RESUMO
Behavioral adaptation in aging may become impaired from abnormal expression of amygdalar corticotropin-releasing hormone (CRH) and/or CRH-binding protein (CRH-BP). In this study, we serially sectioned the amygdala in 4-, 12-, and 24-month-old Fischer 344 rats following perfusion with 4% paraformaldehyde. We determined the amount of CRH and CRH-BP containing cells as well as the density of fibers expressing CRH or CRH-BP utilizing densitometric methods. Images were digitized using Zeiss Axiovision software and densitometrically analyzed using Scion Image. Both sides were analyzed in sections cut at 30 mum thickness. Cell counts of CRH-BP containing cells in the basolateral and lateral nucleus of the amygdala were lower in 24-month-old rats vs. 4-month-old rats, respectively (mean cells/section +/- SE): 31 +/- 6 vs. 72 +/- 10 (n = 3; P < 0.05 via ANOVA and Fisher's PLSD). There was a trend for cell counts of CRH containing cells in the central nucleus of the amygdala to be lower in 24-month-old rats vs. 4-month-old rats, respectively 28 +/- 7 vs. 47 +/- 9 (n = 3; P = 0.07 via ANOVA). Densitometric analysis of the number of CRH-BP positive fibers revealed no age differences in CeA; however, with regards to CRH-positive fibers, both 4- and 12-month rats had greater CeA CRH immunoreactivity relative to 24-month-old rats (Ps < 0.05 via ANOVA and Fisher's PLSD). These changes may contribute to impaired adaptations to stress, cognitive decline, and other pathophysiological processes during aging.
Assuntos
Envelhecimento/fisiologia , Tonsila do Cerebelo/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Fatores Etários , Análise de Variância , Animais , Contagem de Células , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica/métodos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Clinical investigation of melatonin agonists has been hampered by side effects such as hypothermia, hypotension and bradycardia. The availability of a melatonin agonist devoid of these side effects would improve our understanding of the mechanisms by which melatonin agonists affect sleep. This study investigated the pharmacokinetics, pharmacodynamics and safety of the melatonin agonist beta-methyl-6-chloromelatonin at doses up to 100 mg in healthy volunteers. The design was a single blind, across subjects, placebo controlled, group wise dose escalation using doses of 20, 35, 50 and 100 mg beta-methyl-6-chloromelatonin. Eight subjects received one dose of study drug or placebo. Pharmacokinetic analysis showed a consistent Tmax across all doses with a mean of 1.12 +/- 0.11 hr for all groups (mean +/- SD). The half-life was also consistent across dose, with a mean of 1.04 +/- 0.04 hr. Maximum plasma concentrations increased with increasing dose with values of 44.83 +/- 29.79, 100.3 +/- 41.08, 79.84 +/- 26.36 and 410.3 +/- 129.4 ng/ml at doses of 20, 35, 50 and 100 mg, respectively. Area under the curve showed similar increases. No consistent changes in vital signs occurred as a function of dose or time after study drug. The incidence of all adverse events, the severity of the event or the event's relationship to treatment did not increase with higher doses of beta-methyl-6-chloromelatonin. Sleepiness was reported after all doses of beta-methyl-6-chloromelatonin. beta-methyl-6-chloromelatonin appears safe and well tolerated at doses up to 100 mg. These doses are not associated with hypothermia, bradycardia or hypotension. A melatonin agonist lacking these side effects should allow investigation of the direct soporific effects of melatonin agonists.
Assuntos
Melatonina/agonistas , Melatonina/farmacologia , Adolescente , Adulto , Área Sob a Curva , Pressão Sanguínea/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Melatonina/efeitos adversos , Melatonina/análogos & derivados , Melatonina/farmacocinética , Pessoa de Meia-Idade , Método Simples-Cego , Sono/efeitos dos fármacosRESUMO
Treating older patients with neurodegenerative disorders involves numerous challenges. The older patient population is expected to increase appreciably in the coming years; thus, there will be increasing numbers of these individuals requiring treatment. As a result, the appropriate choice of psychopharmacologic agents becomes an important decision in treating older patients with atypical antipsychotics. The atypical antipsychotic medications are replacing the high-potency conventional antipsychotics in the long-term care setting because of the lower risks of side effects. For instance, atypical antipsychotics have lower rates of extrapyramidal side effects and tardive dyskinesia. Double-blind placebo-controlled trials examining the use of risperidone and olanzapine have been published and indicate that both agents safely and effectively reduce agitation symptoms in long-term care patients with neurodegenerative disorders. For instance, based on these studies, the doses that appear efficacious in treating behavioral agitation in dementia are 0.5 to 1.5 mg per day of risperidone and 5 to 10 mg per day of olanzapine. In addition, there are open-label studies examining the use of quetiapine, which suggest that this agent is also safe and efficacious in patients with dementia. Doses used range approximately from 25 to 350 mg per day. Very few studies are available examining the newest atypical antipsychotics, ziprasidone and aripiprazole, in patients with neurodegenerative disorders. These studies do suggest that ziprasidone and aripiprazole are worth further study in the long-term care setting.
