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1.
Hypertension ; 74(4): 809-816, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31446799

RESUMO

Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (r=0.435, P=0.007), a negative correlation was noted in adenomas with no mutation detected (r=-0.548, P=0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (P=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference P<0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (P<0.0001), G151E (P=0.008), and L168R (P<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a KCNJ5 mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel.


Assuntos
Glândulas Suprarrenais/metabolismo , Proliferação de Células/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Glândulas Suprarrenais/patologia , Adulto , Idoso , Apoptose/fisiologia , Linhagem Celular , Feminino , Humanos , Hiperaldosteronismo/patologia , Masculino , Pessoa de Meia-Idade , Mutação
3.
Eur J Endocrinol ; 181(1): 69-78, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096184

RESUMO

Objective: The pathophysiology of aldosterone-producing adenomas (APAs) has been intensively investigated using genetic and epigenetic approaches. However, the role of miRNAs in APA is not fully understood. The present study profiled miRNAs in APAs as an exploratory approach to elucidate their pathophysiological roles in APAs. Design: Tissues of APAs and other adrenocortical adenomas were obtained from patients who underwent adrenalectomy. Methods: Candidate miRNAs differentially detected from samples were examined by whole miRNA sequencing. The expression of candidate miRNAs in APA tissues were further validated by real-time quantitative polymerase chain reaction (qPCR). Further, differential miRNA expression between APAs with and without KCNJ5 somatic mutations was examined. Prediction of miRNA target genes was performed by bioinformatics analysis. For specific miRNAs, correlation analysis between the levels of their target genes and CYP11B2 was analyzed in APA tissues. Results: Our study determined differential expression of six miRNAs in APA or APA with KCNJ5 mutations. We further demonstrated that miR299 levels were negatively correlated with mRNA levels of CACNB2, which encodes the beta-subunit of the L-type calcium channel. Additionally, we found significant correlations among miR299, CACNB2, and CYP11B2 levels in APA tissues. Conclusions: Our study suggests the possible pathophysiological involvement of specific miRNAs in calcium signaling and aldosterone hypersecretion in APAs. Further studies, including in vitro analyses, are required to clarify these findings.


Assuntos
Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Citocromo P-450 CYP11B2/genética , Perfilação da Expressão Gênica , MicroRNAs/fisiologia , Adrenalectomia , Adenoma Adrenocortical/metabolismo , Idoso , Canais de Cálcio Tipo L/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Expressão Gênica/fisiologia , Humanos , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade , Mutação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real
4.
Hypertension ; 73(6): 1283-1290, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006333

RESUMO

Peripheral 18-oxocortisol (18oxoF) level could contribute to the detection of aldosterone-producing adenoma (APA) in patients with primary aldosteronism. However, peripheral 18oxoF varies among such patients, which is a big drawback concerning its clinical application. We studied 48 cases of APA, 35 harboring KCNJ5 mutation, to clarify the significance of clinical and pathological parameters about peripheral 18oxoF. Peripheral 18oxoF concentration ranged widely from 0.50 to 183.13 ng/dL and correlated positively with intratumoral areas stained positively for steroidogenic enzymes ( P<0.0001). The peripheral 18oxoF level also correlated significantly with that of circulating aldosterone ( P<0.0001) but not with that of cortisol, a precursor of 18oxoF. However, a significant correlation was detected between peripheral 18oxoF and intratumoral glucocorticoids ( P<0.05). In addition, peripheral 18oxoF correlated positively with the number of hybrid cells double positive for 11ß-hydroxylase and aldosterone synthase ( P<0.0001). Comparing between the cases with and those without KCNJ5 mutation, the KCNJ5-mutated group demonstrated a significantly higher concentration of peripheral 18oxoF (28.4±5.6 versus 3.0±0.9 ng/dL, P<0.0001) and a larger intratumoral environment including the hybrid cells ( P<0.001), possibly representing a deviation from normal aldosterone biosynthesis. After multivariate analysis, KCNJ5 mutation status turned out to be the most associated factor involved in 18oxoF synthesis in APA ( P<0.0001). Results of our present study first revealed that enhanced 18oxoF synthesis in APA could come from a functional deviation of aldosterone biosynthesis from the normal zona glomerulosa and the utility of peripheral 18oxoF measurement could be influenced by the prevalence of KCNJ5 mutation in an APA.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , DNA de Neoplasias/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hidrocortisona/análogos & derivados , Mutação/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Análise Mutacional de DNA , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Hidrocortisona/biossíntese , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
5.
Circulation ; 139(18): 2157-2169, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30764634

RESUMO

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.


