RESUMO
Di (2ethylhexyl) phthalate (DEHP), an environmental pollutant, is widely used as a plasticizer and causes serious pollution in the ecological environment. As previously reported, exposure to DEHP may cause thyroid dysfunction of the hypothalamicpituitarythyroid (HPT) axis. However, the underlying role of DEHP remains to be elucidated. The present study performed intragastrical administration of DEHP (150, 300 and 600 mg/kg) once a day for 90 consecutive days. DEHPstimulated oxidative stress increased the thyroid follicular cavity diameter and caused thyrocyte oedema. Furthermore, DEHP exposure altered mRNA and protein levels. Thus, DEHP may perturb TH homeostasis by affecting biosynthesis, biotransformation, biotransportation, receptor levels and metabolism through disruption of the HPT axis and activation of the thyroidstimulating hormone (TSH)/TSH receptor signaling pathway. These results identified the formerly unappreciated endocrinedisrupting activities of phthalates and the molecular mechanisms of DEHPinduced thyrotoxicity.
Assuntos
Dietilexilftalato/toxicidade , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Glândula Tireoide/efeitos dos fármacos , Animais , Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/crescimento & desenvolvimento , Sistema Hipotálamo-Hipofisário/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plastificantes/toxicidade , Ratos Wistar , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Glândula Tireoide/crescimento & desenvolvimento , Glândula Tireoide/metabolismo , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismoRESUMO
CONTEXT: Congenital isolated TSH deficiency (i-TSHD) is a rare form of congenital hypothyroidism. Five genes (IGSF1, IRS4, TBL1X, TRHR, and TSHB) responsible for the disease have been identified, although their relative frequencies and hypothalamic/pituitary unit phenotypes have remained to be clarified. OBJECTIVES: To define the relative frequencies and hypothalamic/pituitary unit phenotypes of congenital i-TSHD resulting from single gene mutations. PATIENTS AND METHODS: Thirteen Japanese patients (11 boys and 2 girls) with congenital i-TSHD were enrolled. IGSF1, IRS4, TBL1X, TRHR, and TSHB were sequenced. For a TBL1X mutation (p.Asn382del), its pathogenicity was verified in vitro. For a literature review, published clinical data derived from 74 patients with congenital i-TSHD resulting from single-gene mutations were retrieved and analyzed. RESULTS: Genetic screening of the 13 study subjects revealed six mutation-carrying patients (46%), including five hemizygous IGSF1 mutation carriers and one hemizygous TBL1X mutation carrier. Among the six mutation carriers, one had intellectual disability and the other one had obesity, but the remaining four did not show nonendocrine phenotypes. Loss of function of the TBL1X mutation (p.Asn382del) was confirmed in vitro. The literature review demonstrated etiology-specific relationship between serum prolactin (PRL) levels and TRH-stimulated TSH levels with some degree of overlap. CONCLUSIONS: The mutation screening study covering the five causative genes of congenital i-TSHD was performed, showing that the IGSF1 defect was the leading genetic cause of the disease. Assessing relationships between serum PRL levels and TRH-stimulated TSH levels would contribute to predict the etiologies of congenital i-TSHD.
Assuntos
Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/patologia , Imunoglobulinas/genética , Programas de Rastreamento/métodos , Proteínas de Membrana/genética , Mutação , Tireotropina/deficiência , Adolescente , Adulto , Biomarcadores/análise , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Proteínas Substratos do Receptor de Insulina/genética , Masculino , Linhagem , Prognóstico , Receptores do Hormônio Liberador da Tireotropina/genética , Tireotropina/sangue , Tireotropina/genética , Transducina/genética , Adulto JovemRESUMO
BACKGROUND/AIMS: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs). METHODS: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474), or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. RESULTS: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3) and thyroxine (T4). Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr) was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO) was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3) and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1) were also induced by triclosan. CONCLUSION: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.
