RESUMO
Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.
Assuntos
Córtex Suprarrenal/química , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Córtex Suprarrenal/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Fixação de TecidosRESUMO
Despite being a highly conserved protein, the precise role of the mitochondrial translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), remains elusive. The void created by studies that overturned a presumptive model that described TSPO/PBR as a mitochondrial cholesterol transporter for steroidogenesis has been filled with evidence that it can affect mitochondrial metabolic functions across different model systems. We previously reported that TSPO/PBR deficient steroidogenic cells upregulate mitochondrial fatty acid oxidation and presented a strong positive correlation between TSPO/PBR expression and tissues active in triglyceride metabolism or lipid storage. Nevertheless, the highlighting of inconsistencies in prior work has provoked reprisals that threaten to stifle progress. One frequent factoid presented as being supportive of a cholesterol import function is that there are no steroid-synthesizing cell types without high TSPO/PBR expression. In this study, we examine the hamster adrenal gland that is devoid of lipid droplets in the cortex and largely relies on de novo cholesterol biosynthesis and uptake for steroidogenesis. We find that Tspo expression in the hamster adrenal is imperceptible compared to the mouse. This observation is consistent with a substantially low expression of Cpt1a in the hamster adrenal, indicating minimal mitochondrial fatty acid oxidation capacity compared to the mouse. These findings provide further reinforcement that the much sought-after mechanism of TSPO/PBR function remains correlated with the extent of cellular triglyceride metabolism. Thus, TSPO/PBR could have a homeostatic function relevant only to steroidogenic systems that manage triglycerides associated with lipid droplets.
Assuntos
Glândulas Suprarrenais/metabolismo , Expressão Gênica , Mesocricetus/metabolismo , Receptores de GABA-A/genética , Triglicerídeos/metabolismo , Córtex Suprarrenal/química , Glândulas Suprarrenais/ultraestrutura , Animais , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Lipídeos/análise , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/química , Mitocôndrias/metabolismo , Ovário/metabolismo , Receptores de GABA-A/análise , Receptores de GABA-A/fisiologia , Especificidade da Espécie , Esteroides/biossínteseRESUMO
PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.
Assuntos
Corticosteroides/metabolismo , Aldosterona/biossíntese , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/metabolismo , Metaboloma , Adenoma/complicações , Córtex Suprarrenal/química , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/fisiopatologia , Corticosteroides/análise , Aldosterona/análise , Cromatografia Líquida , Citocromo P-450 CYP11B2/biossíntese , Citocromo P-450 CYP11B2/sangue , Humanos , Hiperaldosteronismo/sangue , Hiperaldosteronismo/fisiopatologia , Hipertensão/etiologia , Imuno-Histoquímica , Valor Preditivo dos Testes , Prognóstico , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Adrenocortical tumors (ACT) are uncommon in the pediatric age group. Using the standard Weiss criteria in pediatric tumors leads to overdiagnosis. This has led to the development of newer systems such as Weineke criteria. Ki67 labeling index aids in differentiating adenomas from carcinomas. We aim to evaluate the diagnostic and prognostic role of Ki67 labeling index, along with immunoexpression of steroidogenic factor-1, insulin like growth factor 2 and p57, in pediatric ACTs diagnosed using Weineke criteria. METHODS: We have studied 25 cases of pediatric ACTs. Immunohistochemical staining for Ki67, SF-1, IGF2 and p57 was done in all cases and the result was correlated with the morphological diagnosis using the Weineke criteria. RESULTS: Ki67 labeling index showed complete concordance with the morphological diagnosis. SF-1 and IGF2 showed similar correlation with the diagnosis, with IGF-2 proving to be a more specific marker. Increased Ki67, SF-1 and IGF2 immunostaining also correlated with worse survival. p57 was more specific in determining benign status of a tumor. CONCLUSION: SF-1 and IGF2 are highly sensitive markers of malignancy in pediatric ACTs and can be used in combination with Ki67 expression for optimal diagnostic and prognostic assessment of pediatric ACTs. TYPE OF STUDY: Prognosis study. LEVEL OF EVIDENCE: Level II.