Assuntos
Fibrilação Atrial , Bloqueio Atrioventricular , Bradicardia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Doenças Genéticas Inatas , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/metabolismo , Bloqueio Atrioventricular/patologia , Bloqueio Atrioventricular/fisiopatologia , Benzopiranos/farmacologia , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/patologia , Bradicardia/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , 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 , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Doenças Genéticas Inatas/fisiopatologia , Humanos , Masculino , Xenopus laevis , Peixe-Zebra
6.
Elife ; 72018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30556810

RESUMO

Dopamine (D2) receptors provide autoinhibitory feedback onto dopamine neurons through well-known interactions with voltage-gated calcium channels and G protein-coupled inwardly-rectifying potassium (GIRK) channels. Here, we reveal a third major effector involved in D2R modulation of dopaminergic neurons - the sodium leak channel, NALCN. We found that activation of D2 receptors robustly inhibits isolated sodium leak currents in wild-type mice but not in NALCN conditional knockout mice. Intracellular GDP-ßS abolished the inhibition, indicating a G protein-dependent signaling mechanism. The application of dopamine reliably slowed pacemaking even when GIRK channels were pharmacologically blocked. Furthermore, while spontaneous activity was observed in nearly all dopaminergic neurons in wild-type mice, neurons from NALCN knockouts were mainly silent. Both observations demonstrate the critical importance of NALCN for pacemaking in dopaminergic neurons. Finally, we show that GABA-B receptor activation also produces inhibition of NALCN-mediated currents. Therefore, we identify NALCN as a core effector of inhibitory G protein-coupled receptors.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais Iônicos/genética , Proteínas do Tecido Nervoso/genética , Receptores de Dopamina D2/metabolismo , Receptores de GABA-B/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Canais de Cálcio Tipo N/genética , Dopamina/farmacologia , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Expressão Gênica , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Canais Iônicos/deficiência , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtomia , Proteínas do Tecido Nervoso/deficiência , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Receptores de Dopamina D2/genética , Receptores de GABA-B/genética , Tionucleotídeos/farmacologia , Técnicas de Cultura de Tecidos , Valina/análogos & derivados , Valina/farmacologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo
7.
Hypertension ; 72(4): 874-880, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30354720

RESUMO

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.


Assuntos
Aldosterona , Canais de Cálcio Tipo L/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo , Hipertensão , Zona Glomerulosa , Aldosterona/biossíntese , Aldosterona/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Hiperaldosteronismo/complicações , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Hiperplasia , Hipertensão/etiologia , Hipertensão/metabolismo , Imuno-Histoquímica , Masculino , Mutação , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
8.
Hypertension ; 72(3): 632-640, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30354756

RESUMO

Aldosterone-producing adenomas (APAs) harbor marked intratumoral heterogeneity in terms of morphology, steroidogenesis, and genetics. However, an association of biological significance of morphologically identified tumor cell subtypes and genotypes is virtually unknown. KCNJ5 mutation is most frequently detected and generally considered a curable phenotype by adrenalectomy. Therefore, to explore the biological significance of KCNJ5 mutation in APA based on intracellular hormonal activities, 35 consecutively selected APAs (n=18; KCNJ5 mutated, n=17; wild type) were quantitatively examined in the whole tumor areas by newly developed digital image analysis incorporating their histological and ultrastructural features (14 cells from 2 KCNJ5-mutated APAs and 15 cells from 1 wild type) and CYP11B2 immunoreactivity. Results demonstrated that KCNJ5-mutated APAs had significantly lower nuclear/cytoplasm ratio and more abundant clear cells than wild type. CYP11B2 immunoreactivity was not significantly different between these genotypes, but a significant correlation was detected between the proportion of clear cells and CYP11B2 immunoreactivity in all of the APAs examined. CYP11B2 was predominantly immunolocalized in clear cells in KCNJ5-mutated APAs. Quantitative ultrastructural analysis revealed that KCNJ5-mutated APAs had significantly more abundant and smaller-sized mitochondria with well-developed cristae than wild type, whereas wild type had more abundant lipid droplets per unit area despite the small number of the cases examined. Our results did provide the novel insights into the morphological features of APA based on their biological significance. KCNJ5-mutated APAs were characterized by predominance of enlarged lipid-rich clear cells possibly resulting in increased neoplastic aldosterone biosynthesis.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/ultraestrutura , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/ultraestrutura , Adulto , Citocromo P-450 CYP11B2/metabolismo , Feminino , Humanos , Gotículas Lipídicas/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura
9.
Hypertens Res ; 41(12): 1045-1053, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30323262