Assuntos
Hipotireoidismo/patologia , Iodeto Peroxidase/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Glândula Tireoide/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/metabolismo , Imidazóis/farmacologia , Fígado/enzimologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Piridinas/farmacologia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do Hormônio Liberador da Tireotropina/antagonistas & inibidores , Receptores do Hormônio Liberador da Tireotropina/genética , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/sangue , Tiroxina/sangue , Triclosan/toxicidade , Tri-Iodotironina/sangue , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Fatigue affects most cancer patients and has numerous potential causes, including cancer itself and cancer treatment. Cancer-related fatigue (CRF) is not relieved by rest, can decrease quality of life, and has no FDA-approved therapy. Thyrotropin-releasing hormone (TRH) has been proposed as a potential novel treatment for CRF, but its efficacy against CRF remains largely untested. Thus, we tested the TRH analog, taltirelin (TAL), in mouse models of CRF. To model fatigue, we used a mouse model of chemotherapy, a mouse model of radiation therapy, and mice bearing colon 26 carcinoma tumors. We used the treadmill fatigue test to assess fatigue-like behavior after treatment with TAL. Additionally, we used wild-type and TRH receptor knockout mice to determine which TRH receptor was necessary for the actions of TAL. Tumor-bearing mice displayed muscle wasting and all models caused fatigue-like behavior, with mice running a shorter distance in the treadmill fatigue test than controls. TAL reversed fatigue-like behavior in all three models and the mouse TRH1 receptor was necessary for the effects of TAL. These data suggest that TAL may be useful in alleviating fatigue in all cancer patients and provide further support for evaluating TAL as a potential therapy for CRF in humans.
Assuntos
Fadiga/tratamento farmacológico , Nootrópicos/uso terapêutico , Hormônio Liberador de Tireotropina/análogos & derivados , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Linhagem Celular Tumoral , Neoplasias do Colo/complicações , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Fadiga/etiologia , Feminino , Fluoruracila/efeitos adversos , Raios gama/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Hormônio Liberador da Tireotropina/genética , Hormônio Liberador de Tireotropina/uso terapêuticoRESUMO
IGSF1 (Immunoglobulin Superfamily 1) gene defects cause central hypothyroidism and macroorchidism. However, the pathogenic mechanisms of the disease remain unclear. Based on a patient with a full deletion of IGSF1 clinically followed from neonate to adulthood, we investigated a common pituitary origin for hypothyroidism and macroorchidism, and the role of IGSF1 as regulator of pituitary hormone secretion. The patient showed congenital central hypothyroidism with reduced TSH biopotency, over-secretion of FSH at neonatal minipuberty and macroorchidism from 3 years of age. His markedly elevated inhibin B was unable to inhibit FSH secretion, indicating a status of pituitary inhibin B resistance. We show here that IGSF1 is expressed both in thyrotropes and gonadotropes of the pituitary and in Leydig and germ cells in the testes, but at very low levels in Sertoli cells. Furthermore, IGSF1 stimulates transcription of the thyrotropin-releasing hormone receptor (TRHR) by negative modulation of the TGFß1-Smad signaling pathway, and enhances the synthesis and biopotency of TSH, the hormone secreted by thyrotropes. By contrast, IGSF1 strongly down-regulates the activin-Smad pathway, leading to reduced expression of FSHB, the hormone secreted by gonadotropes. In conclusion, two relevant molecular mechanisms linked to central hypothyroidism and macroorchidism in IGSF1 deficiency are identified, revealing IGSF1 as an important regulator of TGFß/Activin pathways in the pituitary.
Assuntos
Ativinas/metabolismo , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Hipotireoidismo/patologia , Imunoglobulinas/genética , Proteínas de Membrana/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Análise Mutacional de DNA , Subunidade beta do Hormônio Folículoestimulante/genética , Seguimentos , Deleção de Genes , Humanos , Hipotireoidismo/genética , Recém-Nascido , Masculino , Camundongos , Hipófise/metabolismo , Hipófise/patologia , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Receptores do Hormônio Liberador da Tireotropina/genética , Proteínas Smad/metabolismo , Testículo/metabolismo , Testículo/patologiaRESUMO
Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of longterm steroid treatment and adrenal insufficiency. Although the function of the hypothalamohypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9weekold male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcriptionquantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone ß were significantly elevated in the pituitary gland of autotransplanted rats when compared with shamoperated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the shamoperated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with shamoperated rats. The authors identified significant alterations in the function of not only the hypothalamuspituitaryadrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.
