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1.
Am J Physiol Cell Physiol ; 321(1): C158-C175, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34038243

RESUMO

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.


Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canal de Potássio Kv1.4/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Zona Glomerulosa/metabolismo , Adolescente , Adulto , Aldosterona/biossíntese , Ácido Araquidônico/farmacologia , Autopsia , Criança , Colforsina/farmacologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Expressão Gênica , Humanos , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacos
2.
J Clin Endocrinol Metab ; 106(5): 1389-1397, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33524149

RESUMO

BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.


Assuntos
Córtex Suprarrenal/patologia , Córtex Suprarrenal/fisiologia , Envelhecimento/fisiologia , Adolescente , Córtex Suprarrenal/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Estudos de Casos e Controles , Citocromo P-450 CYP11B2/metabolismo , Citocromos b5/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Progesterona Redutase/metabolismo , Esteroide 11-beta-Hidroxilase/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Adulto Jovem , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia , Zona Reticular/metabolismo , Zona Reticular/patologia
3.
Bull Exp Biol Med ; 170(1): 101-105, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33231799

RESUMO

The study is aimed at elucidation of ultrastructural mechanisms underlying impaired aldosterone synthesis by glomerulosa cells in Wistar rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal development. Analysis of rat zona glomerulosa histology and function during the pubertal and postpubertal periods showed that exposure to endocrine disrupter DDT disturbs its development and reduced the production of aldosterone. Electron microscopy showed that changes in the aldosterone synthesis are related to impaired reorganization of the mitochondrial apparatus, one of the leading factors in the regulation of steroidogenesis, in glomerulosa cells in DDT-exposed rats during puberty.


Assuntos
Aldosterona/sangue , DDT/toxicidade , Disruptores Endócrinos/toxicidade , Mitocôndrias/ultraestrutura , Efeitos Tardios da Exposição Pré-Natal/patologia , Zona Glomerulosa/ultraestrutura , Animais , Feminino , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Ratos , Ratos Wistar , Maturidade Sexual/efeitos dos fármacos , Maturidade Sexual/fisiologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
4.
Hypertension ; 76(6): 1769-1777, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33070662

RESUMO

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.


Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Dieta Hipossódica/métodos , Sódio na Dieta/farmacologia , Esteroides/metabolismo , Transcriptoma/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Animais , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Masculino , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/genética , Sódio na Dieta/administração & dosagem , Sódio na Dieta/metabolismo , Suínos , Transcriptoma/genética , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo
5.
Endocrinology ; 161(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32785697

RESUMO

The physiological stimulation of aldosterone production in adrenocortical glomerulosa cells by angiotensin II and high plasma K+ depends on the depolarization of the cell membrane potential and the subsequent Ca2+ influx via voltage-activated Ca2+ channels. Germline mutations of the low-voltage activated T-type Ca2+ channel CACNA1H (Cav3.2) have been found in patients with primary aldosteronism. Here, we investigated the electrophysiology and Ca2+ signaling of adrenal NCI-H295R cells overexpressing CACNA1H wildtype and mutant M1549V in order to understand how mutant CACNA1H alters adrenal cell function. Whole-cell patch-clamp measurements revealed a strong activation of mutant CACNA1H at the resting membrane potential of adrenal cells. Both the expression of wildtype and mutant CACNA1H led to a depolarized membrane potential. In addition, cells expressing mutant CACNA1H developed pronounced action potential-like membrane voltage oscillations. Ca2+ measurements showed an increased basal Ca2+ activity, an altered K+ sensitivity, and abnormal oscillating Ca2+ changes in cells with mutant CACNA1H. In addition, removal of extracellular Na+ reduced CACNA1H current, voltage oscillations, and Ca2+ levels in mutant cells, suggesting a role of the partial Na+ conductance of CACNA1H in cellular pathology. In conclusion, the pathogenesis of stimulus-independent aldosterone production in patients with CACNA1H mutations involves several factors: i) a loss of normal control of the membrane potential, ii) an increased Ca2+ influx at basal conditions, and iii) alterations in sensitivity to extracellular K+ and Na+. Finally, our findings underline the importance of CACNA1H in the control of aldosterone production and support the concept of the glomerulosa cell as an electrical oscillator.