RESUMO

Blood pressure (BP) responses to dietary sodium intervention vary among individuals. The inwardly rectifying potassium channel (Kir) is a potassium-selective ion channel that allows potassium ions to move more easily into rather than out of the cell. We aimed to investigate the associations of Kir genes with BP responses to dietary sodium intervention. A 7-day low-sodium intervention followed by a 7-day high-sodium intervention was conducted among 1906 participants. BP measurements were obtained at baseline and during each dietary intervention. Both single-marker and gene-based analyses were performed to explore the associations between Kir gene variants and BP responses to dietary sodium interventions. The genetic risk score (GRS) was used to assess the cumulative effect of the variants on the BP response to the sodium interventions. During the low-sodium intervention, markers rs858009, rs2835904, and rs860795 in KCNJ6 were significantly associated with the systolic BP (SBP) response (P = 8.82 × 10-6, 3.32 × 10-6, and 2.39 × 10-4, respectively), whereas rs858009 and rs2835904 were significantly correlated with the mean arterial pressure (MAP) response (P = 1.64 × 10-4 and 2.72 × 10-4, respectively). Marker rs2836023 showed a significant association with the SBP response (P = 5.72 × 10-5) to the high-sodium intervention. The GRS stratified by quartile grouping or as a continuous variable was associated with the BP response to the sodium interventions. Gene-based analyses consistently revealed that KCNJ6 was significantly associated with the BP response to the sodium interventions. These findings suggest that KCNJ6 may contribute to variation of BP responses to dietary sodium interventions. Future studies are warranted to confirm these findings and to identify functional variants for salt sensitivity.


Assuntos
Pressão Sanguínea/genética , Dieta Hipossódica , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hipertensão/dietoterapia , Hipertensão/genética , Polimorfismo de Nucleotídeo Único , Adulto , Determinação da Pressão Arterial , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Sódio na Dieta
10.
PLoS One ; 13(9): e0204447, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30240440

RESUMO

G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M1R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M2R plays an important role in co-localization.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Muscarínicos/metabolismo , Sequência de Aminoácidos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Células HEK293 , Humanos , Mutação , Transporte Proteico , Receptores Muscarínicos/química
11.
Proc Natl Acad Sci U S A ; 115(40): E9479-E9488, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30228121

RESUMO

GABABR-dependent activation of G protein-gated inwardly rectifying potassium channels (GIRK or KIR3) provides a well-known source of inhibition in the brain, but the details on how this important inhibitory pathway affects neural circuits are lacking. We used sorting nexin 27 (SNX27), an endosomal adaptor protein that associates with GIRK2c and GIRK3 subunits, to probe the role of GIRK channels in reward circuits. A conditional knockout of SNX27 in both substantia nigra pars compacta and ventral tegmental area (VTA) dopamine neurons leads to markedly smaller GABABR- and dopamine D2R-activated GIRK currents, as well as to suprasensitivity to cocaine-induced locomotor sensitization. Expression of the SNX27-insensitive GIRK2a subunit in SNX27-deficient VTA dopamine neurons restored GIRK currents and GABABR-dependent inhibition of spike firing, while also resetting the mouse's sensitivity to cocaine-dependent sensitization. These results establish a link between slow inhibition mediated by GIRK channels in VTA dopamine neurons and cocaine addiction, revealing a therapeutic target for treating addiction.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/toxicidade , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Locomoção/efeitos dos fármacos , Animais , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/patologia , Neurônios Dopaminérgicos/patologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Camundongos , Camundongos Knockout , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
12.
J Clin Endocrinol Metab ; 103(10): 3869-3876, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085035

