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1.
Gynecol Endocrinol ; 34(5): 437-441, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29187003

RESUMO

The effects of androgens on gonadotropin-releasing hormone (GnRH) secretion in females have not been fully established. To clarify the direct effects of androgens on hypothalamic reproductive factors, we evaluated the effects of chronic testosterone administration on hypothalamic GnRH regulatory factors in ovariectomized (OVX) female rats. Both testosterone and estradiol reduced the serum luteinizing hormone levels of OVX female rats, indicating that, as has been found for estrogen, testosterone suppresses GnRH secretion via negative feedback. Similarly, the administration of testosterone or estradiol suppressed the hypothalamic mRNA levels of kisspeptin and neurokinin B, both of which are positive regulators of GnRH, whereas it did not affect the hypothalamic mRNA levels of the kisspeptin receptor or neurokinin-3 receptor. On the contrary, the administration of testosterone, but not estradiol, suppressed the hypothalamic mRNA expression of prodynorphin, which is a negative regulator of GnRH. The administration of testosterone did not alter the rats' serum estradiol levels, indicating that testosterone's effects on hypothalamic factors might be induced by its androgenic activity. These findings suggest that as well as estrogen, androgens have negative feedback effects on GnRH in females and that the underlying mechanisms responsible for these effects are similar, but do not completely correspond, to the mechanisms underlying the effects of estrogen on GnRH.


Assuntos
Dinorfinas/metabolismo , Hipotálamo/efeitos dos fármacos , Kisspeptinas/metabolismo , Neurocinina B/metabolismo , Testosterona/farmacologia , Animais , Dinorfinas/genética , Estradiol/farmacologia , Feminino , Hipotálamo/metabolismo , Kisspeptinas/genética , Leptina/sangue , Hormônio Luteinizante/sangue , Neurocinina B/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores de Kisspeptina-1/genética , Receptores de Kisspeptina-1/metabolismo , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/metabolismo
2.
Am J Physiol Gastrointest Liver Physiol ; 313(5): G361-G372, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28774868

RESUMO

G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferases in the cytoplasm, and, thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiological responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR-miRNA interactions related to gastrointestinal pathophysiology, such as inflammatory bowel diseases, irritable bowel syndrome, and gastrointestinal cancers. Given that the presence of different types of cells in the gastrointestinal tract suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis, we also discuss how GPCR-miRNA interactions regulate gene expression at the cellular level and subsequently modulate gastrointestinal pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identify novel molecular pathways leading to the discovery of potential biomarkers for gastrointestinal diseases.


Assuntos
Gastroenteropatias , MicroRNAs/genética , Receptores Acoplados a Proteínas-G/fisiologia , Comunicação Celular/fisiologia , Epigênese Genética/fisiologia , Gastroenteropatias/genética , Gastroenteropatias/fisiopatologia , Trato Gastrointestinal/fisiologia , Trato Gastrointestinal/fisiopatologia , Expressão Gênica , Humanos , Receptores de Interleucina-6/genética , Receptores da Neurocinina-3/genética , Transdução de Sinais/fisiologia
3.
Clin Endocrinol (Oxf) ; 87(6): 748-756, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28802064

RESUMO

OBJECTIVE: Patients with mutations of neurokinin B (NKB) and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin. DESIGN: An open label study wherein men (n = 6) were administered the NK3R antagonist MLE4901 40 mg orally twice a day for 7 days. Kisspeptin-10 (0.3 µg/kg iv bolus) was given before and on day 7 of NK3R antagonist treatment. PATIENTS: Subjects were healthy men. MEASUREMENTS: Reproductive hormones were measured before and during the NK3R antagonist administration, including frequent sampling on two occasions for analysis of pulsatile LH secretion. RESULTS: LH, FSH and testosterone secretion were decreased during NK3R antagonist administration. LH showed a biphasic response, being reduced after 24 hours of treatment (4.5 ± 0.6 IU/L pretreatment to 1.7 ± 0.2 IU/L, P < .05), with partial recovery thereafter, but it was again decreased on day 7 (2.5 ± 0.6 IU/L, P < .05 vs pretreatment). FSH secretion was also suppressed, with a similar temporal pattern to that of LH. Testosterone secretion was decreased from 24 hours (18.4 ± 1.6 pretreatment vs 5.6 ± 1.5 nmol/L, P < .01) and remained suppressed throughout the treatment period. Analysis of LH pulsatility showed that both basal and pulsatile LH secretion were markedly suppressed but there was no detected change in LH pulse frequency. Kisspeptin-10 stimulated LH secretion, with similar responses before and during NK3R antagonist administration. CONCLUSIONS: These data demonstrate a central role for NKB/NK3R in the physiological regulation of reproductive function in men, and that this is functionally upstream of kisspeptin-mediated GnRH secretion.