Assuntos
Córtex Suprarrenal/transplante , Sistema Hipotálamo-Hipofisário/metabolismo , Adrenalectomia , Animais , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/genética , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Hipotálamo/metabolismo , Masculino , Hipófise/metabolismo , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo , Transplante Autólogo , Regulação para Cima , Urocortinas/genética , Urocortinas/metabolismoRESUMO
Autism spectrum disorder (ASD) is characterized by ritualistic-repetitive behaviors and impaired verbal/non-verbal communication. Many ASD susceptibility genes implicated in neuronal pathways/brain development have been identified. The Lebanese population is ideal for uncovering recessive genes because of shared ancestry and a high rate of consanguineous marriages. Aims here are to analyze for published ASD genes and uncover novel inherited ASD susceptibility genes specific to the Lebanese. We recruited 36 ASD families (ASD: 37, unaffected parents: 36, unaffected siblings: 33) and 100 unaffected Lebanese controls. Cytogenetics 2.7 M Microarrays/CytoScan™ HD arrays allowed mapping of homozygous regions of the genome. The CNTNAP2 gene was screened by Sanger sequencing. Homozygosity mapping uncovered DPP4, TRHR, and MLF1 as novel candidate susceptibility genes for ASD in the Lebanese. Sequencing of hot spot exons in CNTNAP2 led to discovery of a 5 bp insertion in 23/37 ASD patients. This mutation was present in unaffected family members and unaffected Lebanese controls. Although a slight increase in number was observed in ASD patients and family members compared to controls, there were no significant differences in allele frequencies between affecteds and controls (C/TTCTG: γ2 value = 0.014; p = 0.904). The CNTNAP2 polymorphism identified in this population, hence, is not linked to the ASD phenotype.
Assuntos
Transtorno do Espectro Autista/genética , Dipeptidil Peptidase 4/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas/genética , Receptores do Hormônio Liberador da Tireotropina/genética , Adolescente , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Consanguinidade , Proteínas de Ligação a DNA , Feminino , Predisposição Genética para Doença , Hereditariedade , Homozigoto , Humanos , Líbano , Masculino , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Análise de Sequência de DNA , Adulto JovemRESUMO
The transduction pathway mediating the inhibitory effect that TRH exerts on r-ERG channels has been thoroughly studied in GH3 rat pituitary cells but some elements have yet to be discovered, including those involved in a phosphorylation event(s). Using a quantitative phosphoproteomic approach we studied the changes in phosphorylation caused by treatment with 1µM TRH for 5min in GH3 cells. The activating residues of Erk2 and Erk1 undergo phosphorylation increases of 5.26 and 4.87 fold, respectively, in agreement with previous reports of ERK activation by TRH in GH3 cells. Thus, we studied the possible involvement of ERK pathway in the signal transduction from TRH receptor to r-ERG channels. The MEK inhibitor U0126 at 0.5µM caused no major blockade of the basal r-ERG current, but impaired the TRH inhibitory effect on r-ERG. Indeed, the TRH effect on r-ERG was also reduced when GH3 cells were transfected with siRNAs against either Erk1 or Erk2. Using antibodies, we found that TRH treatment also causes activating phosphorylation of Rsk. The TRH effect on r-ERG current was also impaired when cells were transfected with any of two different siRNAs mixtures against Rsk1. However, treatment of GH3 cells with 20nM EGF for 5min, which causes ERK and RSK activation, had no effect on the r-ERG currents. Therefore, we conclude that in the native GH3 cell system, ERK and RSK are involved in the pathway linking TRH receptor to r-ERG channel inhibition, but additional components must participate to cause such inhibition.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Somatotrofos/metabolismo , Animais , Linhagem Celular , Canais de Potássio Éter-A-Go-Go/genética , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Ratos , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Somatotrofos/citologia , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismoRESUMO
The hypothalamic-pituitary thyroid (HPT) axis modulates energy homeostasis. Its activity decreases in conditions of negative energy balance but the effects of chronic exercise on the axis are controversial and unknown at hypothalamic level. Wistar male rats were exposed for up to 14 days to voluntary wheel running (WR), or pair-feeding (PF; 18% food restriction), or to repeated restraint (RR), a mild stressor. WR and RR diminished food intake; body weight gain decreased in the 3 experimental groups, but WAT mass and serum leptin more intensely in the WR group. WR, but not RR, produced a delayed inhibition of central markers of HPT axis activity. At day 14, in WR rats paraventricular nucleus-pro-TRH mRNA and serum TSH levels decreased, anterior pituitary TRH-receptor 1 mRNA levels increased, but serum thyroid hormone levels were unaltered, which is consistent with decreased secretion of TRH and clearance of thyroid hormones. A similar pattern was observed if WR animals were euthanized during their activity phase. In contrast, in PF animals the profound drop of HPT axis activity included decreased serum T3 levels and hepatic deiodinase 1 activity; these changes were correlated with an intense increase in serum corticosterone levels. WR effects on HPT axis were not associated with changes in the activity of the hypothalamic-pituitary adrenal axis, but correlated positively with serum leptin levels. These data demonstrate that voluntary WR adapts the status of the HPT axis, through pathways that are distinct from those observed during food restriction or repeated stress.