Assuntos
Glândulas Suprarrenais/fisiopatologia , Canais de Cálcio Tipo T/genética , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Aldosterona/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Canais de Cálcio Tipo T/metabolismo , Cricetinae , Cricetulus , Humanos , Hiperaldosteronismo/patologia , Hiperaldosteronismo/fisiopatologia , Potenciais da Membrana , Mutação , Técnicas de Patch-Clamp , Sódio/metabolismo , Células Tumorais Cultivadas , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia , Zona Glomerulosa/fisiopatologia
6.
Nat Commun ; 11(1): 1679, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245948

RESUMO

Aldosterone-producing zona glomerulosa (zG) cells of the adrenal gland arrange in distinct multi-cellular rosettes that provide a structural framework for adrenal cortex morphogenesis and plasticity. Whether this cyto-architecture also plays functional roles in signaling remains unexplored. To determine if structure informs function, we generated mice with zG-specific expression of GCaMP3 and imaged zG cells within their native rosette structure. Here we demonstrate that within the rosette, angiotensin II evokes periodic Cav3-dependent calcium events that form bursts that are stereotypic in form. Our data reveal a critical role for angiotensin II in regulating burst occurrence, and a multifunctional role for the rosette structure in activity-prolongation and coordination. Combined our data define the calcium burst as the fundamental unit of zG layer activity evoked by angiotensin II and highlight a novel role for the rosette as a facilitator of cell communication.


Assuntos
Aldosterona/metabolismo , Angiotensina II/metabolismo , Cálcio/metabolismo , Zona Glomerulosa/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Técnicas de Cultura de Tecidos
7.
Nat Commun ; 11(1): 1680, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245949

RESUMO

Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of ß-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas ß-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in ß-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.


Assuntos
Junções Aderentes/metabolismo , Morfogênese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Zona Glomerulosa/crescimento & desenvolvimento , beta Catenina/metabolismo , Junções Aderentes/genética , Junções Aderentes/ultraestrutura , Neoplasias das Glândulas Suprarrenais/cirurgia , Animais , Animais Recém-Nascidos , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo , Zona Glomerulosa/ultraestrutura , beta Catenina/genética
8.
Int J Mol Sci ; 21(2)2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31963151

RESUMO

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein ßarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. ß-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the ßAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the ßAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol's effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the ß-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a ßAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.


Assuntos
Aldosterona/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Western Blotting , Linhagem Celular , Quinase 2 de Receptor Acoplado a Proteína G/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina/genética , Receptores Adrenérgicos beta/genética
9.
Biochem Biophys Res Commun ; 524(1): 184-189, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31982132

RESUMO

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated ß3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced ß3-adrenergic receptor expression. Hypoxia-mediated ß3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.


Assuntos
Córtex Suprarrenal/patologia , Aldosterona/biossíntese , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Hipóxia/metabolismo , Hipóxia/patologia , Córtex Suprarrenal/efeitos dos fármacos , Animais , Síndrome Cardiorrenal/complicações , Catecolaminas/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Colesterol/metabolismo , Modelos Animais de Doenças , Humanos , Hipóxia/complicações , Masculino , Fosforilação/efeitos dos fármacos , Ratos Endogâmicos Dahl , Receptores Adrenérgicos beta 3/metabolismo , Esterol Esterase/metabolismo , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
10.
Hypertension ; 75(3): 634-644, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31957522

RESUMO

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.