Assuntos
Gonadotropinas/metabolismo , Receptores da Neurocinina-3/antagonistas & inibidores , Receptores da Neurocinina-3/metabolismo , Testosterona/metabolismo , Adulto , Hormônio Foliculoestimulante/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Hipogonadismo/genética , Hipogonadismo/metabolismo , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Mutação/genética , Neurocinina B/genética , Receptores da Neurocinina-3/genética , Tiadiazóis/farmacologia , Adulto Jovem
4.
Lancet ; 389(10081): 1809-1820, 2017 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-28385352

RESUMO

BACKGROUND: Hot flushes affect 70% of menopausal women and often severely impact physical, psychosocial, sexual, and overall wellbeing. Hormone replacement therapy is effective but is not without risk. Neurokinin B signalling is increased in menopausal women, and has been implicated as an important mediator of hot flushes. METHODS: This phase 2, randomised, double-blind, placebo-controlled, single-centre, crossover trial assessed the effectiveness of an oral neurokinin 3 receptor antagonist (MLE4901) on menopausal hot flushes. Eligible participants were healthy women aged 40-62 years, having seven or more hot flushes in every 24 h of which some were reported as being severe or bothersome, who had not had a menstrual period for at least 12 months, and who had not been taking any medication shown to improve menopausal flushes in the preceding 8 weeks. Participants received 4 weeks of MLE4901 (40 mg, orally, twice daily) and placebo (orally, twice daily) in random order separated by a 2 week washout period. Randomisation was completed by a central computer, and participants were allocated to treatment number in numerical order. The primary outcome was the total number of hot flushes during the final week of both treatment periods. Analyses were by intention to treat and per protocol using generalised linear mixed models and standard crossover analysis. All analyses were prespecified in the study protocol. The trial is registered at ClinicalTrials.gov, number NCT02668185. FINDINGS: 68 women were screened between Feb 3 and Oct 10, 2016, of which 37 were randomly assigned and included in an intention-to-treat analysis. 28 participants completed the trial and were included in a per-protocol analysis. MLE4901 significantly reduced the total weekly number of hot flushes by 45 percentage points (95% CI 22-67) compared with the placebo (intention-to-treat adjusted means: placebo 49·01 [95% CI 40·81-58·56] vs MLE4901 19·35 [15·99-23·42]; adjusted estimate of difference 29·66 [17·39-42·87], p<0·0001). Treatment was well tolerated. Three participants developed a transaminase rise (alanine aminotransferase 4·5-5·9 times the upper limit of normal) with a normal bilirubin 28 days after starting MLE4901, which normalised within 90 days. INTERPRETATION: Treatment with a neurokinin 3 receptor antagonist (MLE4901) could be practice changing as it safely and effectively relieves hot flush symptoms without the need for oestrogen exposure. Larger scale studies of longer duration are now indicated. FUNDING: UK Medical Research Council and National Institute for Health Research.


Assuntos
Fogachos/tratamento farmacológico , Menopausa/fisiologia , Receptores da Neurocinina-3/antagonistas & inibidores , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Terapia de Reposição Hormonal/efeitos adversos , Terapia de Reposição Hormonal/métodos , Fogachos/etiologia , Humanos , Menopausa/genética , Menopausa/psicologia , Pessoa de Meia-Idade , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/uso terapêutico , Resultado do Tratamento
5.
Menopause ; 24(3): 252-261, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28231077

