RESUMO
Nicotinic cholinergic receptors play a role in cardiovascular regulation in the lower brain stem. Herein, we present evidence that l-3,4-dihydroxyphenylalanine (DOPA), a putative neurotransmitter in the central nervous system, is involved in the depressor response to microinjection of nicotine into the nucleus tractus solitarii (NTS). Microinjection of nicotine into the medial area of the NTS led to decreases in arterial blood pressure and heart rate in anesthetized rats. Mecamylamine, a nicotinic receptor antagonist, microinjected into NTS, blocked the depressor and bradycardic responses to nicotine. Nicotine-induced depressor and bradycardic responses were blocked by DOPA cyclohexyl ester (DOPA CHE), an antagonist for DOPA. DOPA CHE did not modify the action of carbachol on excitatory postsynaptic potential in rat cortical slices. These results suggest that endogenous DOPA is involved in nicotine-induced depressor responses in the NTS of anesthetized rats.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Levodopa/análogos & derivados , Levodopa/antagonistas & inibidores , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Núcleo Solitário/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , Levodopa/farmacologia , Masculino , Mecamilamina/farmacologia , Microinjeções , Antagonistas Nicotínicos/farmacologia , Ratos , Ratos WistarRESUMO
L-3,4-dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic L-amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Dopaminérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Levodopa/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Benserazida/farmacologia , Contagem de Células/métodos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Lateralidade Funcional , Hidrazinas/farmacologia , Imuno-Histoquímica/métodos , Masculino , Feixe Prosencefálico Mediano/lesões , Atividade Motora/efeitos dos fármacos , Oxidopamina/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose.
Assuntos
Córtex Cerebral/metabolismo , Glucose/farmacologia , Hipotálamo Médio/metabolismo , Neurônios/metabolismo , Animais , Córtex Cerebral/citologia , Desoxiglucose/farmacologia , Eletrodos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Hipotálamo Médio/citologia , Técnicas In Vitro , Ácido Iodoacético/farmacologia , Masculino , Florizina/farmacologia , Ácido Pirúvico/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estimulação QuímicaRESUMO
To examine the sex difference in the 24-h profile of the acetylcholine (ACh) release in the hippocampus, in addition to the effects of housing conditions on this profile, we performed an in vivo microdialysis study in intact male and cycling female rats that had been living in large (diameter=35 cm) or small (diameter=19 cm) cylindrical cages. Each rat was individually housed in a cage for 4 days. On the day of the experiment, the dialysate was collected from the dorsal hippocampus at 20-min intervals and sequential blood samples were simultaneously obtained at 2-h intervals, under the freely moving condition for more than 24 h. ACh in the dialysates was measured by the high performance liquid chromatography system, while the corticosterone concentration in the serum was measured by radiostereoassay. Although the ACh release showed a clear daily rhythm in both sexes of rats, the amount of ACh released in female rats was significantly lower than that in males. Furthermore, the housing in the small cage significantly attenuated the ACh release during the dark phase in male rats, but not in female rats. Conversely, the serum corticosterone concentration showed a clear daily rhythm and the mean concentration of serum corticosterone in female rats was significantly higher than that in male rats. Housing in the small cage did not affect the corticosterone rhythm in either sex. These results reveal a sex difference in the 24-h profile of the ACh release, which suggests vulnerability of the cholinergic system in male rats depending on its housing conditions.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Abrigo para Animais , Caracteres Sexuais , Córtex Suprarrenal/metabolismo , Análise de Variância , Animais , Ritmo Circadiano/fisiologia , Corticosterona/sangue , Feminino , Masculino , Microdiálise/métodos , Ratos , Ratos WistarRESUMO
We have reported in the past that female rats fed a powdered diet showed better spatial learning and memory functions than female rats a fed pelleted diet. In the present study, we examined the effects of feeding with powdered diet on acetylcholine release in the hippocampus in both sexes of rats. After weaning (3 weeks of age), rats were fed either standard pelleted diet or powdered diet, and after maturation (9-12 weeks of age), they were used in an in vivo microdialysis study, in which no eserine (a cholinesterase inhibitor) was added to the perfusate. The dialysate was collected from the dorsal hippocampus at 20-min intervals under freely moving conditions for more than 24 h. Acetylcholine in the dialysate was measured by high performance liquid chromatography. As we reported previously, the acetylcholine release showed a clear daily rhythm in both sexes, and males showed significantly greater acetylcholine release in the hippocampus than females in rats fed pelleted diet. Conversely, in rats fed powdered diet, no sex difference in the acetylcholine release was observed, since feeding with powdered diet significantly increased the acetylcholine release only in females. To further examine the number of cholinergic neurons in the medial septum and horizontal limb of the diagonal band of Broca, immunocytochemistry for choline acetyltransferase was performed in both sexes of rats fed either standard pelleted diet or powdered diet. However, neither sex nor feeding conditions affect the number of choline acetyltransferase immunoreactive cells in the areas. These results suggest that powdered diet after weaning enhances spontaneous acetylcholine release in the hippocampus in female rats without changes in the number of cholinergic neurons in the areas. It is possible that this effect of feeding contributes to improve the performance in spatial learning and memory functions in female rats fed powdered diet.