Assuntos
Adenoma/patologia , Neoplasias do Córtex Suprarrenal/patologia , Aldosterona/biossíntese , Hiperaldosteronismo/metabolismo , Zona Glomerulosa/patologia , Adenoma/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Divisão Celular , Citocromo P-450 CYP11B2/análise , Análise de Fourier , Humanos , Hiperaldosteronismo/etiologia , Redes e Vias Metabólicas , Proteínas de Neoplasias/análise , Fenótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esteroide 11-beta-Hidroxilase/análise , Zona Glomerulosa/metabolismo
11.
Mol Cell Endocrinol ; 501: 110657, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31751625

RESUMO

Activating mutations in the KCNJ5 gene are responsible for the significant number of aldosterone-producing adenomas. To elucidate the molecular mechanisms underlying KCNJ5 expression, we characterized the entire human KCNJ5 gene. The gene spanned approximately 29.8 kb and contained three exons and two introns. The strongest expression of KCNJ5 mRNA was observed in the adrenal gland. The promoter region contained a putative binding site for SF-1 at -1782 bp. A construct containing -2444 bp of the promoter region exhibited the strongest promoter activity in adrenal H295R cells, and the introduction of a mutation in the SF-1 binding site almost completely abolished promoter activity. Furthermore, deletion mutation, EMSA, and knockdown analyses revealed that SF-1 bound to this element and was functional. Immunochemistry showed that KCNJ5 was predominantly expressed in the zona glomerulosa, while SF-1 was ubiquitously expressed in the adrenal cortex. These results demonstrated that SF-1 mediates the expression of human KCNJ5 in the adrenal cortex.


Assuntos
Córtex Suprarrenal/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Regiões Promotoras Genéticas/fisiologia , Fator Esteroidogênico 1/metabolismo , Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/metabolismo , Linhagem Celular Tumoral , Genômica , Células HeLa , Células Hep G2 , Humanos , Mutação/fisiologia , RNA Mensageiro/metabolismo , Zona Glomerulosa/metabolismo
12.
Cell Calcium ; 84: 102104, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31706065

RESUMO

CaV3.2 calcium channels play important roles in both neural excitability and aldosterone secretion. Recent clinical studies found four germline mutations (S196 L, M1549I, V1951E and P2083 L) in CaV3.2 channels. All four mutations caused primary aldosteronism (PA), while only the M1549I mutation resulted in obvious neural malfunctions besides PA. In human, there are two major CaV3.2 channel gene (CACNA1H) splice variants, either with or without exon 26. In this study, we tested the expression of the two CACNA1H splice variants in zona glomerulosa (ZG) cells of human adrenal cortex and the possibility that CaV3.2 (-26) and CaV3.2 (+26) channels have different functional responses to the four PA mutations. We found that human ZG cells only express long form CaV3.2(+26) channels. The M1549I mutation slowed the inactivation of CaV3.2(+26) more than 5 fold, and CaV3.2(-26) more than 2 fold. The S196 L, V1951E and P2083 L mutations accelerated channel recovery from inactivation for CaV3.2(+26), but not CaV3.2(-26) channels. All four mutations resulted in gain of function of CaV3.2(+26) channels, leading to overproduction of aldosterone. In conclusion, the four PA mutations caused more profound changes on CaV3.2 (+26) currents than on CaV3.2 (-26) currents, and except the M1549I mutation, the S196 L, V1951E and P2083 L have little effect on the electrophysiological properties of CaV3.2(-26) currents, which may partially explain the limitation of the phenotype associated with the V1951E, S196 L and P2083 L germline mutations to PA.


Assuntos
Aldosterona/metabolismo , Canais de Cálcio Tipo T/metabolismo , Hiperaldosteronismo/metabolismo , Zona Glomerulosa/metabolismo , Processamento Alternativo , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Células Cultivadas , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Humanos , Hiperaldosteronismo/genética , Especificidade de Órgãos , Fenótipo , Mutação Puntual/genética , Isoformas de Proteínas/genética , Zona Glomerulosa/patologia
13.
Hypertension ; 74(5): 1152-1159, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31564164

RESUMO

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10-24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.