Assuntos
Adaptação Fisiológica , Sistema Hipotálamo-Hipofisário/fisiopatologia , Atividade Motora , Estresse Fisiológico , Estresse Psicológico/fisiopatologia , Glândula Tireoide/fisiopatologia , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Restrição Calórica/efeitos adversos , Regulação da Expressão Gênica , Sistema Hipotálamo-Hipofisário/metabolismo , Leptina/sangue , Masculino , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Adeno-Hipófise/metabolismo , Ratos , Ratos Wistar , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Restrição Física/efeitos adversos , Estresse Psicológico/sangue , Glândula Tireoide/metabolismo , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismo , Tireotropina/sangue , Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismoRESUMO
We used somatolactotroph GH3 cells to examine changes in response to stimulation with thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) after sustained treatment with these peptides. TRH and PACAP increased prolactin promoter activity in mock- and PACAP type 1 receptor (PAC1R)-transfected cells. When the cells were pretreated with TRH for 48 h, the response of the prolactin promoter to both TRH and PACAP was diminished. Similarly, in PAC1R-transfected GH3 cells pretreated with PACAP, the effects of TRH and PACAP on the prolactin promoter were eliminated. The stimulation of prolactin mRNA expression by TRH and PACAP was eliminated by prolonged pretreatment with these peptides in PAC1R-transfected cells. Both the serum response element (SRE) promoters and cAMP response element (CRE) promoters were activated by TRH and PACAP in either mock- or PAC1R-transfected cells. Pretreatment for 48 h with TRH also eliminated the effects of TRH and PACAP on the SRE and CRE promoters, and pretreatment of PAC1R-transfected cells with PACAP for 48 h reduced the responses of the SRE and CRE promoters to TRH and PACAP. These observations demonstrated that sustained stimulation with TRH and PACAP desensitizes their own and each other's receptors.
Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Prolactina/biossíntese , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores do Hormônio Liberador da Tireotropina/genética , Hormônio Liberador de Tireotropina/fisiologia , Animais , Células Cultivadas , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Adeno-Hipófise/citologia , Cultura Primária de Células , Prolactina/genética , Regiões Promotoras Genéticas , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Elemento de Resposta SéricaRESUMO
Thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH(2)) has multiple, but transient, homeostatic functions in the brain. It is hydrolyzed in vitro by pyroglutamyl peptidase II (PPII), a narrow specificity ectoenzyme with a preferential localization in the brain, but evidence that PPII controls TRH communication in the brain in vivo is scarce. We therefore studied in male Wistar rats the distribution of PPII mRNA in the septum and the consequence of PPII inhibition on the analeptic effect of TRH injected into the medial septum. Twelve to 14% of cell profiles expressed PPII mRNA in the medial septum-diagonal band of Broca; in this region the specific activity of PPII was relatively high. Twenty to 35% of PPII mRNA-labeled profiles were positive for TRH-receptor 1 (TRH-R1) mRNA. The intramedial septum injection of TRH reduced, in a dose-dependent manner, the duration of ethanol-induced loss of righting reflex (LORR). Injection of the PPII inhibitor pGlu-Asn-Pro-7-amido-4-methylcoumarin into the medial septum enhanced the effect of TRH. The injection of a phosphinic TRH analog, a higher-affinity inhibitor of PPII, diminished the duration of LORR by itself. In contrast, the intraseptal injection of pGlu-Asp-Pro-NH(2), a peptide that did not inhibit PPII activity, or an inhibitor of prolyl oligopeptidase did not change the duration of LORR. We conclude that in the medial septum PPII activity may limit TRH action, presumably by reducing the concentration of TRH in the extracellular fluid around cells coexpressing PPII and TRH-R1.
