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
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
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
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
The interaction between a glandular target and hypothalamic dopaminergic neurons during development was studied by cocultivating dispersed fetal hypothalamic and adult intermediate lobe rat cells in serum-free medium. In such conditions, hypothalamic neurons aggregated around intermediate lobe cells and were interconnected by well-developed neurites. They could be grown in coculture at lower density than alone. In regard to dopaminergic activity, both tyrosine hydroxylase content and accumulation of [(3)H]DA in the cells were significantly increased in coculture after 6 days in vitro (DIV). These effects persisted until 18 DIV, but were progressively reduced with time. Other neuronal activities (choline acetyltransferase activity or thyroliberin content) were not modified by coculture. Furthermore, dopaminergic activity was not stimulated when hypothalamic neurons were grown in the presence of anterior pituitary cells, nor mesencephalic neurons together with intermediate lobe cells, suggesting selectivity of intermediate lobe cells on hypothalamic DA neurons. After radioautographic labeling of DA neurons at 6 DIV, morphometric analysis revealed that the presence of intermediate lobe cells increased the number of branching points and the total neuritic length, without changing the number of DA neurons, the size of cell bodies, nor the number of neurites emerging from the soma. Cocultured DA neurons at 6 DIV exhibited morphological features of more differentiated neurons, as estimated by morphological analysis of 12 DIV control neurons. Thus, intermediate lobe cells induce in vitro clustering of fetal hypothalamic neuronal somata and accelerate specifically hypothalamic dopaminergic neuron maturation.
RESUMO
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.