Assuntos
Neoplasias do Córtex Suprarrenal , Córtex Suprarrenal , Inibidor de Quinase Dependente de Ciclina p57 , Fator de Crescimento Insulin-Like II , Fator Esteroidogênico 1 , Córtex Suprarrenal/química , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/química , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Criança , Inibidor de Quinase Dependente de Ciclina p57/análise , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like II/análise , Fator de Crescimento Insulin-Like II/metabolismo , Prognóstico , Fator Esteroidogênico 1/análise , Fator Esteroidogênico 1/metabolismoRESUMO
Environmental pollutants may act as endocrine disruptors in animals. Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) enter the food chain and may accumulate in the fatty animal tissues, including adrenals. To our knowledge, no previous study has investigated their presence in the human normal adrenal (NA) cortex and aldosterone-producing adenomas (APA). Surgical fragments of APA from 11 patients and NA from 8 kidney donors were analyzed for 16 PCBs congeners and 10 OCPs. A Matrix Solid-Phase Dispersion (MSPD) method for simultaneous determination of the target compounds in cortex homogenates was developed. A gas-chromatography triple quadrupole mass spectrometry (Triple Quad GC-MS) system was used for the analysis. Data were analyzed using Random Forest and Wilcoxon's rank-sum test. OCPs and PCBs were found in specimens from both types. A subset of pollutants characterized APA more than NA. Higher concentrations (µg g-1 ) in APA were observed for α-, ß-, and γ- Hexachlorocyclohexane (HCH) (1.48 ± 3.32 vs. 0.17 ± 0.19, P = 0.028; 2.81 ± 2.10 vs. 0.96 ± 0.98, P = 0.011; 2.16 ± 4.85 vs. 0.17 ± 0.26, P = 0.004, respectively), as well as for Hexachlorobenzene (HCB) and for PCBs 28, 52 and 101 (3.41 ± 3.11 vs. 0.97 ± 1.06, P = 0.021; 2.34 ± 4.68 vs. 0.25 ± 0.22, P = 0.039; 0.58 ± 1.19 vs. 0.06 ± 0.02, P = 0.002; 0.26 ± 0.43 vs. 0.05 ± 0.00, P = 0.001, respectively). Environmental organochlorine pollutants were shown to be present in the human normal and abnormal adrenal cortex, deserving future investigation on their possible role as adrenal endocrine disruptors in human disease. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Córtex Suprarrenal/química , Poluentes Ambientais/análise , Hidrocarbonetos Clorados/análise , Praguicidas/análise , Bifenilos Policlorados/análise , Adenoma/química , Adulto , Idoso , Aldosterona/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
Female laboratory macaques were studied under a variety of treatment protocols to determine if immunoreactive luteinizing hormone/gonadal chorionic gonadotropin (LH/CG) receptors were present in the adrenal cortex. All adrenal tissues revealed an absence of immunoreactivity in the in the medulla while staining was present in all three outer zones of the cortex. Increased staining was observed in the zonae reticularis with least staining in the zonae glomerulosa. Moderate and variable staining was found in the zonae fasciculata. These results demonstrate that LH/CG receptors in the adrenal cortex may be more common in higher primates than previously recognized and help explain some aspects of the endocrine changes observed in mid-aged women during the menopausal transition when circulating LH concentrations are rising.