RESUMO

OBJECTIVE: Vasomotor symptoms (VMS, ie, hot flashes or night sweats) are reported by many, but not all, women. The extent to which VMS are genetically determined is unknown. We evaluated the relationship of genetic variation and VMS. METHODS: In this observational study, we accessed data from three genome-wide association studies (GWAS) (SNP Health Association Resource cohort [SHARe], WHI Memory Study cohort [WHIMS+], and Genome-Wide Association Studies of Treatment Response in Randomized Clinical Trials [GARNET] studies, total n = 17,695) of European American, African American, and Hispanic American postmenopausal women aged 50 to 79 years at baseline in the Women's Health Initiative Study. We examined genetic variation in relation to VMS (yes/no) in each study and using trans-ethnic inverse variance fixed-effects meta-analysis. A total of 11,078,977 single-nucleotide polymorphisms (SNPs) met the quality criteria. RESULTS: After adjustment for covariates and population structure, three SNPs (on chromosomes 3 and 11) were associated with VMS at the genome-wide threshold of 5 × 10 in the African American SHARe GWAS, but were not associated in the other cohorts. In the meta-analysis, 14 SNPs, all located on chromosome 4 in the tachykinin receptor 3 (TACR3) locus, however, had P < 5 × 10. These SNPs' effect sizes were similar across studies/participants' ancestry (odds ratio ∼1.5). CONCLUSIONS: Genetic variation in TACR3 may contribute to the risk of VMS. To our knowledge, this is the first GWAS to examine SNPs associated with VMS. These results support the biological hypothesis of a role for TACR3 in VMS, which was previously hypothesized from animal and human studies. Further study of these variants may lead to new insights into the biological pathways involved in VMS, which are poorly understood.


Assuntos
Fogachos/genética , Polimorfismo de Nucleotídeo Único , Pós-Menopausa/genética , Receptores da Neurocinina-3/genética , Sudorese/genética , Afro-Americanos/genética , Idoso , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Hispano-Americanos/genética , Humanos , Pessoa de Meia-Idade , Razão de Chances , Pós-Menopausa/etnologia
6.
Sci Rep ; 6: 36102, 2016 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-27786296

RESUMO

In our previous study, NKB/NK3R system has been shown to act at the pituitary level to up-regulate SLα synthesis and secretion in grass carp. However, whether NK3R expression can serve as a regulatory target at the pituitary level and contribute to NKB interactions with other SLα regulators is still unclear. In current study, using grass carp pituitary cells as a model, we have a novel finding that co-treatment of SLα/SLß with carp TAC3 gene products, could induce a noticeable enhancement in SLα mRNA expression and these potentiating effects occurred with a parallel rise in NK3R transcript level after SLα/SLß treatment. Interestingly, the stimulatory effects of SLα/SLß on NK3R gene expression could be further potentiated by co-treatment with IGF-I/-II and simultaneous exposure of carp pituitary cells to SLα/SLß and IGF-I/-II in the presence of TAC3 gene products was found to markedly elevated SLα mRNA expression (20 fold increase) and this synergistic stimulation was mediated by cAMP/PKA-, PLC/PKC- and Ca2+ -dependent cascades functionally coupled with NK3R activation. These findings suggest that local release of SLα via functional interactions with IGF-I/-II and TAC3/NK3R system may constitute a potent stimulatory signal for SLα gene expression in the carp pituitary via up-regulation of NK3R expression.


Assuntos
Carpas/metabolismo , Proteínas de Peixes/metabolismo , Hipófise/metabolismo , Hormônios Hipofisários/farmacologia , Receptores da Neurocinina-3/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Peixes/genética , Homeostase , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Neurocinina B/metabolismo , Hipófise/citologia , Hipófise/efeitos dos fármacos , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores da Neurocinina-3/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Fosfolipases Tipo C/metabolismo
7.
Neuropsychopharmacology ; 41(11): 2714-22, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27238620

RESUMO

Recently we determined that activation of the tachykinin 2 (Tac2) pathway in the central amygdala (CeA) is necessary and sufficient for the modulation of fear memories. The Tac2 pathway includes the Tac2 gene, which encodes the neuropeptide neurokinin B and its corresponding receptor neurokinin 3 receptor (NK3R). In this study, using Tac2-cre and Tac2-GFP mice, we applied a combination of in vivo (optogenetics) and multiple in vitro techniques to further explore the mechanisms of action within the Tac2 pathway. In transgenic mice that express ChR2 solely in Tac2 neurons, in vivo optogenetic stimulation of CeA Tac2-expressing neurons during fear acquisition enhanced fear memory consolidation and drove action potential firing in vitro. In addition, Tac2-CeA neurons were shown to co-express striatal-enriched protein tyrosine phosphatase, which may have an important role in regulating Nk3R signaling during fear conditioning. These data extend our current understanding for the underlying mechanism(s) for the role of the Tac2 pathway in the regulation of fear memory, which may serve as a new therapeutic target in the treatment of fear-related disorders.


