and TNBS in rats.
Assuntos
Colite Ulcerativa , Colite , Periplaneta , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Hormônio Adrenocorticotrópico/uso terapêutico , Animais , Colite/patologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colo/patologia , Modelos Animais de Doenças , Enema , Etanol/farmacologia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos , Ácido Trinitrobenzenossulfônico/metabolismoRESUMO
Background: The expression and overexpression of luteinizing hormone (LH) receptors in the canine adrenal gland cortex have been reported. Therefore, it was hypothesized that a LH-dependent form of Cushing's syndrome (CS) could exist in dogs. Aim: To assess whether the adrenal gland post-ovariectomy (OVx) exhibits a greater response to adrenocorticotrophin (ACTH) stimulation; to evaluate whether the adrenal gland responds to human chorionic gonadotropin (hCG) stimulation by increasing the release of cortisol; and to consider whether hCG stimulus testing would be useful as a diagnosis for possible cases of LH-dependent CS. Methods: Cortisol concentrations were measured from healthy female dogs (n=16) at baseline and following ACTH stimulation before and 2 months after gonadectomy (OVx). Cortisol concentrations were also measured for female dogs with CS (n = 14) following administration of hCG (5000 IU). A post-hCG cortisol concentration greater than 140 nmol/l was used to define dogs with LH-dependent Cushing's syndrome. Results: In normal female dogs, both pre- and post-stimulation cortisol concentrations increased following OVx (p = 0.002 and p = 0.0003, respectively). In female dogs with CS, cortisol concentrations increased following stimulation with hCG in 57% (8/14; p = 0.002). Age at the time of OVx was associated (p = 0.015) with the cortisol response to hCG [8 (5-9) years vs. 3.5 (2-6) years, p = 0.0013). Conclusion: Based on these results, an LH-dependent form of CS occurs in spayed female dogs, and that it is more likely to occur when female dogs are spayed later in life.
Assuntos
Córtex Suprarrenal , Síndrome de Cushing , Hormônio Adrenocorticotrópico/farmacologia , Animais , Gonadotropina Coriônica/farmacologia , Cães , Feminino , HidrocortisonaRESUMO
CONTEXT: The low-dose (1 µg) ACTH test (LDT) is widely used to assess central adrenal insufficiency (CAI); however, the serum cortisol cutoff value is controversial. Salivary cortisol (SC) may be a more accurate measurement for CAI. OBJECTIVE: To assess a new maximum cutoff value of serum cortisol after LDT in pediatric patients, taking into account serum and SC measurements. DESIGN AND SETTING: Prospective study in a pediatric tertiary referral center. WORKING HYPOTHESIS: The combined analysis of serum and SC response to LDT might improve LDT for CAI diagnosis. PARTICIPANT AND OUTCOME MEASUREMENT: A total of 145 pediatric patients underwent LDT. Serum and SC levels were measured. A central adrenal sufficient (CAS) response was established according to the reference serum cortisol cutoff value of ≥497 nmol/L. RESULTS: The LDT study showed central adrenal sufficiency in 72 patients and CAI in 73 patients. Considering the lower quartile of maximum SC value (21 nmol/L) in the CAS group, an intermediate CAI (InCAI) group and a real CAI (RCAI) group were defined. Regarding the median maximum value of serum cortisol levels in the InCAI group, a new serum cortisol cutoff value of 450 nmol/L was established. Furthermore, 91% of the patients in the RCAI group were below this cutoff value. CONCLUSION: The combined evaluation of maximum serum and SC levels to LDT might be useful to define an InCAI group and to avoid unnecessary hormone replacement therapy. However, rigorous patient follow-up is required.
Assuntos
Insuficiência Adrenal/diagnóstico , Hormônio Adrenocorticotrópico/farmacologia , Hidrocortisona/sangue , Sistema Hipófise-Suprarrenal/fisiopatologia , Glândulas Salivares/metabolismo , Adolescente , Insuficiência Adrenal/sangue , Hormônio Adrenocorticotrópico/sangue , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pediatria , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Estudos Prospectivos , Curva ROC , Sensibilidade e EspecificidadeRESUMO
The participation of aberrant receptors and intra-adrenal ACTH in hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells. Therefore, we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules containing steroidogenic cells, aberrant receptors and intra-adrenal ACTH. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and proliferative capacity without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures; these effects need to be explored further.
Assuntos
Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Proteínas Supressoras de Tumor/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Idoso , Proteínas do Domínio Armadillo , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Hiperplasia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/genética , Pró-Opiomelanocortina/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Receptor Tipo 2 de Melanocortina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Sequência de DNA , Coloração e Rotulagem , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Proteínas Supressoras de Tumor/genética , Vasopressinas/farmacologiaRESUMO
Glucocorticoids (GCs) such as cortisol and corticosterone are important steroid hormones with different functions in intermediate metabolism, development, cell differentiation, immune response and reproduction. In response to physiological and immunological stress, adrenocorticotropic hormone (ACTH) acts on the adrenal gland by stimulating the synthesis and secretion of GCs. However, there is increasing evidence that GCs may also be synthesized by extra-adrenal tissues. Here, we examined the gene and protein expression of the enzyme 11ß-hydroxylase P450c11 (CYP11B1), involved in the conversion of 11-deoxycortisol to cortisol, in the different components of the bovine ovary and determined the functionality of CYP11B1 in vitro CYP11B1 mRNA was expressed in granulosa and theca cells in small, medium and large antral ovarian follicles, and CYP11B1 protein was expressed in medium and large antral follicles. After stimulation by ACTH, we observed an increased secretion of cortisol by the wall of large antral follicles. We also observed a concentration-dependent decrease in the concentration of cortisol in response to metyrapone, an inhibitor of CYP11B1. This decrease was significant at 10-5 µM metyrapone. In conclusion, this study demonstrated for the first time the presence of CYP11B1 in the bovine ovary. This confirms that there could be a local synthesis of GCs in the bovine ovary and therefore a potential endocrine responder to stress through these hormones.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/enzimologia , Esteroide 11-beta-Hidroxilase/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Bovinos , Células Cultivadas , Feminino , Hormônios/farmacologia , Hidrocortisona/metabolismo , Folículo Ovariano/citologia , Esteroide 11-beta-Hidroxilase/genéticaRESUMO
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/Fe(2+) 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.
Assuntos
Glândulas Suprarrenais/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.