Assuntos
Córtex Suprarrenal/química , Córtex Suprarrenal/metabolismo , Hormônio Luteinizante/metabolismo , Receptores do LH/análise , Receptores do LH/metabolismo , Fatores Etários , Animais , Feminino , Macaca mulatta , Ovariectomia , Distribuição AleatóriaRESUMO
We describe the pathology of 5 patients with germline PRKACA copy number gain and Cushing syndrome: 4 males and 1 female, aged 2 to 43 years, including a mother and son. Imaging showed normal or slightly enlarged adrenal glands in 4 patients and a unilateral mass in the fifth. Biochemically, the patients had corticotropin-independent hypercortisolism. Four underwent bilateral adrenalectomy; unilateral adrenalectomy was performed in the patient with the adrenal mass. Pathologically, 3 patients, including the 1 with the tumor (adenoma), had primary pigmented nodular adrenocortical disease with extranodular cortical atrophy and mild intracapsular and extracapsular extension of cortical cells. The other 2 patients had cortical hyperplasia and prominent capsular and extracapsular micronodular cortical hyperplasia. Immunoperoxidase staining revealed differences for synaptophysin, inhibin-A, and Ki-67 (nuclei) in the atrophic cortices (patients 1, 2, and 3) and hyperplastic cortices (patients 4 and 5) and for Ki-67 (nuclei) and vimentin in the extracortical nodules in the 2 groups of patients. ß-Catenin stained the cell membrane, cytoplasm, and nuclei of the adenoma. The patients were well at follow-up (1-23 years); 24-hour urinary cortisol excretion was elevated in the patient who had unilateral adrenalectomy.
Assuntos
Córtex Suprarrenal/patologia , Síndrome de Cushing/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Variações do Número de Cópias de DNA , Amplificação de Genes , Dosagem de Genes , Córtex Suprarrenal/química , Córtex Suprarrenal/diagnóstico por imagem , Córtex Suprarrenal/cirurgia , Adrenalectomia , Adulto , Atrofia , Biomarcadores/análise , Biópsia , Criança , Pré-Escolar , Síndrome de Cushing/diagnóstico por imagem , Síndrome de Cushing/enzimologia , Síndrome de Cushing/patologia , Síndrome de Cushing/cirurgia , Feminino , Predisposição Genética para Doença , Humanos , Hiperplasia , Imuno-Histoquímica , Inibinas/análise , Antígeno Ki-67/análise , Imageamento por Ressonância Magnética , Masculino , Fenótipo , Sinaptofisina/análise , Fatores de Tempo , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Vimentina/análise , Adulto JovemRESUMO
The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from the coelomic epithelium of the urogenital ridge. Neither the intercellular signals that regulate cortical development and maintenance nor the lineage relationships within the adrenal are well defined. We have explored adrenal Shh activity and found that Shh is expressed in relatively undifferentiated steroidogenic cells, which signal to the overlying capsule and subjacent nonsteroidogenic mesenchyme cells that we also find are progenitors of steroidogenic lineages. Shh-expressing cells also generate all steroidogenic cell types, but not nonsteroidogenic ones. Shh mutant adrenals have a thin capsule and small cortex. Our findings both support a novel dual lineage, Shh-independent and Shh-dependent, model of adrenocortical development, and identify distinct populations of adrenocortical progenitor and candidate stem cells.