Assuntos
Aminopeptidases/antagonistas & inibidores , Estimulantes do Sistema Nervoso Central/farmacologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Septo do Cérebro/efeitos dos fármacos , Septo do Cérebro/enzimologia , Hormônio Liberador de Tireotropina/farmacologia , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Masculino , Peptídeos/farmacologia , Prolil Oligopeptidases , Ácido Pirrolidonocarboxílico/antagonistas & inibidores , Ácido Pirrolidonocarboxílico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Reflexo de Endireitamento/genética , Septo do Cérebro/metabolismo , Serina Endopeptidases/farmacologiaRESUMO
Two GPCRs (G-protein-coupled receptors), TRHR (thyrotropin-releasing hormone receptor) and beta(2)AR (beta(2)-adrenergic receptor), are regulated in distinct manners. Following agonist binding, TRHR undergoes rapid phosphorylation attributable to GRKs (GPCR kinases); beta(2)AR is phosphorylated by both second messenger-activated PKA (protein kinase A) and GRKs with slower kinetics. TRHR co-internalizes with arrestin, whereas beta(2)AR recruits arrestin, but internalizes without it. Both receptors are dephosphorylated following agonist removal, but TRHR is dephosphorylated much more rapidly while it remains at the plasma membrane. We generated chimaeras swapping the C-terminal domains of these receptors to clarify the role of different receptor regions in phosphorylation, internalization and dephosphorylation. beta(2)AR with a TRHR cytoplasmic tail (beta(2)AR-TRHR) and TRHR with a beta(2)AR tail (TRHR-beta(2)AR) signalled to G-proteins normally. beta(2)AR-TRHR was phosphorylated well at the PKA site in the third intracellular loop, but poorly at GRK sites in the tail, whereas TRHR-beta(2)AR was phosphorylated strongly at GRK sites in the tail (Ser(355)/Ser(356) of the beta(2)AR). Both chimaeric receptors exhibited prolonged, but weak, association with arrestin at the plasma membrane, but high-affinity arrestin interactions and extensive co-internalization of receptor with arrestin required a phosphorylated TRHR tail. In contrast, swapping C-terminal domains did not change the rates of phosphorylation and dephosphorylation or the dependence of TRHR dephosphorylation on the length of agonist exposure. Thus the interactions of GPCRs with GRKs and phosphatases are determined not simply by the amino acid sequences of the substrates, but by regions outside the cytoplasmic tails.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Arrestina/metabolismo , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Quinases de Receptores Acoplados a Proteína G/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Isoproterenol/farmacologia , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Ligação Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores do Hormônio Liberador da Tireotropina/agonistas , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: The investigation of expression and functional relevance of G-protein coupled receptors in primary aldosteronism (PA) by molecular and clinical studies. PATIENTS AND METHODS: Tissues of 14 aldosterone-producing adenomas (APA), of one unilateral adrenal hyperplasia and of six healthy adult adrenal glands; 12 patients with confirmed PA due to APA; (n=5), idiopathic hyperaldosteronism (n=7) and 8 control subjects (C). The tissues were subjected to a quantitative PCR for determination of mRNA expression levels of AT2R1, GIPR, MC2R, GnRHR, LHR, TRHR, TSHR, glucagon-R, V1aR, V2R, and 5-HT4R. The patients and controls were enrolled in a test protocol consisting of stimulation by posture, mixed meal, ACTH, GnRH, TRH, glucagon, vasopressin, and metoclopramide (MCP). Three patients could be analyzed by both studies. A positive response was defined as an aldosterone increase of more than 50% following stimulation. RESULTS: All the tissues revealed AT2R1, MC2R, AVPR, and 5-HT4R mRNA expression. LHR mRNA was found in normal adrenals and 13 adrenal tumors. By contrast with normal adrenals tumorous adrenal tissue expressed GnRHR mRNA (4/15) and TSHR mRNA (1/15). Both the patients and controls responded to posture, ACTH, glucagon, AVP, and MCP. Specific responses were seen in one patient following TRH and three patients following GnRH stimulation. CONCLUSIONS: We provide evidence for peptide hormone responsiveness to various peptide hormones in patients with PA, including GnRH and TRH. A good correlation between clinical and molecular testing could be observed, making an involvement of the receptor expressed in PA possible.