Assuntos
Acetilcolina/metabolismo , Dieta , Hipocampo/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Cromatografia Líquida de Alta Pressão , Feixe Diagonal de Broca/metabolismo , Feminino , Hipocampo/química , Imuno-Histoquímica , Masculino , Microdiálise , Neurônios/metabolismo , Gravidez , Ratos , Septo do Cérebro/metabolismo , Caracteres Sexuais , DesmameRESUMO
We employed morphological and electrophysiological methods in order to elucidate mechanisms which are responsible for communication between cellular oscillators in the cultured rat suprachiasmatic nucleus, the site of the endogenous biological clock that regulates circadian rhythms in mammals. When a gap junction-permeable dye, Lucifer Yellow, was injected into single neurons in the suprachiasmatic nucleus culture, the dye was transferred to neighboring cells in a gap junction blocker-sensitive manner. Optical imaging of neural activity evoked by electrical stimulation in the culture revealed that the spread of depolarization was inhibited by gap junction blockers but not by a blocker of voltage-dependent Na(+) channels. Depolarization propagation was inhibited by muscimol, a GABA(A) receptor agonist, in a dose-dependent manner and the inhibition was reversed by bicuculline, a GABA(A) receptor antagonist. Furthermore, muscimol inhibited dye-transfer between neurons in the suprachiasmatic nucleus culture in a dose-dependent fashion.These independent lines of evidence suggest that the gap junction communication is involved in interneuronal communication in the suprachiasmatic nucleus slice culture and that the coupling state between neurons is not static but dynamically regulated via GABA(A) receptor systems.
Assuntos
Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Núcleo Supraquiasmático/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Bicuculina/farmacologia , Técnicas de Cultura , Eletrofisiologia , Corantes Fluorescentes , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Halotano/farmacologia , Isoquinolinas , Muscimol/farmacologia , Octanóis/farmacologia , Ratos , Ratos Wistar , Tetrodotoxina/farmacologiaRESUMO
Pituitary adenylate cyclase-activating peptide is densely distributed in the suprachiasmatic nucleus, which functions as the circadian pacemaker. A receptor for pituitary adenylate cyclase-activating peptide, denoted as PAC(1), exists in six variant forms. We used reverse transcriptase-polymerase chain reaction to identify the PAC(1) variants that are expressed in the suprachiasmatic nucleus. Dominant variant forms of PAC(1) in the suprachiasmatic nucleus were PAC(1)short, PAC(1)hip, and PAC(1)hop1. By in situ hybridization, we examined 24-h profiles of mRNAs for the identified receptor variants in the suprachiasmatic nucleus in constant darkness and during the light-dark cycle. In constant darkness there were clear circadian rhythms in PAC(1)short mRNA with a peak at circadian time 4 but no rhythmicity was observed in PAC(1)hip mRNA or PAC(1)hop1 mRNA. In light-dark cycles, on the other hand, PAC(1)hip mRNA displayed a bimodal rhythm with troughs at zeitgeber time 4 and 16 but PAC(1)hop1 mRNA stayed constant during the day. These results suggest that PAC(1) splice variants are differentially regulated in the rat suprachiasmatic nucleus.