Assuntos
Córtex Suprarrenal/crescimento & desenvolvimento , Linhagem da Célula , Proteínas Hedgehog/fisiologia , Transdução de Sinais , Células-Tronco/citologia , Esteroides/biossíntese , Córtex Suprarrenal/química , Animais , Diferenciação Celular , Proteínas Hedgehog/análise , Fatores de Transcrição Kruppel-Like/análise , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Proteína GLI1 em Dedos de ZincoRESUMO
DESIGN AND METHODS: The adrenal cortex is not considered to be an intrinsic part of the diffuse neuroendocrine system, but adrenocortical neoplasms possess neuroendocrine properties. In this study, we examined synaptophysin (SYP) and neural cell adhesion molecule (NCAM) expression in adrenocortical adenomas in relation to adrenal function. RESULTS: Immunohistochemical analysis showed that 50.7 and 98.6% of the cortical adenomas showed SYP and NCAM immunoreactivities respectively. There was no apparent difference in NCAM immunoreactivity among the adenomas. However, the immunostaining for SYP was significantly stronger in cortisol-producing adenomas (CPA) than in aldosterone-producing adenomas (APA), nonfunctioning adenomas (NFA), showing no clinical or endocrinological abnormality, or adenomas associated with preclinical Cushing's syndrome (preCS). Western blotting and real-time PCR demonstrated that the expression level of SYP protein and mRNA was significantly higher in CPA than in APA or NFA. Additionally, the SYP mRNA level showed a positive correlation with CYP17A1 mRNA. In addition to the plasma membrane, mitochondria, and smooth endoplasmic reticulum, SYP immunoreactivity was detected in the Golgi area, which is known to be involved in the regulation of mitochondrial cholesterol and the transport of steroid intermediates. It was unexpected that the ratio of positive cells for SYP in preCS was less than that in APA and NFA. However, further examination is required, because the number of preCS cases we investigated was very small. CONCLUSIONS: We propose that SYP expression in adrenocortical cells may be involved in some aspect of adrenal function such as transport or secretion of glucocorticoids.
Assuntos
Córtex Suprarrenal/química , Neoplasias das Glândulas Suprarrenais/química , Esteroide 17-alfa-Hidroxilase/biossíntese , Sinaptofisina/análise , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Humanos , Moléculas de Adesão de Célula Nervosa/biossíntese , RNA Mensageiro/metabolismo , Sinaptofisina/biossínteseRESUMO
Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-micro (PKC-micro) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP(3)) and calcium ([Ca(2+)](i)). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-alpha/betaII, -epsilon, and -micro but not PKC-delta, -theta, or -zeta/lambda of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-epsilon phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP(3) and [Ca(2+)](i). Inhibition of PKC-epsilon translocation by the PKC-epsilon-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP(3) and [Ca(2+)](i). We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-epsilon phosphorylation and [Ca(2+)](i) elevation.
Assuntos
Aldosterona/metabolismo , Angiotensina II/farmacologia , Cálcio/fisiologia , Proteína Quinase C-épsilon/fisiologia , Receptores de Dopamina D4/fisiologia , Transdução de Sinais/fisiologia , Córtex Suprarrenal/química , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/fisiologia , Neoplasias do Córtex Suprarrenal , Linhagem Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP11B2/genética , Humanos , Inositol 1,4,5-Trifosfato/análise , Fosforilação , RNA Mensageiro/análise , Receptores de Dopamina D4/genéticaRESUMO
Expression of a previously cloned secretory protein named adrenocortical zonation factor 1 (AZ-1, also called Tin-ag-RP or lipocalin 7) is tightly linked with the zonal differentiation of adrenocortical cells. It is also present in vascular smooth muscle (VSM), although its function has remained unknown. In this study, the location of AZ-1 was specified to the basal laminae along adrenocortical sinusoidal capillaries and surrounding VSM cells in the arterial system, consistent with the fact that AZ-1 was extractable under denaturing conditions as a 52 kDa polypeptide. Purified recombinant AZ-1 exhibited abilities to bind to fibronectins via the first type III repeat (anastellin) and to collagens with affinities in submicromolar ranges. AZ-1 immobilized on substratum or bound to collagens or anastellin promoted adhesion and spreading of adrenocortical cells. Although VSM cells spread on AZ-1 slowly, AZ-1 bound to anastellin facilitated the spreading. The adhesion activity of AZ-1 was mediated by a subset of integrins, including alpha(1)beta(1), alpha(2)beta(1), and alpha(5)beta(1), in a cell type-specific manner. Collectively with the putative role of AZ-1 in the adrenocortical zonation, we propose that AZ-1 potentially regulates functions of adrenocortical and VSM cells by modulating cell-matrix interactions.