RESUMO

Context: Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown. Objective: To investigate the prevalence and spectrum of somatic mutations in APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)‒guided next-generation sequencing (NGS) approach. Methods: Formalin-fixed paraffin-embedded adrenal tissue from white American patients with primary aldosteronism who underwent adrenalectomy at the University of Michigan was used. Genomic DNA was isolated from 75 APAs (identified by CYP11B2 IHC). NGS was performed to identify somatic mutations by sequencing the entire coding region of a panel of genes mutated in APAs. Results: Somatic mutations were identified in 66 of 75 APAs (88%). Of the APAs with somatic mutations, six were smaller than coexisting CYP11B2-negative adrenocortical adenomas. The most frequently mutated gene was KCNJ5 (43%), followed by CACNA1D (21%), ATP1A1 (17%), ATP2B3 (4%), and CTNNB1 (3%). In addition to identification of previously reported mutations, we identified five previously unreported mutations (two in KCNJ5, one in ATP1A1, one in ATP2B3, and one in CACNA1D genes). KCNJ5 mutations were more frequent in women (70% vs 24% in men). Conclusion: Comprehensive NGS of CYP11B2-expressing adrenal tumors identified somatic mutations in aldosterone-driving genes in 88% of APAs, a higher rate than in previous studies using conventional approaches.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Mutação , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/cirurgia , Adrenalectomia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Adenoma Adrenocortical/cirurgia , Adulto , Canais de Cálcio Tipo L/genética , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPase Trocadora de Sódio-Potássio/genética
13.
Cell Physiol Biochem ; 49(1): 65-77, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30134221

RESUMO

BACKGROUND/AIMS: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. METHODS: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K+ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. RESULTS: Global HDAC inhibition increased histone acetylation and prolonged APD90 in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K+ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K+ channel remodeling. CONCLUSION: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Animais , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
14.
Curr Gene Ther ; 18(4): 246-251, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29984652

RESUMO

Duchenne muscular dystrophy is a disorder with variable expression caused by framedisrupting mutations in the dystrophin gene. It is characterized by progressive muscle weakness and dilated cardiomyopathy. In-frame dystrophin mutations cause a clinically moderate disorder named Becker muscular dystrophy. Our aim was to study the clinical and genetic characteristics of a family with inherited cardiomyopathy and Becker muscular dystrophy. The index case was diagnosed with psychomotor retardation at 5 years of age. Asymmetric left ventricular hypertrophy and a long QT interval were evidenced at the age of 12. Mild muscular weakness was developed subsequently. Three genetic variants were identified in the index case: p.Arg891Alafs*160 in the MYBPC3 gene, p.Thr263Met in the KCNJ5 gene, and p.Ser2437_Ile2554delinsPhe in the DMD gene. The latter was expected to generate an in-frame deletion of exons 51 and 52 of the dystrophin gene. A family study revealed that the father and 3 uncles were carriers of the MYBPC3 mutation. The mother and a maternal grandfather were carriers of the other 2 variants. The 80-year-old grandfather, who had the dystrophin mutation, showed no sign of cardiomyopathy or muscular weakness. The deletion of exons 51 and 52 in the DMD gene, which has been proposed as one of the therapeutic strategies for Duchenne, is consistent with a normal life expectancy and the absence of myopathic symptoms in hemizygous males.


Assuntos
Proteínas de Transporte/genética , Distrofina/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Distrofia Muscular de Duchenne/genética , Mutação , Penetrância , Deleção de Sequência , Idoso de 80 Anos ou mais , Pré-Escolar , Feminino , Humanos , Masculino , Distrofia Muscular de Duchenne/patologia , Fenótipo , Prognóstico
15.
J Clin Endocrinol Metab ; 103(9): 3477-3485, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30020487