Assuntos
Processamento Alternativo/genética , Ritmo Circadiano/genética , Neurônios/metabolismo , RNA Mensageiro/genética , Receptores do Hormônio Hipofisário/genética , Núcleo Supraquiasmático/metabolismo , Animais , Northern Blotting , Hibridização In Situ , Masculino , Neurônios/citologia , Estimulação Luminosa , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Núcleo Supraquiasmático/citologiaRESUMO
GABA is a potent regulator of gonadotropin-releasing hormone neurons in the hypothalamus. To determine the profile of GABA release in the medial preoptic area where the gonadotropin surge generator resides, an in vivo microdialysis study was performed in cyclic female rats. The microdialysis samples were collected and sequential blood samples (150 microl each) were also obtained, at 1-h intervals. During estrus and diestrus 1, GABA release in the medial preoptic area was relatively low. A small increase in the GABA release began in the afternoon of diestrus 1 and attained its peak in the morning of diestrus 2, but declined in the afternoon of that day. The GABA release markedly increased from late in the night of diestrus 2 through the morning of proestrus, when it attained its peak, and thereafter it declined sharply until the critical period of proestrus. A distinct preovulatory luteinizing hormone surge was observed in the afternoon of proestrus in all proestrous rats. From these results we suggest that the preovulatory elevation of the GABA release from the night through to the morning of proestrus, followed by a sharp decline, is closely associated with the onset of the preovulatory luteinizing hormone surge in cyclic female rats. The present study is the first to report the 4-day profile of GABA release in the medial preoptic area during the estrous cycle.
Assuntos
Ciclo Estral/metabolismo , Área Pré-Óptica/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Ritmo Circadiano , Diestro/metabolismo , Estradiol/sangue , Ciclo Estral/sangue , Estro/metabolismo , Feminino , Hormônio Luteinizante/sangue , Microdiálise , Proestro/metabolismo , Radioimunoensaio , Ratos , Ratos Endogâmicos , Ratos WistarRESUMO
We describe a procedure for detection of low-abundance cellular RNAs by in situ hybridization histochemistry, using single-stranded DNA probes produced by amplified primer extension labeling with Taq polymerase. We have used this approach to detect a number of high- and low-abundance RNA species and have found it to be a simple and reproducible method of obtaining sensitive probes for in situ hybridization studies. For example, DNA probes generated by amplified primer extension labeling can detect low-abundance heteronuclear RNAs in individual neurons. Since this procedure does not involve recombinant DNA technology or microbiological facilities, it should prove useful to a wide variety of investigators studying the regulation of gene expression at the cellular level.
Assuntos
Sondas de DNA , DNA de Cadeia Simples , Hibridização In Situ/métodos , RNA Nuclear Heterogêneo/análise , RNA Mensageiro/análise , Animais , Sequência de Bases , DNA Polimerase Dirigida por DNA , Amplificação de Genes , Humanos , Dados de Sequência Molecular , RNA Nuclear Heterogêneo/genética , RNA Mensageiro/genética , Taq PolimeraseRESUMO
Our previous study provided a new 2-dimensional echocardiographic technique to detect peripheral right coronary artery aneurysms in Kawasaki disease, with use of the subcostal approach. An additional study was performed to detect peripheral left coronary artery aneurysms. Because the left anterior descending artery runs along the anterior interventricular sulcus and the left circumflex artery along the mitral valve ring, these regions were searched for coronary aneurysms by use of the subcostal imaging approach. Among 143 patients with Kawasaki disease, 44 left coronary aneurysms were visualized in 22 patients. Three aneurysms at the origin of the obtuse marginal artery and 1 in the further peripheral site of the left circumflex artery were observed in 3 patients. Two aneurysms at the origin of the second diagonal branch of the peripheral left anterior descending artery were detected. These echocardiographic studies were done prospectively, and their features coincided well in size, shape, and anatomic location with confirmatory angiographic appearances.
Assuntos
Aneurisma/etiologia , Doença das Coronárias/etiologia , Ecocardiografia/métodos , Doenças Linfáticas/complicações , Síndrome de Linfonodos Mucocutâneos/complicações , Aneurisma/patologia , Criança , Pré-Escolar , Angiografia Coronária , Doença das Coronárias/patologia , Vasos Coronários/patologia , Feminino , Humanos , Lactente , MasculinoRESUMO
In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB-injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL-injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL-injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported.