Assuntos
Córtex Suprarrenal/citologia , Proteínas de Transporte/fisiologia , Adesão Celular/fisiologia , Integrinas/fisiologia , Músculo Liso Vascular/citologia , Proteínas de Neoplasias/fisiologia , Córtex Suprarrenal/química , Animais , Aorta/química , Proteínas de Transporte/isolamento & purificação , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Integrina alfa1beta1/fisiologia , Integrina alfa2beta1/fisiologia , Integrina alfa5beta1/fisiologia , Lipocalinas , Camundongos , Proteínas de Neoplasias/isolamento & purificação , Desnaturação ProteicaRESUMO
CITED2 gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.
Assuntos
Córtex Suprarrenal/metabolismo , Proteínas de Ligação a DNA/análise , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Proteínas Repressoras/análise , Transativadores/análise , Córtex Suprarrenal/química , Córtex Suprarrenal/embriologia , Carcinoma Adrenocortical , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Adulto , Linhagem Celular Tumoral , Células Cultivadas , Colforsina/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Desenvolvimento Embrionário , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica/métodos , Microscopia de Fluorescência , RNA Mensageiro/análise , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transativadores/genética , Transativadores/metabolismo , Transfecção/métodosRESUMO
Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.
Assuntos
Córtex Suprarrenal/química , Cardanolídeos/isolamento & purificação , Cardenolídeos/isolamento & purificação , Hipertensão/induzido quimicamente , Saponinas/isolamento & purificação , Córtex Suprarrenal/citologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Pressão Sanguínea/efeitos dos fármacos , Bufanolídeos/imunologia , Bufanolídeos/urina , Cardanolídeos/administração & dosagem , Cardanolídeos/farmacologia , Cardenolídeos/administração & dosagem , Cardenolídeos/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Esquema de Medicação , Ensaio de Imunoadsorção Enzimática , Injeções Intraperitoneais , Masculino , Espectrometria de Massas , Ratos , Ratos Wistar , Saponinas/administração & dosagem , Saponinas/farmacologiaRESUMO
We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81-88 sequence of the precursor of human interleukin-1alpha ([3H]GKVLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (Kd 2.2 +/- 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10-20 microg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 microg/animal, three times) or significantly decreases (at a dose of 10 microg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.
Assuntos
Corticosteroides/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Interleucina-1alfa/farmacologia , Fragmentos de Peptídeos/farmacologia , Substâncias Protetoras/farmacologia , Receptores da Corticotropina/agonistas , Estresse Fisiológico/prevenção & controle , Administração Intranasal , Córtex Suprarrenal/química , Córtex Suprarrenal/metabolismo , Corticosteroides/análise , Hormônio Adrenocorticotrópico/química , Sequência de Aminoácidos , Animais , Humanos , Interleucina-1alfa/química , Interleucina-1alfa/metabolismo , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/metabolismo , Ratos , Ratos Endogâmicos , Receptores da Corticotropina/metabolismoRESUMO
In mammals, the master clock of the suprachiasmatic nuclei (SCN) and subordinate clocks found throughout the body coordinate circadian rhythms of behavior and physiology. We characterize the clock of the adrenal, an important endocrine gland that synchronizes physiological and metabolic rhythms. Clock gene expression was detected in the outer adrenal cortex prefiguring a role of the clock in regulating gluco- and mineral corticoid biogenesis. In Per2/Cry1 double mutant mice, which lack a circadian clock, hypothalamus/pituitary/adrenal axis regulation was defective. Organ culture and tissue transplantation suggest that the adrenal pacemaker gates glucocorticoid production in response to adrenocorticotropin (ACTH). In vivo the adrenal circadian clock can be entrained by light. Transcriptome profiling identified rhythmically expressed genes located at diverse nodes of steroid biogenesis that may mediate gating of the ACTH response by the adrenal clock.