RESUMO

Context: Aldosterone biosynthesis is regulated principally by ACTH and gene mutations as well as by angiotensin II and serum potassium. In addition, previous studies have reported the potential effects of KCNJ5 mutations in aldosterone-producing adenoma (APA) on cardiovascular diseases. However, responsiveness to ACTH in APAs according to potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5) mutations remains unknown. Objective: To investigate KCNJ5 genotype-specific differences in aldosterone biosynthesis in response to ACTH stimulation. Design and Setting: A cross-sectional study through retrieval of clinical records. Participants: One hundred forty-one patients aged ≥20 years with APA were examined. Main Outcome Measures: Associations between KCNJ5 mutations and clinical parameters reflecting the renin-angiotensin system [saline infusion test (SIT)] and ACTH pathways [dexamethasone suppression test (DST)]. Results: KCNJ5 mutations were detected in 107 cases. In the crude comparison, patients with mutations in KCNJ5 had higher plasma aldosterone concentrations (PACs) both at baseline and after the SIT. PAC after the DST showed a significant inverse association with KCNJ5 genotypes after controlling for age, sex, tumor size, and PAC after the SIT. Immunohistochemical analysis of 101 cases revealed more abundant immunoreactivity of CYP11B1 and CYP17 in the KCNJ5-mutated group than in the KCNJ5 wild-type group. Conclusion: This report of marked suppression of PAC by dexamethasone in patients with KCNJ5-mutated APAs indicates that such APAs respond to endogenous ACTH more readily than APAs in nonmutated cases. Further molecular and epidemiologic studies are required to validate our results and clarify the clinical effectiveness of the DST for predicting KCNJ5 mutations before adrenalectomy.


Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Adenoma Adrenocortical/tratamento farmacológico , Aldosterona/biossíntese , Antineoplásicos Hormonais/administração & dosagem , Dexametasona/administração & dosagem , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Hormônio Adrenocorticotrópico/sangue , Adulto , Aldosterona/sangue , Estudos Transversais , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Sistema Renina-Angiotensina/efeitos dos fármacos , Resultado do Tratamento
16.
Epilepsy Res ; 145: 82-88, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29929098

RESUMO

OBJECTIVE: Infantile spasms (IS) is a catastrophic childhood seizure disorder that is characterized by extensor and/or flexor spasms, cognitive deterioration and a characteristic EEG abnormality. The latter consists of a pattern of a spike-wave followed by an electrodecremental response (EDR), which is a flattening of the EEG waveform amplitude. The mechanism/circuitry that underpins IS is unknown. Children with Down Syndrome (DS) are particularly vulnerable to IS. The standard mouse model of DS is the Ts65Dn mutant mouse (Ts). Using the Ts mouse, we have created an animal model of IS in DS. This model entails the treatment of Ts mice with a GABABR agonist with a resultant recapitulation of the semiological, electrographic, and pharmacological phenotype of IS. One of the genes triplicated in Ts mice is the kcnj6 gene which codes for the G-protein inwardly rectifying potassium channel 2 (GIRK2) protein. We have shown that over expression of GIRK2 in Ts brain is necessary for the production of the GABABR agonist induced IS phenotype in the Ts mouse. Here, we ask the question whether the excess GIRK2 is sufficient for the production of the GABABR agonist induced IS phenotype. METHODS: To address this question, we used kcnj6 triploid mice, and compared the number of spasms via video analysis and EDR events via EEG to that of the WT mice. RESULTS: We now show that GABARR agonist-treated kcnj6 triploid mice failed to show susceptibility to the IS phenotype. Therefore, over expression of GIRK2 in the brain is necessary, but not sufficient to confer susceptibility to the GABABR agonist-induced IS phenotype in the Ts model of DS. SIGNIFICANCE: It is therefore likely that GIRK2 is working in concert with another factor or factors that are altered in the Ts brain in the production of the GABABR agonist-induced IS phenotype.


Assuntos
Síndrome de Down/genética , Síndrome de Down/patologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , 2-Amino-5-fosfonovalerato/uso terapêutico , Animais , Anticonvulsivantes/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Síndrome de Down/tratamento farmacológico , Eletroencefalografia , Embrião de Mamíferos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Genótipo , Hipocampo/patologia , Humanos , Técnicas In Vitro , Lactente , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Peptídeo Hidrolases/metabolismo , Quinoxalinas/farmacologia , Oxibato de Sódio/farmacologia , Espasmos Infantis/etiologia , Trissomia/genética
17.
Neuroscience ; 384: 152-164, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29852244