Assuntos
Relógios Biológicos/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/biossíntese , Neurônios/efeitos dos fármacos , Pentobarbital/farmacologia , Proestro/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Relógios Biológicos/fisiologia , Diestro/efeitos dos fármacos , Diestro/fisiologia , Eletrodos Implantados , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Infusões Intravenosas , Hormônio Luteinizante/sangue , Hormônio Luteinizante/metabolismo , Naloxona/administração & dosagem , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Periodicidade , Proestro/fisiologia , Ratos , Ratos WistarRESUMO
Whether nicotine inhibits the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator to suppress pulsatile LH secretion, and whether this suppression of LH secretion by nicotine is mediated by opioid neurons, were studied in ovariectomized rats by examining changes in LH secretion and the multiunit activity (MUA) of the medial basal hypothalamus. Intravenous (i.v.) injection of nicotine (nicotine bitartrate, 100 micrograms) significantly increased the interval between characteristic increases (volleys) in MUA and LH pulses. This inhibitory effect of nicotine on the GnRH pulse generator activity was not blocked by the prior injection of an opiate receptor antagonist naloxone (naloxone hydrochlolide, 2 mg/kg bw), which was effective in significantly decreasing the interval between MUA volleys. The results suggest that nicotine alters the activity of the GnRH pulse generator, and that cholinergic neurons appear to be directly involved in suppressing pulsatile secretion of LH.
Assuntos
Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Luteinizante/metabolismo , Nicotina/farmacologia , Animais , Feminino , Hipotálamo Médio/fisiologia , Injeções Intravenosas , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Nicotina/administração & dosagem , Ovariectomia , Periodicidade , Ratos , Ratos WistarRESUMO
Many vasoactive intestinal polypeptide (VIP) neurons are distributed in the suprachiasmatic nucleus (SCN) and most of them arborize throughout the nuclei. We determined temporal patterns of VIP receptor (VPAC2 receptor) mRNA in the rat SCN in constant darkness and light-dark cycles, by means of in situ hybridization histochemistry. Amounts of VIP precursor mRNA in the SCN were also quantified in adjacent sections to compare its temporal profile with that of VPAC2 receptor mRNA. In constant darkness no circadian rhythms in VIP precursor mRNA or VPAC2 receptor mRNA in the SCN were detected. In light-dark cycles VIP precursor mRNA and VPAC2 receptor mRNA in the SCN showed robust but different daily rhythms. VPAC2 receptor mRNA decreased over the course of the light period and then gradually recovered during the dark period. VIP precursor mRNA was also reduced in response to light but the reduction was transient. This difference in the nature of light responsiveness between VIP mRNA and VPAC2 receptor mRNA suggests that pre- and postsynaptic events of the VIP transduction system represent different stages of light information processing in the SCN.
Assuntos
Precursores de Proteínas/genética , RNA Mensageiro/análise , Receptores de Peptídeo Intestinal Vasoativo/genética , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/química , Peptídeo Intestinal Vasoativo/genética , Análise de Variância , Animais , Northern Blotting , Histocitoquímica , Hibridização In Situ , Masculino , Estimulação Luminosa , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
To identify cellular sites of prolactin receptor messenger RNA synthesis in the rat brain, we used a combined reverse transcriptase-polymerase chain reaction protocol to generate single stranded DNA probes for in situ hybridization. The results of these experiments identify the epithelial cells of the choroid plexus as a major site of prolactin receptor gene expression in the rat central nervous system.
Assuntos
Plexo Corióideo/metabolismo , RNA Mensageiro/biossíntese , Receptores da Prolactina/genética , Animais , Plexo Corióideo/citologia , Epitélio/metabolismo , Feminino , Ovário/fisiologia , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , RatosRESUMO
Using in situ hybridization with multiple probes to the rat preproenkephalin gene, we have identified a novel population of cells in the reticular thalamic nucleus and basal forebrain which express RNA derived from the preproenkephalin gene. These cells contain nuclear RNA from downstream of an alternate transcription start site in intron A of the preproenkephalin gene (Kilpatrick et al., Mol. Cell Biol., 10 (1990) 3717-3726), while in the same cells preproenkephalin exon 2 RNA is undetectable. The results suggest that in this population of cells, preproenkephalin gene transcription initiates from the intron A initiation site, and is regulated by an additional mechanism which results in the accumulation of nuclear preproenkephalin intron A-derived heteronuclear RNA. The anatomical distribution of these cells indicates that they may be involved in the control of cerebral cortical function.