Assuntos
Corticosteroides/metabolismo , Córtex Suprarrenal/química , Córtex Suprarrenal/metabolismo , Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Corticosteroides/análise , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Criptocromos , Flavoproteínas/genética , Flavoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Circadianas Period , Transdução de Sinais , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CONTEXT: Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland. OBJECTIVE: The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland. DESIGN: In vitro studies were conducted on cultured human adrenocortical cells. SETTING: This study was conducted in a university research laboratory. PATIENTS: Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied. MAIN OUTCOME MEASURE: Cortisol secretion from cultured adrenocortical cells was measured. RESULTS: NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis. CONCLUSION: Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Hidrocortisona/metabolismo , Neurotensina/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Adaptadoras de Transporte Vesicular , Córtex Suprarrenal/química , Hormônio Adrenocorticotrópico/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neurotensina/genética , Fragmentos de Peptídeos/genética , RNA Mensageiro/análise , Receptores de Neurotensina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Congenital lipoid adrenal hyperplasia (lipoid CAH) is an autosomal recessive disorder characterized by severe adrenal insufficiency and male sex reversal. Lipoid CAH is caused by mutations in two proteins that are essential for all steroid biosynthesis, the steroidogenic acute regulatory (StAR) protein and cytochrome P450scc. In this review, we discuss the clinical presentation and mechanisms behind the pathology of this fatal disorder.
Assuntos
Hiperplasia Suprarrenal Congênita/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Mutação , Fosfoproteínas/genética , Córtex Suprarrenal/química , Córtex Suprarrenal/embriologia , Córtex Suprarrenal/crescimento & desenvolvimento , Hiperplasia Suprarrenal Congênita/complicações , Hiperplasia Suprarrenal Congênita/epidemiologia , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Química Encefálica , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Ovário/química , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Fosfoproteínas/análise , Fosfoproteínas/fisiologia , Esteroides/biossíntese , Testículo/química , Testículo/embriologia , Testículo/crescimento & desenvolvimentoRESUMO
OBJECTIVES: Adrenocortical carcinoma (ACC) is a rare malignant neoplasm with extremely poor prognosis. The molecular mechanisms of adrenocortical tumorigenesis are still not well understood. The comparative analysis by cDNA microarrays of gene-expression patterns of benign and malignant adrenocortical tumors allows us to identify new tumor-suppressor genes and proto-oncogenes underlying adrenocortical tumorigenesis. DESIGN AND METHODS: Total RNA from fresh-frozen tissue of 10 ACC and 10 benign adrenocortical adenomas was isolated after histologic confirmation of neoplastic cellularity of at least 85%. The reference consisted of pooled RNA of 10 normal adrenal cortex samples. Amplified RNA of tumor and reference was used to synthesize Cy3- and Cy5-fluorescently labeled cDNA in a flip-color technique. D-chips containing 11 540 DNA spots were hybridized and scanned and the images were analyzed by ImaGene 3.0 software. RESULTS: The comparative analysis of gene expression revealed many genes with more than fourfold expression difference between ACC and normal tissue (42 genes), cortical adenoma and normal tissue (11 genes), and ACC and cortical adenoma (21 genes) respectively. As confirmed by real-time PCR, the IGF2 gene was significantly upregulated in ACCs versus cortical adenomas and normal cortical tissue. Genes that were downregulated in adrenocortical tumors included chromogranin B and early growth response factor 1. CONCLUSIONS: Comprehensive expression profiling of adrenocortical tumors by the cDNA microarray technique is a very powerful tool to elucidate the molecular steps associated with the tumorigenesis of these ill-defined neoplasms. To evaluate the role of identified genes, further detailed analyses, including correlation with clinical data, are required.