RESUMO

Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to the Keppen-Lubinsky syndrome but without lipodystrophy. Whole-exome sequencing of the patient's DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen-Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação com Ganho de Função , Hipercinese/genética , Transtornos dos Movimentos/genética , Encéfalo/fisiopatologia , Pré-Escolar , Análise Mutacional de DNA , Eletroencefalografia , Feminino , Humanos , Hipercinese/fisiopatologia , Transtornos dos Movimentos/fisiopatologia
18.
Sci Rep ; 8(1): 6257, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29674690

RESUMO

A nanotechnology-based approach for the inhibition of breast cancer cell proliferation is proposed. The innovative solution consists in a platform based on biocompatible piezoelectric nanoparticles able to target and remotely stimulate HER2-positive breast cancer cells. The anti-proliferative effects of the ultrasound-driven piezoelectric nanoparticle-assisted stimulation significantly reduced the proliferation by inducing the cell cycle arrest. Similarly to a low-intensity alternating electric field, chronic piezoelectric stimulation resulted able to inhibit cancer cell proliferation by upregulating the expression of the gene encoding Kir3.2 inward rectifier potassium channels, by interfering on Ca2+ homeostasis, and by affecting the organization of mitotic spindles during mitosis. The proposed platform, even if specific for HER2-positive cells, shows huge potential and versatility for the treatment of different type of cancers.


Assuntos
Neoplasias da Mama/terapia , Proliferação de Células/efeitos dos fármacos , Nanopartículas/uso terapêutico , Receptor ErbB-2/análise , Ondas Ultrassônicas , Neoplasias da Mama/patologia , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Nanopartículas/efeitos da radiação , Nanotecnologia/métodos , Fuso Acromático/efeitos dos fármacos
19.
Int J Mol Sci ; 19(4)2018 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-29642543

RESUMO

Primary aldosteronism is the most common form of endocrine hypertension with a prevalence of 6% in the general population with hypertension. The genetic basis of the four familial forms of primary aldosteronism (familial hyperaldosteronism FH types I-IV) and the majority of sporadic unilateral aldosterone-producing adenomas has now been resolved. Familial forms of hyperaldosteronism are, however, rare. The sporadic forms of the disease prevail and these are usually caused by either a unilateral aldosterone-producing adenoma or bilateral adrenal hyperplasia. Aldosterone-producing adenomas frequently carry a causative somatic mutation in either of a number of genes with the KCNJ5 gene, encoding an inwardly rectifying potassium channel, a recurrent target harboring mutations at a prevalence of more than 40% worldwide. Other than genetic variations, gene expression profiling of aldosterone-producing adenomas has shed light on the genes and intracellular signalling pathways that may play a role in the pathogenesis and pathophysiology of these tumors.


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Hiperaldosteronismo/genética , Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Redes Reguladoras de Genes , Humanos , Hiperaldosteronismo/metabolismo , Mutação
20.
Curr Opin Endocrinol Diabetes Obes ; 25(3): 147-154, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29432258

RESUMO

PURPOSE OF REVIEW: Primary aldosteronism is the most common form of secondary hypertension. Early diagnosis and treatment are key to cure of hypertension and prevention of cardiovascular complications. Recent genetic discoveries have improved our understanding on the pathophysiology of aldosterone production and triggered the development of new diagnostic procedures and targeted treatments for primary aldosteronism. RECENT FINDINGS: Different inherited genetic abnormalities distinguish specific forms of familial hyperaldosteronism. Somatic mutations are found not only in aldosterone-producing adenoma (APA), leading to primary aldosteronism, but also in aldosterone producing cell clusters of normal and micronodules from image-negative adrenal glands. Genetic knowledge has allowed the discovery of surrogate biomarkers and specific pharmacological inhibitors. Ageing appears to be associated with dysregulated and relatively autonomous aldosterone production. SUMMARY: New biochemical markers and pharmacological approaches may allow preoperative identification of somatic mutation carriers and use of targeted treatments.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Aldosterona/genética , Hiperaldosteronismo/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/metabolismo , Fatores Etários , Envelhecimento , Aldosterona/fisiologia , Citocromo P-450 CYP11B2/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/terapia , Hipertensão/etiologia , Masculino , Mutação , Fatores Sexuais , Síndrome
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