Assuntos
Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Idoso , Antipsicóticos/efeitos adversos , Aripiprazol , Benzodiazepinas/farmacologia , Benzodiazepinas/uso terapêutico , Dibenzotiazepinas/farmacologia , Dibenzotiazepinas/uso terapêutico , Humanos , Assistência de Longa Duração , Olanzapina , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Qualidade de Vida , Fumarato de Quetiapina , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Risperidona/farmacologia , Risperidona/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Estados UnidosRESUMO
Previous studies of neuronal degeneration induced by the neurotoxin, kainic acid, employed silver stain techniques that are non-quantitative or ELISA measurement of the non-neuronal protein, glial fibrillary acidic protein. As previous studies employed biomarkers that were either non-quantitative or non-neuronal, the present study employed a new neuronally localized biomaker of neuronal damage, cleaved microtubule-associated protein (MAP)-tau (C-tau). The time course of kainate neurotoxicity was quantitatively determined in several brain regions in the present study employing a C-tau specific ELISA. Differences in ELISA determined regional brain levels of C-tau were compared with the density of somatodendritic C-tau labeling qualitatively determined in immunohistochemical anatomical mapping studies of kainic acid-treated animals. Immunoblot studies revealed that the C-tau antibodies employed in the present study were highly specific for proteolytic cleaved C-tau. Immunolabeling of 45 kD-50 kD C-tau proteins was observed only in brain samples from kainic acid-treated but not vehicle-treated rats. Time course studies revealed that C-tau levels determined by ELISA were maximal 3 days after kainic acid with C-tau levels increasing 26-fold in hippocampus, 16-fold in cortex and four-fold in both striatum and hypothalamus. These statistical differences in maximal C-tau levels observed in the ELISA studies were similar to differences qualitatively observed in C-tau immunohistochemical studies. C-tau immunohistochemistry revealed extensive damage in hippocampal regions CA1 and 3, moderate damage in several cortical regions and mild damage in striatum and hypothalamus. Similar cleavage of rat MAP-tau to C-tau has been reported after neuronal degeneration induced by neurotoxic doses of methamphetamine and neuronal degeneration resulting from bacterial meningitis. In humans, C-tau proteolysis has been demonstrated to be a reliable biomarker of neuronal damage in traumatic brain injury and stroke where cerebrospinal C-tau levels are correlated with patient clinical outcome. These data suggest that C-tau proteolysis may prove a reliable species independent biomarker of neuronal degeneration regardless of source of injury.
Assuntos
Morte Celular/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Síndromes Neurotóxicas/metabolismo , Proteínas tau/metabolismo , Animais , Biomarcadores/análise , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Química Encefálica , Líquido Cefalorraquidiano/metabolismo , Maleato de Dizocilpina/farmacologia , Ensaio de Imunoadsorção Enzimática , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Proteínas dos Microtúbulos/metabolismo , Neuregulina-1/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Coloração pela Prata , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Fatores de Tempo , Proteínas tau/químicaRESUMO
Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.
Assuntos
Tonsila do Cerebelo , Hormônio Liberador da Corticotropina/genética , Regulação da Expressão Gênica , Hipotálamo , Transdução de Sinais/fisiologia , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/enzimologia , Tonsila do Cerebelo/metabolismo , Células Cultivadas , Hormônio Liberador da Corticotropina/biossíntese , Hipotálamo/citologia , Hipotálamo/enzimologia , Hipotálamo/metabolismo , Transdução de Sinais/genéticaRESUMO
Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.
Assuntos
Hormônio Liberador da Corticotropina/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Hipotálamo/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Transdução de Sinais , Animais , Antígenos Transformantes de Poliomavirus/genética , Western Blotting , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Hormônio Liberador da Corticotropina/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dexametasona/farmacologia , Expressão Gênica/efeitos dos fármacos , Hipotálamo/química , Fosforilação , Pró-Opiomelanocortina/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/genética , Transfecção , Vasopressinas/genéticaRESUMO
Quantifying the functional consequences of illness in terms of quality of life can enhance our understanding of both mental and physical disorders. However, little is known about the quality of life among older inpatients vs. outpatients with schizophrenia. We present the results of health-related quality of life assessments in 54 middle-aged and elderly long-term inpatients with schizophrenia and a demographically matched outpatient sample. Assessments were performed using the Quality of Well-Being (QWB) scale, along with standard measures of psychopathology and global cognitive impairment. Compared with outpatients, the inpatients had a significantly lower health-related quality of life, as measured by the QWB. In the inpatient and outpatient groups, higher levels of positive symptoms were associated with lower health-related quality of life. Health-related quality of life remained fairly stable among the inpatients who remained hospitalized over 6 months. In both inpatients and outpatients, baseline cognitive status and psychopathology predicted QWB scores at the 6-month follow-up. These findings further support the use of the QWB in severely mentally ill populations; implications for improving health-related quality of life among older patients with schizophrenia are discussed.