Assuntos
Aldosterona/biossíntese , Anoctaminas/genética , Regulação da Expressão Gênica , Hiperaldosteronismo/genética , Hipertensão/fisiopatologia , Transdução de Sinais/genética , Zona Glomerulosa/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Análise de Variância , Comunicação Celular/genética , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Hiperaldosteronismo/patologia , Hipertensão/etiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise Serial de Tecidos , Técnicas de Cultura de Tecidos , Transcriptoma/genética , Regulação para Cima , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/patologia
14.
Nat Commun ; 10(1): 4678, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615979

RESUMO

Human primary aldosteronism (PA) can be caused by mutations in several ion channel genes but mouse models replicating this condition are lacking. We now show that almost all known PA-associated CLCN2 mutations markedly increase ClC-2 chloride currents and generate knock-in mice expressing a constitutively open ClC-2 Cl- channel as mouse model for PA. The Clcn2op allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca2+ concentration. Clcn2op mice display typical features of human PA, including high serum aldosterone in the presence of low renin activity, marked hypertension and hypokalemia. These symptoms are more pronounced in homozygous Clcn2op/op than in heterozygous Clcn2+/op mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2+/op zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2op mice are a valuable model to study the pathological mechanisms underlying this disease.


Assuntos
Canais de Cloreto/genética , Modelos Animais de Doenças , Hiperaldosteronismo/genética , Hipertensão/genética , Camundongos , Zona Glomerulosa/metabolismo , Animais , Técnicas de Introdução de Genes , Heterozigoto , Homozigoto , Hiperaldosteronismo/complicações , Hipertensão/etiologia , Hipopotassemia/etiologia , Hipopotassemia/genética , Mutação
15.
Bull Exp Biol Med ; 167(4): 568-573, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31502134

RESUMO

We analyzed the expression of transcriptional factor Oct4 in rat adrenal cortical cells during postnatal development. It was found that Oct4 is expressed by typical cortical cells of the zona glomerulosa, zona fasciculata, and zona reticularis in pubertal and postpubertal periods. The maximum number of Oct4+ cells was found in the zona glomerulosa. An inverse correlation between the number of Oct4+ glomerulosa cells and serum level of aldosterone both in pubertal and postpubertal periods was revealed. After puberty, the number of Oct4+ glomerulosa cells directly correlated with the number of Ki-67+ cells. A hypothesis was put forward that Oct4 is involved in postnatal morphogenesis, regeneration, and functioning of the adrenal cortex.


Assuntos
Córtex Suprarrenal/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Córtex Suprarrenal/citologia , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/metabolismo , Animais , Fator 3 de Transcrição de Octâmero/genética , Ratos , Ratos Wistar , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo
16.
PLoS One ; 14(9): e0222005, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31479491

RESUMO

The involvement of secretin (SCT) and its receptor (SCTR) in angiotensin II (ANGII)-mediated osmoregulation by forming SCTR/ angiotensin II type 1 receptor (AT1R) heteromer is well established. In this study, we demonstrated that SCTR/AT1R complex can mediate ANGII-induced aldosterone secretion/release through potentiating calcium mobilization. Through IHC and cAMP studies, we showed the presence of functional SCTR and AT1R in the primary zona glomerulosa (ZG) cells of C57BL/6N (C57), and functional AT1R and non-functional SCTR in SCTR knockout (SCTR-/-) mice. Calcium mobilization studies revealed the important role of SCTR on ANGII-mediated calcium mobilization in adrenal gland. The fluo4-AM loaded primary adrenal ZG cells from the C57 mice displayed a dose-dependent increase in intracellular calcium influx ([Ca2+]i) when exposed to ANGII but not from the SCTR-/- ZG cells. Synthetic SCTR transmembrane (TM) peptides STM-II/-IV were able to alter [Ca2+]i in C57 mice, but not the mice with mutated STM-II/-IV (STM-IIm/IVm) peptides. Through enzyme immunoassay (EIA), we measured the aldosterone release from primary ZG cells of both C57 and SCTR-/- mice by exposing them to ANGII (10nM). SCTR-/- ZG cells showed impaired ANGII-induced aldosterone secretion compared to the C57 mice. TM peptide, STM-II hindered the aldosterone secretion in ZG cells of C57 mice. These findings support the involvement of SCTR/AT1R heterodimer complex in aldosterone secretion/release through [Ca2+]i.