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Testes Genéticos , Hiperaldosteronismo/genética , Receptores Acoplados a Proteínas G/genética , Adulto , Idoso , Aldosterona/sangue , Humanos , Pessoa de Meia-Idade , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Melanocortina/genética , Receptores dos Hormônios Gastrointestinais/genética , Receptores de Glucagon/genética , Receptores do LH/genética , Receptores LHRH/genética , Receptores 5-HT4 de Serotonina/genética , Receptores da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores de Vasopressinas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: Thyrotropin-releasing hormone (TRH) is expressed in rodent and human adult pancreata and in mouse pancreas during embryonic development. However, expression of TRH receptors (TRHRs) in the pancreas is controversial. We sought to provide evidence that the TRH/TRHR system might play a role in fetal development. METHODS: We used quantitative reverse transcription-polymerase chain reaction to measure TRH and TRHR messenger RNA (mRNA). To study the effects of TRHR expression in a pancreatic progenitor population, we expressed TRHRs in human islet-derived precursor cells (hIPCs) by infection with adenoviral vector AdCMVmTRHR. Thyrotropin-releasing hormone receptor signaling was measured as inositol phosphate production and intracellular calcium transients. Thyrotropin-releasing hormone receptor expression was measured by [H]methyl-TRH binding. Apoptosis was monitored by release of cytochrome c from mitochondria. RESULTS: We show that TRH mRNA is expressed in human fetal and adult pancreata, and that TRHR mRNA is expressed in fetal human pancreas but not in adult human pancreas. Thyrotropin-releasing hormone receptors expressed in hIPCs were shown to signal normally. Most importantly, TRH treatment for several days stimulated apoptosis in hIPCs expressing approximately 400,000 TRHRs per cell. CONCLUSIONS: These findings suggest a possible role for TRH/TRHR signaling in pancreatic precursors to promote programmed cell death, a normal constituent of morphogenesis during embryonic development in humans.
Assuntos
Apoptose , Pâncreas/citologia , Pâncreas/embriologia , Receptores do Hormônio Liberador da Tireotropina/fisiologia , Humanos , RNA Mensageiro/análise , Receptores do Hormônio Liberador da Tireotropina/análise , Receptores do Hormônio Liberador da Tireotropina/genética , Hormônio Liberador de Tireotropina/genéticaRESUMO
Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326-345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4-S5 linker.
Assuntos
Canais de Potássio Éter-A-Go-Go/fisiologia , Receptores do Hormônio Liberador da Tireotropina/fisiologia , Sequência de Aminoácidos , Animais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Dados de Sequência Molecular , Oócitos/fisiologia , Receptores do Hormônio Liberador da Tireotropina/genética , Xenopus laevisRESUMO
TRH neurons of the hypothalamic paraventricular nucleus (PVN), regulate pituitary-thyroid axis (HPT). Fasting activates expression of orexigenic peptides from the arcuate nucleus, increases corticosterone while reduces leptin, and pro-TRH mRNA levels despite low serum thyroid hormone concentration (tertiary hypothyroidism). TRH synthesis is positively regulated by anorexigenic peptides whose expression is reduced in fasting. The model of dehydration-induced anorexia (DIA) leads to decreased voluntary food intake but peptide expression in the arcuate is similar to forced-food restriction (FFR), where animals remain hungered. We compared the response of HPT axis of female Wistar rats submitted to DIA (2.5% saline solution, food ad libitum, 7 days) with FFR (provided with the amount of food ingested by DIA) and naïve (N) group fed ad libitum, as well as their response to acute cold exposure. Pro-TRH and pro-CRH mRNA levels in the PVN were measured by RT-PCR, TRH content, serum concentration of TSH and thyroid hormones by radioimmunoassay. DIA rats reduced 80% their food consumption compared to N, decreased PVN pro-CRH expression, serum estradiol and leptin levels, increased corticosterone similar to FFR. HPT axis of DIA animals failed to adapt: FFR presented tertiary hypothyroidism and DIA, primary. Response to cold stimulation leading to increased pro-TRH mRNA levels and TRH release was preserved under reduced energy availability in FFR rats but not in DIA, although the dynamics of hormonal release differed: TSH release augmented only in naïve; thyroxine in all but highest in DIA, and triiodothyronine in FFR and DIA suggesting a differential regulation of deiodinases.