Assuntos
Tonsila do Cerebelo/fisiologia , Medo , Aprendizagem/fisiologia , Precursores de Proteínas/genética , Transdução de Sinais/genética , Taquicininas/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Antipsicóticos/farmacologia , Channelrhodopsins , Condicionamento Clássico/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Locomoção/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organofosfatos/metabolismo , Piperidinas/farmacologia , Polímeros/metabolismo , Proteína Quinase C-delta/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/metabolismo , Taquicininas/metabolismo
8.
Hum Reprod ; 31(6): 1363-74, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27094476

RESUMO

STUDY QUESTION: What is the exact prevalence of Kisspeptin Receptor (KISS1R) mutations in the population of patients with normosmic congenital hypogonadotrophic hypogonadism (nCHH) by comparison with other genes, involved in gonadotrophin-releasing hormone (GnRH) release or action? SUMMARY ANSWER: KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases and were less prevalent than GnRH Receptor (GNRHR) mutations. WHAT IS KNOWN ALREADY: The respective prevalence of each of the genetic causes of nCHH is unclear. Large series of patients are very rare and suffer from heterogeneity of the population of CHH studied. STUDY DESIGN, SIZE, DURATION: Patients with nCHH were consecutively enrolled in a single French referral centre and were gradually tested for KISS1R between January 2006 and April 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 603 patients with nCHH (399 men and 204 women) were diagnosed at the Bicêtre Hospital and underwent KISS1R analysis. The GNRHR, tachykinin receptor 3 (TACR3), gonadotrophin-releasing hormone 1 (GNRH1), tachykinin 3 (TAC3) and KISS1 genes were also sequenced. Functional characterization of KISS1R mutations included a study of signal transduction using a reporter gene (serum response element-luciferase (SRE-Luc) involved in the mitogen-activated protein (MAP) kinase pathway. MAIN RESULTS AND THE ROLE OF CHANCE: We detected 15 KISS1R variants (10 novel), in 12 of the 603 patients (2.0%, 95% CI [0.9-3.1]. KISS1R mutations were less prevalent than GNRHR (4.7%) and TACR3 (2.6%) mutations but more prevalent than GNRH1 (1.5%), TAC3 (1.0%) and KISS1 (0%) mutations. KISS1R mutants were present in the biallelic state in 8 of the 12 patients concerned. Among 5 men with biallelic KISS1R mutations, 4 had either micropenis or cryptorchidism. In vitro analysis of the 5 new variants present in the biallelic state (C95W, Y103*, C115W, P176R and A287E) showed a loss of function. LIMITATIONS, REASONS FOR CAUTION: The prevalence of TACR3, GNRH1, TAC3 and KISS1 mutations was calculated from a smaller number of nCHH patients than KISS1R and GNRHR. This should prompt caution concerning the reported prevalence of mutations in these four genes. WIDER IMPLICATIONS OF THE FINDINGS: We show that KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases. Together, the genes analysed here were mutated in fewer than 15% of patients, suggesting a role of other genes in nCHH. The presence of cryptorchidism and/or micropenis in the majority of men with biallelic KISS1R mutations strongly suggests that this gene is essential for prenatal GnRH secretion. STUDY FUNDING, COMPETING INTERESTS: This work was supported in part by grants from Paris-Sud University (Bonus Qualité Recherche, and Attractivité grants) to J.B., French Ministry of Health, Hospital Clinical Research Program on Rare Diseases. Assistance Publique Hôpitaux de Paris, Programme Hospitalier de Recherche Clinique (PHRC # P081212 HYPOPROTEO) to J.Y. C.P. was supported by student fellowships 'Année Recherche' from Agence Régionale de Santé Provence Alpes Côtes d'Azur. The authors have nothing to disclose.