Assuntos
Aldosterona/metabolismo , Angiotensina II/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Sinalização do Cálcio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Osmorregulação/genética , Osmorregulação/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Receptor Tipo 1 de Angiotensina/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficiência , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/deficiência , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo
17.
FASEB J ; 33(9): 10218-10230, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31208233

RESUMO

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.


Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Processamento de Proteína Pós-Traducional/fisiologia , Sumoilação/fisiologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/ultraestrutura , Neoplasias do Córtex Suprarrenal/patologia , Hormônio Adrenocorticotrópico/administração & dosagem , Animais , Complexo de Carney/metabolismo , Linhagem Celular Tumoral , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Preparações de Ação Retardada , Dexametasona/análogos & derivados , Dexametasona/farmacologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Zona Fasciculada/efeitos dos fármacos , Zona Fasciculada/metabolismo , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo , beta Catenina/deficiência , beta Catenina/genética
18.
J Endocrinol ; 240(2): 111-122, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30400034

RESUMO

Human risk allele carriers of lysine-specific demethylase 1 (LSD1) and LSD1-deficient mice have salt-sensitive hypertension for unclear reasons. We hypothesized that LSD1 deficiency causes dysregulation of aldosterone's response to salt intake resulting in increased cardiovascular risk factors (blood pressure and microalbumin). Furthermore, we determined the effect of biological sex on these potential abnormalities. To test our hypotheses, LSD1 male and female heterozygote-knockout (LSD1+/-) and WT mice were assigned to two age groups: 18 weeks and 36 weeks. Plasma aldosterone levels and aldosterone production from zona glomerulosa cells studied ex vivo were greater in both male and female LSD1+/- mice consuming a liberal salt diet as compared to WT mice consuming the same diet. However, salt-sensitive blood pressure elevation and increased microalbuminuria were only observed in male LSD1+/- mice. These data suggest that LSD1 interacts with aldosterone's secretory response to salt intake. Lack of LSD1 causes inappropriate aldosterone production on a liberal salt diet; males appear to be more sensitive to this aldosterone increase as males, but not females, develop salt sensitivity of blood pressure and increased microalbuminuria. The mechanism responsible for the cardiovascular protective effect in females is uncertain but may be related to estrogen modulating the effect of mineralocorticoid receptor activation.


Assuntos
Aldosterona/metabolismo , Pressão Sanguínea/fisiologia , Histona Desmetilases/deficiência , Zona Glomerulosa/metabolismo , Fatores Etários , Albuminúria/etiologia , Albuminúria/genética , Albuminúria/metabolismo , Animais , Pressão Sanguínea/genética , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Feminino , Histona Desmetilases/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco , Fatores Sexuais , Cloreto de Sódio na Dieta/efeitos adversos , Zona Glomerulosa/citologia
19.
Proc Natl Acad Sci U S A ; 115(52): E12265-E12274, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30541888

RESUMO

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


Assuntos
Córtex Suprarrenal/enzimologia , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Transdução de Sinais , Córtex Suprarrenal/metabolismo , Animais , Diferenciação Celular , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esteroides/metabolismo , Zona Fasciculada/citologia , Zona Fasciculada/enzimologia , Zona Fasciculada/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/enzimologia , Zona Glomerulosa/metabolismo
20.
Medicina (Kaunas) ; 54(5)2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30463213

RESUMO

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


Assuntos
Cafeína/efeitos adversos , Bebidas Energéticas/efeitos adversos , Gotículas Lipídicas , Taurina/efeitos adversos , Zona Fasciculada/metabolismo , Zona Fasciculada/patologia , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia , Animais , Apoptose , Caspase 3/biossíntese , Colágeno/biossíntese , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Antígeno Ki-67/biossíntese , Masculino , Ratos , Ratos Wistar , Zona Fasciculada/citologia , Zona Glomerulosa/citologia
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