Assuntos
Adaptação Fisiológica , Regulação do Apetite/fisiologia , Privação de Alimentos/fisiologia , Neurônios/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Aminopeptidases/metabolismo , Animais , Anorexia/etiologia , Anorexia/metabolismo , Ansiedade/complicações , Ansiedade/metabolismo , Composição Corporal , Temperatura Baixa , Corticosterona/sangue , Desidratação/complicações , Desidratação/metabolismo , Modelos Animais de Doenças , Feminino , Sistema Hipotálamo-Hipofisário/metabolismo , Análise por Pareamento , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Adeno-Hipófise/metabolismo , Precursores de Proteínas/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Fatores Sexuais , Estresse Fisiológico/complicações , Estresse Fisiológico/metabolismo , Glândula Tireoide/metabolismo , Hormônios Tireóideos/sangueAssuntos
Hipopituitarismo/etiologia , Mutação , Prolactina/deficiência , Receptores do Hormônio Liberador da Tireotropina/genética , Tireotropina/deficiência , Animais , Diagnóstico Diferencial , Humanos , Hipopituitarismo/diagnóstico , Hipopituitarismo/fisiopatologia , Hipopituitarismo/terapia , Prognóstico , Receptores do Hormônio Liberador da Tireotropina/química , Receptores do Hormônio Liberador da Tireotropina/fisiologia , Tiroxina/administração & dosagemRESUMO
Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic beta-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic beta-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated alphabetagamma-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse beta-cell line, betaTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in betaTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic beta-cell biology.
Assuntos
Receptores ErbB/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Hormônio Liberador de Tireotropina/farmacologia , Animais , Anticorpos/farmacologia , Sequência de Bases , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/imunologia , Fator de Crescimento Epidérmico/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Expressão Gênica/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Células Secretoras de Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Pirimidinas/farmacologia , Receptores do Hormônio Liberador da Tireotropina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
Thyrotropin releasing hormone (TRH) stimulates an increase in plasma cortisol in horses with pars intermedia dysfunction (PPID, Cushing's disease). A similar phenomenon is observed in humans with Cushing's disease or Nelson's syndrome. The mechanism of the response in humans is not known, but an alteration in receptor expression, selectivity or responsiveness in abnormal corticotropes has been proposed. Horses with PPID, unlike humans, almost exclusively have adenomas of pars intermedia (PI) rather than pars distalis (PD) origin. Therefore, the mechanism responsible for the TRH response observed in horses likely differs. We proposed that TRH directly stimulates the PI in normal and PPID-affected horses to release proopiomelanocortin (POMC) derived peptides. Using alpha-melanocyte stimulating hormone (alpha-MSH) as a marker of a PI response and ACTH as a marker of a PD response, we were able to demonstrate a marked increase in plasma concentration of alpha-MSH and a modest, but significant increase in ACTH after TRH treatment in normal horses. The ability of TRH to directly stimulate release of POMC peptides was confirmed using PI and PD tissue explants. The presence of TRH receptor mRNA in PI tissue from both normal and PPID horses was confirmed using reverse transcriptase polymerase chain reaction. We conclude that TRH triggers the release of POMC-derived peptides from the PI through the direct stimulation of TRH receptors normally expressed on melanotropes. The increase in plasma cortisol following TRH in horses with PPID is likely attributable to the release of ACTH from the hyperplastic PI.