RESUMO
Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.
Assuntos
Tonsila do Cerebelo/fisiologia , Regulação da Expressão Gênica/fisiologia , Excitação Neurológica , Hormônio Liberador de Tireotropina/fisiologia , Animais , Sequência de Bases , Primers do DNA , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores do Hormônio Liberador da Tireotropina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABA(A) receptors' agonists and antagonists were tested at 1 h (+/-ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol's inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol's inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons.
Assuntos
Aminopeptidases/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Etanol/farmacologia , Sistema Límbico/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/análogos & derivados , Hormônio Liberador de Tireotropina/metabolismo , Transtornos do Sistema Nervoso Induzidos por Álcool/enzimologia , Transtornos do Sistema Nervoso Induzidos por Álcool/genética , Transtornos do Sistema Nervoso Induzidos por Álcool/fisiopatologia , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Química Encefálica/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Esquema de Medicação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A , Sistema Límbico/enzimologia , Sistema Límbico/fisiopatologia , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/enzimologia , Vias Neurais/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Ácido Pirrolidonocarboxílico/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia , Hormônio Liberador de Tireotropina/genéticaRESUMO
The effect of chronic ethanol consumption during pregnancy and lactation on thyrotropin releasing hormone (TRH) metabolism was investigated in the hypothalamus and limbic areas of female rats and their weaned pups. Pregnant female rats received ethanol or isocaloric glucose solution during pregnancy either alone, or also during the 3 weeks of lactation. Thyrotropin (TSH) and corticosterone levels were measured in serum; TRH and TRH-gly concentrations were determined in hypothalamus, hippocampus, n.accumbens, frontal cortex and amygdala of dams and pups at 21 days after parturition. Ethanol or glucose consumption during pregnancy and lactation produced a decrease in TSH levels compared with control animals fed at libitum; water replacement during lactation normalized TSH levels only in glucose-fed dams. Pups from ethanol or pair-fed dams showed low weight and increased TSH levels compared with normal rats. Variations in TRH metabolism were detected in limbic areas. Chronic ethanol caused a decrease in the levels of TRH in the hippocampus and frontal cortex of dams. In contrast, glucose chronic ingestion increased TRH content specifically in n.accumbens and amygdala of dams. Most of the variations in TRH content of limbic areas of pups were not specific for glucose or ethanol treatment and correlated with the deleterious effect of the mother's thyroid condition, although some differences were observed depending on pup's gender. These results support the involvement of TRHergic neurons in the limbic system of the female rat exposed to alcohol or glucose during pregnancy and lactation.
Assuntos
Etanol/farmacologia , Glucose/farmacologia , Hipotálamo/efeitos dos fármacos , Sistema Límbico/efeitos dos fármacos , Hormônio Liberador de Tireotropina/metabolismo , Animais , Peso Corporal , Etanol/administração & dosagem , Feminino , Glucose/administração & dosagem , Hipotálamo/citologia , Hipotálamo/metabolismo , Sistema Límbico/citologia , Sistema Límbico/metabolismo , Masculino , Neurônios/metabolismo , Gravidez , Ratos , Ratos WistarRESUMO
Little is known about the temporal relationship and the sequential steps for peptide biosynthesis during the terminal differentiation of the peptide phenotype in central nervous system. Analysis of the TRH phenotype in primary cultures of rat fetal day 17 hypothalamic cells has shown that TRH levels start increasing only after a week in culture, in contrast with in vivo data showing a steady increase during late fetal life. The purpose of this study was to compare the developmental patterns of TRH and pro-TRH mRNA levels in vitro to determine whether the initial low and steady levels of TRH are due to deficient transcription. Pro-TRH mRNA levels were detected by semi-quantitative RT-PCR through the development of primary cultures of serum-supplemented hypothalamic fetal cells from 17 day old embryos. Pro-TRH mRNA levels per dish increased steadily since the beginning of the culture. In contrast, TRH levels per dish were low and stable during the first week increasing afterwards, but remaining low compared to equivalent in vivo values. Pro-TRH mRNA levels per hypothalamus increased between fetal day 17 and postnatal 14, suggesting that the in vitro pattern of pro-TRH mRNA development mimics that occurring in vivo. These data show that pro-TRH gene expression does not limit TRH accumulation in vitro suggesting that the transcriptional and post-transcriptional programs leading to peptide accumulation are established independently.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Neurônios/fisiologia , Precursores de Proteínas/genética , Hormônio Liberador de Tireotropina/genética , Animais , Células Cultivadas , Feminino , Feto/citologia , Hipotálamo/embriologia , Técnicas In Vitro , Neurônios/citologia , Gravidez , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