Assuntos
Hipogonadismo/genética , Mutação , Receptores de Kisspeptina-1/genética , Adolescente , Adulto , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Receptores LHRH/genética , Receptores da Neurocinina-3/genética , Transdução de Sinais
9.
Neuroendocrinology ; 103(3-4): 230-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26088945

RESUMO

INTRODUCTION: Many missense variants in G protein-coupled receptors (GPCRs) involved in the neuroendocrine regulation of reproduction have been identified by phenotype-driven or large-scale exome sequencing. Computational functional prediction analysis is commonly performed to evaluate their impact on receptor function. METHODS: To assess the performance and outcome of functional prediction analyses for these GPCRs, we performed a statistical analysis of the prediction performance of SIFT and PolyPhen-2 for variants with documented biological function as well as variants retrieved from Ensembl. We obtained missense variants with documented biological function testing from patients with reproductive disorders from a comprehensive literature search. Missense variants from individuals with known reproductive disorders were retrieved from the Human Gene Mutation Database. Missense variants from the general population were retrieved from the Ensembl genome database. RESULTS: The accuracies of SIFT and PolyPhen-2 were 83 and 85%, respectively. The performance of both prediction tools was greater in predicting loss-of-function variants (SIFT: 92%; PolyPhen-2: 95%) than in predicting variants that did not affect function (SIFT: 54%; PolyPhen-2: 57%). Concordance between SIFT and PolyPhen-2 did not improve accuracy. Surprisingly, approximately half of the variants retrieved from Ensembl were predicted as loss-of-function variants by SIFT (47%) and PolyPhen-2 (54%). CONCLUSION: Our findings provide new guidance for interpreting the results and limitations of computational functional prediction analyses for GPCRs and will help to determine which variants require biological function testing. In addition, our findings raise important questions regarding the link between genotype and phenotype in the general population.


Assuntos
Biologia Computacional , Mutação de Sentido Incorreto/genética , Sistemas Neurossecretores/fisiopatologia , Receptores Acoplados a Proteínas-G/genética , Reprodução/genética , Feminino , Humanos , Infertilidade Masculina/genética , Masculino , Técnicas de Diagnóstico Molecular , Fenótipo , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes , PubMed/estatística & dados numéricos , Receptores de Kisspeptina-1 , Receptores LHRH/genética , Receptores da Neurocinina-3/genética , Receptores de Peptídeos/genética , Software
10.
Nat Commun ; 6: 7756, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26239645

RESUMO

More than 100 loci have been identified for age at menarche by genome-wide association studies; however, collectively these explain only ∼3% of the trait variance. Here we test two overlooked sources of variation in 192,974 European ancestry women: low-frequency protein-coding variants and X-chromosome variants. Five missense/nonsense variants (in ALMS1/LAMB2/TNRC6A/TACR3/PRKAG1) are associated with age at menarche (minor allele frequencies 0.08-4.6%; effect sizes 0.08-1.25 years per allele; P<5 × 10(-8)). In addition, we identify common X-chromosome loci at IGSF1 (rs762080, P=9.4 × 10(-13)) and FAAH2 (rs5914101, P=4.9 × 10(-10)). Highlighted genes implicate cellular energy homeostasis, post-transcriptional gene silencing and fatty-acid amide signalling. A frequently reported mutation in TACR3 for idiopathic hypogonatrophic hypogonadism (p.W275X) is associated with 1.25-year-later menarche (P=2.8 × 10(-11)), illustrating the utility of population studies to estimate the penetrance of reportedly pathogenic mutations. Collectively, these novel variants explain ∼0.5% variance, indicating that these overlooked sources of variation do not substantially explain the 'missing heritability' of this complex trait.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Autoantígenos/genética , Imunoglobulinas/genética , Laminina/genética , Proteínas de Membrana/genética , Menarca/genética , Proteínas/genética , Proteínas de Ligação a RNA/genética , Receptores da Neurocinina-3/genética , Adolescente , Adulto , Fatores Etários , Idoso , Amidas , Cromossomos Humanos X/genética , Códon sem Sentido , Metabolismo Energético/genética , Grupo com Ancestrais do Continente Europeu/genética , Ácidos Graxos , Feminino , Frequência do Gene , Genes Ligados ao Cromossomo X/genética , Variação Genética , Genótipo , Humanos , Hipogonadismo/genética , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Penetrância , Fenótipo , Interferência de RNA , Transdução de Sinais/genética , Adulto Jovem
11.
Neurobiol Aging ; 36(6): 2060-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25976010