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Adenoma/genética , Adenoma/metabolismo , Adolescente , Córtex Suprarrenal/química , Neoplasias do Córtex Suprarrenal/metabolismo , Adulto , Idoso , Aldosterona/biossíntese , Feminino , Humanos , Hidrocortisona/biossíntese , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin alpha, beta(A) and beta(B) subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2-7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [(125)I]inhibin A specifically bound to cells of the adrenal cortex, mainly in the zona reticularis. Scatchard analyses of in vitro [(125)I]inhibin A binding to dispersed rat adrenal cells and AC cells revealed sites of high affinity (K(d)(1) of 0.18 and 0.15 nM, respectively) and low affinity (K(d)(2) of 2.6 and 1.3 nM, respectively. Competition for [(125)I]inhibin A binding by activin A or B (30 nM) was negligible, whereas BMP-2, -6 and -7 competed for between 21 and 33% of specific inhibin A binding (IC(50) between 0.2 and 0.3 nM). Inhibin B crossreaction with inhibin A binding sites was < 8%. Multiple binding protein complexes (molecular weight ranging from 35 to > 220 kDa) were affinity labelled by [(125)I]inhibin A on both the primary rat adrenal and AC cells. The species of > 220 kDa were shown by immunoprecipitation to include betaglycan, the species of 105 kDa is consistent in size with type II receptors for activin/BMP, and that of 62 kDa co-migrates with the inhibin-follistatin complex. In summary, the results show that inhibin A binds selectively and with both high and low affinity to AC cells via multiple binding proteins, including a single betaglycan-like species. The results support the role of glycosylated betaglycan in the high affinity binding of inhibin A, but provide consistent evidence from two independent sources of adrenal cells that inhibin A interacts with several membrane proteins in addition to those currently understood to mediate the anti-activin/BMP actions of inhibin.
Assuntos
Ativinas/metabolismo , Córtex Suprarrenal/química , Comunicação Autócrina/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Inibinas/análise , Peptídeos e Proteínas de Sinalização Intracelular/análise , Córtex Suprarrenal/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoprecipitação , Inibinas/genética , Inibinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Marcação por Isótopo , Masculino , Ligação Proteica , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: In NaCl-loaded Dahl salt-sensitive (DS) rats the transient stimulation of brain endogenous ouabain (EO) precedes the increase in renal excretion of marinobufagenin (MBG), a vasoconstrictor and natriuretic. In hypertensive DS rats, EO raises blood pressure (BP) via an ATII-sensitive pathway. We hypothesized that an NaCl-induced increase in MBG is linked to the EO-stimulated ATII pathway. METHODS: We studied the effects of 3 h of NaCl loading (17 mmol/kg, intraperitoneally) in male DS rats treated with antibodies to MBG or ouabain, or with losartan (25 mg/kg). RESULTS: NaCl loading alone induced a transient stimulation of pituitary EO (22.4 +/- 1.8 versus 12.2 +/- 1.3 pmol/g) and ATII (39.4 +/- 2.8 versus 18.4 +/- 3.2 ng/g), a sustained increase in MBG excretion (5.2 +/- 0.6 versus 1.1 +/- 0.2 pmol/h), a 40% inhibition of the renal sodium pump, a natriuretic response, a 35 mmHg increase in systolic BP, and an increase in adrenocortical ATII and MBG levels and in plasma norepinephrine. The anti-MBG antibody reduced the natriuresis (36%) and BP (40 mmHg), and restored renal sodium pump activity. The anti-ouabain antibody prevented the increase in pituitary ATII, reduced MBG excretion, natriuresis and BP, increased sodium pump activity, and prevented increases in plasma norepinephrine, pituitary and adrenocortical ATII, and adrenocortical MBG. Losartan mimicked the effects of the anti-ouabain antibody, but did not affect the excretion of EO. In adrenocortical cells of DS rats, ATII stimulated MBG secretion, and losartan blocked this effect. CONCLUSIONS: In response to NaCl loading, brain EO, via an AT1 receptor pathway and probably via sympathetic activation, stimulates adrenocortical MBG, which inhibits the renal sodium pump and elevates BP.