Assuntos
Encefalinas/biossíntese , Expressão Gênica , Íntrons , Prosencéfalo/metabolismo , Precursores de Proteínas/biossíntese , RNA Nuclear Heterogêneo/biossíntese , Núcleos Talâmicos/metabolismo , Animais , Sequência de Bases , Southern Blotting , DNA/genética , DNA/isolamento & purificação , Sondas de DNA , Encefalinas/genética , Éxons , Hibridização In Situ , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase/métodos , Precursores de Proteínas/genética , Ratos , Transcrição GênicaRESUMO
Gonadotropin releasing hormone (GnRH) is crucial in regulating the reproductive system of female vertebrates. In the present study we have analyzed the estrogen regulation of the GnRH receptor mRNA at the cellular level in Sprague-Dawley female rats. Northern blot analysis detected 3 species (5.0, 4.5 and 1.4 kb) of GnRH receptor mRNA in pituitary tissues. The GnRH receptor mRNA levels of these 3 species were increased by estrogen. By in situ hybridization we observed a 3.5-fold increase in GnRH receptor mRNA levels after 48 h of estrogen treatment when compared to ovariectomized (OVX) rats, 12 h of estrogen treatment did not change the GnRH mRNA levels. Similar increases in GnRH receptor mRNA levels by estrogen were also found in Wistar-Imamichi female rat pituitary tissue. In situ hybridization analysis identified clusters of anterior pituitary cells that expressed the GnRH receptor mRNA. The estradiol effect depends on increased mRNA levels in these clusters. Moreover, a significant increase in the number of pituitary cells that expressed GnRH receptor was observed after 48 h of estrogen treatment. These findings suggest that the mechanisms for estrogen regulation of GnRH receptor include changing levels of GnRH receptor mRNA in the rat pituitary.
Assuntos
Estrogênios/farmacologia , Adeno-Hipófise/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores LHRH/genética , Animais , Feminino , Hibridização In Situ , Ovário/fisiologia , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
To gain a better understanding of the relationship between the female rat reproductive system and preproenkephalin (PPE) expressing neurons under physiological conditions, we examined changes in PPE mRNA levels in the mediobasal hypothalamus during the rat estrous cycle by means of northern blotting and in situ hybridization histochemistry (ISHH). In the Northern blot studies, we found that PPE mRNA levels in the mediobasal hypothalamus were significantly increased by noon of proestrus compared to those in the morning and stayed high until diestrus day 1, and returned toward low levels on diestrous day 2. In contrast, measured as controls, glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were significantly higher on proestrus regardless of time of day compared to diestrus day 2, and levels of calcineurin mRNA on proestrous and estrous were significantly lower than diestrous day 1 and day 2. ISHH studies revealed that these changes in PPE mRNA levels were specific in the ventromedial hypothalamic nucleus pars ventrolateralis (VMHVL), since we could not see any significant changes in signal in other parts including ventromedial hypothalamic nucleus pars dorsomedialis and arcuate hypothalamic nucleus. In the VMHVL, PPE mRNA levels in the afternoon of proestrous were significantly higher than those in the afternoon of diestrous day 2 whereas no significant change in PPE mRNA was observed in the caudate-putamen. The present study provides additional information relevant to possible implications of PPE gene expression in female reproductive systems, since changes in PPE mRNA levels may be associated with estrogen as well as progesterone or other hormonal concentrations during the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Núcleo Caudado/metabolismo , Encefalinas/biossíntese , Estro/fisiologia , Hipotálamo/metabolismo , Precursores de Proteínas/biossíntese , Animais , Autorradiografia , Encefalinas/metabolismo , Feminino , Hibridização In Situ , Precursores de Proteínas/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Because Fos is thought to be induced in neurons that are activated, we examined whether luteinizing hormone-releasing hormone (LHRH) neurons expressed Fos protein when they were stimulated by an opioid receptor antagonist naloxone (NAL), expecting to identify LHRH neurons which are regulated by opioid neurons directly or indirectly. Further, we examined whether an ovulation-blocking dosage of pentobarbital sodium (PB) would affect the NAL-induced Fos expression. Female rats were infused with naloxone (5 mg/kg/h) for 90 min (10.00-11.30) in the morning of proestrus, during which infusion blood sampling was done, and were killed by i.v. injection with an overdose of PB at 11.30-12.00. Dual immunoperoxidase/immunofluorescence staining for both Fos and LHRH revealed that some LHRH immunoreactive (ir) neurons in the forebrain expressed Fos-ir, associated with an increase in serum LH concentrations, but little co-localization was found in rats in proestrus which were infused with saline as the control. The proportion of LHRH-ir neurons which expressed Fos-ir was about 35-62% in the caudal part of the forebrain including the mediobasal hypothalamus, and this was larger than that (10%) in the rostral part of the forebrain including the preoptic area. PB injection (32 mg/kg bw, i.p.) 15 min prior to the beginning of NAL infusion significantly enhanced the increase in LH secretion due to NAL, and also enhanced Fos-ir expression in LHRH-ir neurons. Together with the well-established fact that PB blocks the LHRH surge generator and our previous findings that NAL stimulates the LHRH pulse generator even in the PB-blocked proestrous rat, these results strongly suggest that the LHRH pulse generator exists in the mediobasal hypothalamus which contains LHRH neurons that are responsive to NAL and express Fos protein.