RESUMO

The neuropeptide neurokinin 3 (NK3) and its receptor modulate cholinergic activity of the basal forebrain (BF) and are implicated in learning and memory. In Alzheimer's disease, the rs2765 single-nucleotide polymorphism (SNP) of the NK3 receptor-coding gene TACR3 was correlated with the right hippocampus volume. Here, we studied the association of the rs2765 SNP with magnetic resonance imaging-based volumes of the BF and hippocampus in a population-based sample of 1967 participants between 21 and 90 years of age. The rs2765 SNP was significantly associated with the most anterior BF volume corresponding to the medial septum/diagonal band, and with a significantly steeper age-related volume decline. The rs2765 SNP was not associated with other BF subvolumes or hippocampus volumes. Apolipoprotein E ε4 showed no correlation with any brain volume or global cognition. Our findings in a large population-based sample suggest an association of an NK3 receptor SNP with age-related decline of rostral cholinergic BF volume.


Assuntos
Prosencéfalo Basal/patologia , Estudos de Associação Genética , Hipocampo/patologia , Tamanho do Órgão/genética , Polimorfismo de Nucleotídeo Único , Receptores da Neurocinina-3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Neurônios Colinérgicos/fisiologia , Cognição/fisiologia , Feminino , Humanos , Aprendizagem/fisiologia , Imagem por Ressonância Magnética , Masculino , Memória/fisiologia , Pessoa de Meia-Idade , Adulto Jovem
12.
Spine (Phila Pa 1976) ; 40(16): 1261-9, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25929203

RESUMO

STUDY DESIGN: Laboratory study. OBJECTIVE: To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SUMMARY OF BACKGROUND DATA: SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. METHODS: Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1ß, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. RESULTS: We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1ß, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. CONCLUSION: NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. LEVEL OF EVIDENCE: 4.


Assuntos
Interleucinas/genética , Disco Intervertebral/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Antagonistas do Receptor de Neuroquinina-1/farmacologia , Substância P/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-8/genética , Masculino , Pessoa de Meia-Idade , Fosforilação , RNA Mensageiro/metabolismo , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-2/genética , Receptores da Neurocinina-3/genética , Fator de Transcrição RelA/metabolismo
13.
J Med Chem ; 58(7): 3060-82, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25738882

RESUMO

Neurokinin-3 receptor (NK3R) has recently emerged as important in modulating the tonic pulsatile gonadotropin-releasing hormone (GnRH) release. We therefore decided to explore NK3R antagonists as therapeutics for sex-hormone disorders that can potentially benefit from lowering GnRH pulsatility with consequent diminished levels of plasma luteinizing hormone (LH) and correspondingly attenuated levels of circulating androgens and estrogens. The discovery and lead optimization of a novel N-acyl-triazolopiperazine NK3R antagonist chemotype achieved through bioisosteric lead change from the high-throughput screening (HTS) hit is described. A concomitant improvement in the antagonist bioactivity and ligand lipophilic efficiency (LLE) parameter were the principal guidelines in the lead optimization efforts. Examples of advanced lead analogues to demonstrate the amenability of this chemotype to achieving a suitable pharmacokinetic (PK) profile are provided as well as pharmacokinetic-pharmacodynamic (PKPD) correlations to analyze the trends observed for LH inhibition in castrated rats and monkeys that served as preliminary in vivo efficacy models.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores da Neurocinina-3/antagonistas & inibidores , Relação Estrutura-Atividade , Androgênios/sangue , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Técnicas de Química Sintética , Cricetulus , Cristalografia por Raios X , Descoberta de Drogas , Canal de Potássio ERG1 , Doenças do Sistema Endócrino/sangue , Doenças do Sistema Endócrino/tratamento farmacológico , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Ligantes , Hormônio Luteinizante/sangue , Masculino , Ratos Sprague-Dawley , Receptores da Neurocinina-3/genética
14.
Endocrinology ; 156(4): 1386-97, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25574869