TRH is hydrolyzed by pyroglutamyl aminopeptidase II (PP II), a highly specific ecto-enzyme which is localized on the surface of lactotrophs. To study whether PP II activity may be rapidly regulated during a burst of prolactin secretion, we used an in vitro model in which primary cultures of adenohypophyseal cells were incubated with 500 nM dopamine (DA) for 24 h prior to treatments. We observed a rapid increase of PP II activity when 100 nM [3-Me-His(2)]-TRH, a TRH agonist, was added at removal of DA. PPII activity was maximal after 20 min of treatment and reduced to time 0 activity at 30 min. Dopamine withdrawal alone, slightly and transiently, modified the enzyme activity: an initial activation at 15 min was followed by a transient inhibition at 20 min. The specific contribution of [3-Me-His(2)]-TRH in this paradigm was a transient enhancement of PP II activity. If DA was not removed, [3-Me-His(2)]-TRH was ineffective. These data demonstrate that during in vitro conditions that mimic a suckling episode, adenohypophyseal PP II activity is rapidly and reversibly adjusted.
Assuntos
Aminopeptidases/metabolismo , Dopamina/farmacologia , Adeno-Hipófise/enzimologia , Hormônio Liberador de Tireotropina/análogos & derivados , Hormônio Liberador de Tireotropina/agonistas , Hormônio Liberador de Tireotropina/farmacologia , Animais , Células Cultivadas , Feminino , Adeno-Hipófise/citologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Ratos WistarRESUMO
External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipotálamo/citologia , Neurônios/enzimologia , Precursores de Proteínas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Feto/citologia , Fibronectinas/análise , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/embriologia , Técnicas In Vitro , Laminina/análise , Neurônios/química , Neurônios/citologia , Pró-Proteína Convertases , Biossíntese de Proteínas/fisiologia , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Ratos Wistar , Tenascina/análise , Hormônio Liberador de Tireotropina/genética , Vitronectina/análiseRESUMO
TRH-like immunoreactivity distinct from TRH is present in various tissues and fluids. In order to determine whether TRH-like molecules are secreted by the hypothalamus, we analyzed tissues and media from hypothalamic slices incubated in Krebs Ringer bicarbonate. Media from basal or high KCl conditions contained 3 TRH-like molecules evidenced by reverse phase high performance liquid chromatography followed by TRH radioimmunoassay. Peak I corresponded to authentic TRH (73% of total immunoreactivity) and peaks II and III had a higher retention time. These additional TRH-like forms were neither detected in hypothalamic tissue nor in tissue or medium from olfactory bulb. Gel filtration analysis of hypothalamic media revealed only one TRH-like peak eluting as TRH, suggesting that the molecular weights of peaks II and III are similar to that of TRH. Peak II retention time was similar to that of pglu-phe-proNH2. We analysed if they could be produced by post secretory metabolism of TRH. Incubation of hypothalamic slices with [3H-Pro]-TRH did not produce radioactive species comigrating with peaks II or III. However, it induced rapid degradation to [3H-Pro]-his-prodiketopiperazine ([3H]-HPDKP). Inhibitor profile suggested that pyroglutamyl aminopeptidase II, but not pyroglutamyl aminopeptidase I, is responsible for [3H]-HPDKP production. These data are consistent with the hypothesis that pyroglutamyl aminopeptidase II is the main aminopeptidase degrading TRH in hypothalamic extracellular fluid. Furthermore, we suggest that the hypothalamus releases additional TRH-like molecules, one of them possibly pglu-phe-proNH2, which may participate in control of adenohypophyseal secretions.
Assuntos
Hipotálamo/metabolismo , Extratos Placentários/metabolismo , Aminopeptidases/metabolismo , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dipeptídeos/metabolismo , Técnicas In Vitro , Masculino , Bulbo Olfatório/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Ratos WistarRESUMO
The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations. To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1-3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways. TRH mRNA relative changes were determined by the RT-PCR technique. One hour incubation with 10(-10)-10(-4) M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10(-10) M, an increase above control at 10(-8)-10(-6) M and a reduction at higher concentrations (10(-5)- 10(-4) M). The stimulatory effect of 10(-8) M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment. Changes in TRH mRNA levels were reflected by enhanced TRH cell content. Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively. An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells. The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment. The stimulatory effect of 10(-7) M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10(-3) M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs. An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone. These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.