Assuntos
Genes fos , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo Médio/metabolismo , Naloxona/farmacologia , Pentobarbital/farmacologia , Proestro/fisiologia , Animais , Feminino , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hipotálamo Médio/citologia , Hipotálamo Médio/efeitos dos fármacos , Técnicas Imunoenzimáticas , Hormônio Luteinizante/metabolismo , Antagonistas de Entorpecentes/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
Alkylphenols, such as p-nonylphenol (NP) and 4-tert-octylphenol (OP) and bisphenol A (BPA) are thought to mimic oestrogens in their action, and are called endocrine disrupters. We examined whether these endocrine disrupters affected progesterone receptor (PR) mRNA expression in the adult female rat neocortex. In one experiment, at 12.00 h, ovariectomized rats were given a subcutaneous injection of 10 mg of NP, 10 mg of OP or 10 mg of BPA, or sesame oil alone as control. Twenty-four hours after injection, the left side of the frontal cortex, parietal cortex and temporal cortex was collected. In a second experiment to study the time-course of the effects of BPA on PR mRNA, the ovariectomized rats were given a subcutaneous injection of 10 mg of BPA and killed 0, 6, 12 and 24 h after injection. In addition to the frontal cortex and temporal cortex, the occipital cortex was also collected. Northern blotting revealed that, in the first experiment, injection of NP, OP or BPA significantly increased PR mRNA expression in the frontal cortex but not in the parietal cortex. In the temporal cortex, BPA significantly decreased PR mRNA, but NP and OP produced no significant changes. The second experiment revealed that, in the frontal cortex, BPA induced a significant increase in PR mRNA expression at 6 h after injection, which lasted until 24 h after injection. In the temporal cortex, PR mRNA expression was significantly decreased 6 h after injection of BPA and was still significantly low 24 h after injection. No significant change was observed in the occipital cortex. These results suggest that, even in adult rats, endocrine disrupters alter the neocortical function by affecting the PR system, although the physiological significance of PR in the affected area is unknown.
Assuntos
Córtex Cerebral/metabolismo , Poluentes Ambientais/toxicidade , Estrogênios não Esteroides/toxicidade , Fenóis/toxicidade , Receptores de Progesterona/metabolismo , Animais , Compostos Benzidrílicos , Northern Blotting , Estrogênios/agonistas , Feminino , Lobo Frontal/metabolismo , Ovariectomia , Lobo Parietal/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de Progesterona/genética , Lobo Temporal/metabolismo , Fatores de Tempo , Distribuição TecidualRESUMO
Butyl benzyl phthalate (BBP) and bisphenol A (BPA), termed endocrine disrupters, are known to mimic oestrogen in their actions, and therefore there is concern about their effect on reproductive functions. Since it is reported that the inhibitory action of oestrogen on the pulsatile secretion of luteinizing hormone (LH) is enhanced under insulin-induced hypoglycaemia, whether this also applies to BBP and BPA was examined in the present study. In adult ovariectomized (OVX) rats, the pulsatile LH secretion 24 h after subcutaneous injection of 10 mg BBP (BBP-treated), 10 mg BPA (BPA-treated) or 100 ng 17beta-oestradiol (E2-treated), all of which were dissolved in sesame oil, was not changed significantly compared to that after the injection of sesame oil only. Furthermore, in oil-treated OVX rats, the pulsatile LH secretion immediately after intravenous injection of insulin (1.0 U) was not changed compared to that after saline injection. In BBP-treated OVX rats, the injection of insulin (1.0 U) significantly decreased the number of LH pulses as in E2-treated OVX rats. The injection of insulin did not significantly affect the amplitude of LH pulses in BBP-, BPA- and E2-treated OVX rats. The results indicate that the oestrogenic action of BBP is significantly enhanced by insulin-induced hypoglycaemia and thus the pulsatile LH secretion is inhibited. We suggest that weak oestrogenic endocrine disrupters may become harmful to reproductive functions even in adult female rats, if acting under a low energy state.