RESUMO

Humans carrying mutations in neurokinin B (NKB) or the NKB receptor fail to undergo puberty due to decreased secretion of GnRH. Despite this pubertal delay, many of these patients go on to achieve activation of their hypothalamic-pituitary-gonadal axis in adulthood, a phenomenon termed reversal, indicating that NKB signaling may play a more critical role for the timing of pubertal development than adult reproductive function. NKB receptor-deficient mice are hypogonadotropic but have no defects in the timing of sexual maturation. The current study has performed the first phenotypic evaluation of mice bearing mutations in Tac2, the gene encoding the NKB ligand, to determine whether they have impaired sexual development similar to their human counterparts. Male Tac2-/- mice showed no difference in the timing of sexual maturation or fertility compared with wild-type littermates and were fertile. In contrast, Tac2-/- females had profound delays in sexual maturation, with time to vaginal opening and first estrus occurring significantly later than controls, and initial abnormalities in estrous cycles. However, cycling recovered in adulthood and Tac2-/- females were fertile, although they produced fewer pups per litter. Thus, female Tac2-/- mice parallel humans harboring NKB pathway mutations, with delayed sexual maturation and activation of the reproductive cascade later in life. Moreover, direct comparison of NKB ligand and receptor-deficient females confirmed that only NKB ligand-deficient animals have delayed sexual maturation, suggesting that in the absence of the NKB receptor, NKB may regulate the timing of sexual maturation through other tachykinin receptors.


Assuntos
Fertilidade/genética , Neurocinina B/genética , Reprodução/genética , Maturidade Sexual/fisiologia , Animais , Estro/genética , Estro/metabolismo , Feminino , Tamanho da Ninhada de Vivíparos/genética , Masculino , Camundongos , Mutação , Neurocinina B/metabolismo , Neurônios/metabolismo , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/metabolismo , Fatores de Tempo
15.
J Pediatr Endocrinol Metab ; 28(1-2): 65-71, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25153567

RESUMO

BACKGROUND AND AIMS: The allele variant of neurokinin B and its receptor genes were thought important in regulating the human reproductive axis in many populations. The aim of this study was to investigate whether the variances in TAC3 and TACR3 genes that encoded neurokinin B (NKB) and its receptor (NK3R), individually, were associated with idiopathic precocious puberty in Chinese girls. METHODS: The genotyping method of polymerase chain reaction-restriction fragment length polymorphism was applied in this study; the distribution of five active single nucleon acid polymorphisms (SNPs) in TAC3 (p.G34R, p. A63P, p.Q66*, p.S99P, and a mutation on 5' UTR) and four sites in TACR3 (A29V or G, G59E, S455G, and A449S or T) genes was analyzed in 267 healthy and 186 idiopathic precocious puberty Chinese girls. RESULTS: Among the nine active SNPs, only A63P in TAC3 gene showed statistical differences; the p value was 0.024. CONCLUSION: A63P in TAC3 gene was statistically associated with the puberty onset time in Chinese girls.


Assuntos
Neurocinina B/genética , Puberdade Precoce/genética , Receptores da Neurocinina-3/genética , Alelos , Substituição de Aminoácidos , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Criança , China/epidemiologia , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Puberdade Precoce/epidemiologia
16.
Neuropharmacology ; 86: 259-72, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25107588

RESUMO

The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3.


Assuntos
Carbamatos/farmacologia , Neurotransmissores/farmacologia , Piperidinas/farmacologia , Receptores da Neurocinina-3/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antipsicóticos/farmacologia , Sítios de Ligação , Carbamatos/farmacocinética , Fármacos do Sistema Nervoso Central/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/fisiologia , Feminino , Gerbillinae , Cobaias , Células HEK293 , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neurotransmissores/farmacocinética , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/fisiologia , Fragmentos de Peptídeos/farmacologia , Piperidinas/farmacocinética , Receptores da Neurocinina-2/antagonistas & inibidores , Receptores da Neurocinina-2/metabolismo , Receptores da Neurocinina-3/agonistas , Receptores da Neurocinina-3/genética , Receptores da Neurocinina-3/metabolismo , Substância P/análogos & derivados , Substância P/farmacologia , Técnicas de Cultura de Tecidos
17.
Neuron ; 83(2): 444-454, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-24976214