Assuntos
AMP Cíclico/fisiologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Hipotálamo/efeitos dos fármacos , RNA Mensageiro/biossíntese , Hormônio Liberador de Tireotropina/genética , Animais , Bucladesina/farmacologia , Células Cultivadas , Hipotálamo/citologia , Hipotálamo/metabolismo , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
PURPOSE: Thyrotropin-releasing hormone (TRH), present in extra hypothalamic brain areas, has been proposed to have neuromodulatory functions and to be susceptible to change by electrical stimulation paradigms. We measured TRH concentrations of several brain areas during kindling development before its establishment and determined whether the changes detected in TRH levels were related to the behavioral stages of kindling, the number of stimulations required to reach these stages and, with the electrophysiological parameters characteristic of this paradigm (amygdaloid afterdischarge (AD) frequency, duration, and propagation). METHODS: Male Wistar rats were implanted stereotaxically with indwelling bipolar electrodes in the basolateral nucleus of the amygdala and with two stainless-steel electrodes epidurally in frontal cortex. Amygdaloid kindling was induced by daily electrical stimulation; AD frequency and duration were recorded and analyzed throughout the development of kindling. TRH was extracted from several regions and quantified by radioimmunoassay (RIA). RESULTS: Modifications in TRH concentrations were detected, depending on the region assayed, from stage II of kindling. A positive correlation was noted between the levels of TRH and the frequency and propagation of AD, but not with the number of stimulations. The rate of change in TRH concentration in relation to AD frequency or duration was highest in frontal cortex followed by hippocampus and amygdala. CONCLUSIONS: A graded response was noted in the increase in TRH concentration dependent on the increase of AD frequency and propagation. The rate of response correlated with the region's epileptogenic susceptibility.
Assuntos
Tonsila do Cerebelo/química , Química Encefálica , Eletroencefalografia , Epilepsia/metabolismo , Excitação Neurológica/metabolismo , Hormônio Liberador de Tireotropina/análise , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiologia , Animais , Estimulação Elétrica , Eletrofisiologia , Epilepsia/fisiopatologia , Lobo Frontal/química , Lobo Frontal/fisiologia , Lateralidade Funcional/fisiologia , Hipocampo/química , Hipocampo/fisiologia , Excitação Neurológica/fisiologia , Masculino , Radioimunoensaio , Ratos , Ratos Wistar , Hormônio Liberador de Tireotropina/biossínteseRESUMO
Primary cultures of hypothalamic cells maintained in the presence of serum were either kept with homologous conditioned medium (CM) (i.e. only half of the medium was removed at each medium change) or without (total medium change). In cultures with homologous CM, TRH levels were increased. The effects of CMs from various intervals of the primary culture were tested. The strongest increases of TRH levels were obtained with CM from cultures enriched with hypothalamic glia.
Assuntos
Hipotálamo/efeitos dos fármacos , Fatores de Crescimento Neural/biossíntese , Neurônios/efeitos dos fármacos , Hormônio Liberador de Tireotropina/biossíntese , Sequência de Aminoácidos , Animais , Contagem de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Citarabina/farmacologia , Hipotálamo/citologia , Hipotálamo/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismoRESUMO
In order to determine the pathway of extracellular metabolism of the thyrotropin releasing hormone (pyroglu-his-proNH(2)) in brain, the topographical organization of pyroglutamate aminopeptidase II on the plasma membrane was investigated. Its activity was only slightly increased when intact brain synaptosomes were lysed by osmotic shock or detergent treatment. Trypsin treatment of intact synaptosomes destroyed 70-80% of enzyme activity without affecting lactate dehydrogenase. Pyroglutamate aminopeptidase II activity was present in primary cultures of foetal mice cortical cells. It was detected in intact cells, was not released by the cells and its activity was not increased by saponin pretreatment. Trypsin treatment of the cells reduced pyroglutamate aminopeptidase II by 70% but did not affect pyroglutamate aminopeptidase I and lactate dehydrogenase. These data support that brain pyroglutamate aminopeptidase II is an ectoenzyme. They suggest that this enzyme could be responsible for thyrotropin releasing hormone extracellular catabolism in brain.