RESUMO

The centromedial amygdala (CeM), a subdivision of the central amygdala (CeA), is believed to be the main output station of the amygdala for fear expression. We provide evidence that the Tac2 gene, expressed by neurons specifically within the CeM, is required for modulating fear memories. Tac2 is colocalized with GAD65 and CaMKIIα but not with PKCd and Enk neurons in the CeM. Moreover, the Tac2 product, NkB, and its specific receptor, Nk3R, are also involved in the consolidation of fear memories. Increased Tac2 expression, through a stress-induced PTSD-like model, or following lentiviral CeA overexpression, are sufficient to enhance fear consolidation. This effect is blocked by the Nk3R antagonist osanetant. Concordantly, silencing of Tac2-expressing neurons in CeA with DREADDs impairs fear consolidation. Together, these studies further our understanding of the role of the Tac2 gene and CeM in fear processing and may provide approaches to intervention for fear-related disorders.


Assuntos
Tonsila do Cerebelo/fisiologia , Medo/fisiologia , Memória/fisiologia , Neurocinina B/metabolismo , Neurônios/fisiologia , Precursores de Proteínas/metabolismo , Receptores da Neurocinina-3/metabolismo , Taquicininas/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Medo/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Neurocinina B/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Piperidinas/farmacologia , Precursores de Proteínas/genética , Receptores da Neurocinina-3/genética , Taquicininas/genética
18.
Endocrinology ; 155(9): 3582-96, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24971612

RESUMO

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.


Assuntos
Carpas/metabolismo , Proteínas de Peixes/metabolismo , Glicoproteínas/metabolismo , Neurocinina B/metabolismo , Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Prolactina/metabolismo , Receptores da Neurocinina-2/metabolismo , Receptores da Neurocinina-3/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Carpas/genética , Clonagem Molecular , Proteínas de Peixes/genética , Glicoproteínas/genética , Modelos Animais , Dados de Sequência Molecular , Neurocinina B/química , Neurocinina B/genética , Filogenia , Hipófise/citologia , Hormônios Hipofisários/genética , Prolactina/genética , Receptores da Neurocinina-2/genética , Receptores da Neurocinina-3/genética , Alinhamento de Sequência
19.
Artigo em Inglês | MEDLINE | ID: mdl-24802197

RESUMO

BACKGROUND: The genetic background of idiopathic central precocious puberty (ICPP) is not well understood, and is thought to arise from the effect of multiple genes. Familial ICPP have been reported suggesting the existence of monogenic causes of ICPP. The neurokinin B (NKB) system has recently been implicated in the regulation of the human reproductive axis. In humans, NKB and its receptor are encoded by the TAC3 and TACR3 genes, respectively. Mutations in these genes have been suggested to be causative for ICPP. METHODS: ICPP was defined by pubertal onset before 8 yrs of age in girls, and a pubertal LH response to GnRH testing. Twenty eight girls with ICPP were included in the study (age at diagnosis was 5.72±2.59; bone age, 6.12±2.81, height at the start of treatment, 0.90±1.48 SD). LHRH test was performed and was pubertal in all subjects (LH 20.35±32.37 mIU/ml; FSH 23.32±15.72 mIU/ml). The coding regions of TAC and TACR3 were sequenced. RESULTS: No rare variants were detected in TAC and TACR3 in the 28 subjects with ICPP. CONCLUSIONS: We confirmed that mutations in TAC and TACR3 are not a common cause for ICPP.


Assuntos
DNA/genética , Mutação , Neurocinina B/genética , Puberdade Precoce/genética , Receptores da Neurocinina-3/genética , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Recém-Nascido , Neurocinina B/metabolismo , Puberdade Precoce/metabolismo , Receptores da Neurocinina-3/metabolismo
20.
FASEB J ; 28(4): 1924-37, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24376026

RESUMO

Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.


Assuntos
Hormônio Liberador de Gonadotropina/deficiência , Mutação de Sentido Incorreto , Receptores da Neurocinina-3/genética , Transdução de Sinais/genética , Animais , Sítios de Ligação/genética , Ligação Competitiva/genética , Western Blotting , Células COS , Membrana Celular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Neurocinina B/genética , Neurocinina B/metabolismo , Fosforilação , Multimerização Proteica , Receptores da Neurocinina-3/química , Receptores da Neurocinina-3/metabolismo
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