Assuntos
Nível de Saúde , Satisfação Pessoal , Qualidade de Vida , Esquizofrenia/reabilitação , Adulto , Doença Crônica , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Feminino , Seguimentos , Hospitalização , Hospitais Psiquiátricos , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Esquizofrenia/complicações , Índice de Gravidade de DoençaRESUMO
Corticotropin-releasing hormone (CRH) is a neuropeptide thought to play a role in appetite regulation. In this report, we used a serial cerebrospinal fluid (CSF) sampling technique to examine the relationship between CSF CRH, plasma ACTH and cortisol and perceptions of hunger and satiety in fasting and sated volunteers. CSF was withdrawn continuously from 11:00 AM to 5:00 PM via an indwelling subarachnoid catheter. Blood was withdrawn every 10 min via an antecubital vein catheter. Fed subjects received a meal at 1:00 PM. Subjects who were fed had lower post-prandial ratings on hunger scales and higher ratings on satiety scales. Fed subjects also had slightly lower levels of CSF CRH after feeding. Furthermore, fed subjects had higher ACTH and cortisol concentrations in the first 3 h; by the fourth h the opposite was true. Our findings do not support the hypothesis that CNS CRH is a central satiety factor in the human. Instead our findings of slightly diminished CSF CRH levels after feeding may be accounted for by the rises in glucocorticoids and their associated negative feedback effects on CNS CRH. Alternatively, our findings could also reflect changes in CRH levels associated with feeding in multiple brain areas and in the spinal cord with the net effect being in the negative direction.
Assuntos
Hormônio Liberador da Corticotropina/líquido cefalorraquidiano , Comportamento Alimentar/fisiologia , Hormônio Adrenocorticotrópico/sangue , Adulto , Análise de Variância , Jejum/sangue , Jejum/líquido cefalorraquidiano , Feminino , Humanos , Hidrocortisona/sangue , Masculino , Período Pós-Prandial/fisiologia , Resposta de Saciedade/fisiologiaRESUMO
Studies examining regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Most in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Furthermore, clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. CRH levels in the amygdala in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated the signal transduction mechanisms responsible for CRH expression. These mechanistic studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Clearly much more investigative work in better understanding CRH regulation in vitro is needed.
Assuntos
Aminas Biogênicas/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Citocinas/metabolismo , Glucocorticoides/metabolismo , Transdução de Sinais/fisiologia , Animais , Monóxido de Carbono/metabolismo , Linhagem Celular , Hormônio Liberador da Corticotropina/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismoRESUMO
BACKGROUND: Little is known about the relationship between endogenous central nervous system (CNS) testosterone and any psychiatric syndrome. The goal of this study was to screen for potential abnormalities in CNS testosterone levels in patients with post-traumatic stress disorder (PTSD) and/or tobacco dependence. METHODS: We sampled cerebrospinal fluid (CSF) via a subarachnoid catheter over six hours and determined hourly basal CSF concentrations of testosterone in 11 combat veterans with PTSD and 12 normal volunteers. Smokers were abstinent for 11-17 h. Testosterone in CSF and matching plasma samples was assayed by radioimmunoassay. RESULTS: A factor analysis for effects of PTSD status, smoking status and sample time revealed significant effects of PTSD or smoking status, but not time, on CSF testosterone. CSF testosterone levels were lower in individuals with PTSD as compared with normal volunteers. When divided by smoking status, abstinent smokers had mean CSF testosterone levels higher than those of non-smokers. A similar analysis of plasma testosterone revealed no significant effects of any factor on plasma testosterone. CONCLUSIONS: These results indicate that CSF testosterone is significantly influenced by PTSD and smoking status. The exposure of the brain to altered levels of testosterone in smokers and patients with PTSD may have pathophysiologic significance in these conditions.
Assuntos
Transtornos de Estresse Pós-Traumáticos/sangue , Transtornos de Estresse Pós-Traumáticos/líquido cefalorraquidiano , Testosterona/sangue , Testosterona/líquido cefalorraquidiano , Tabagismo/sangue , Tabagismo/líquido cefalorraquidiano , Adulto , Feminino , Humanos , Masculino , Escalas de Graduação Psiquiátrica , Fumar/sangue , Fumar/líquido cefalorraquidiano , Fumar/psicologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Tabagismo/psicologiaRESUMO
Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.
Assuntos
Tonsila do Cerebelo/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Neuropeptídeo Y/metabolismo , Transdução de Sinais , Tonsila do Cerebelo/citologia , Animais , Sequência de Bases , Linhagem Celular Transformada , AMP Cíclico/metabolismo , Primers do DNA , Ligação Proteica , Receptores de Neuropeptídeo YRESUMO
Corticotropin-releasing factor (CRF) coordinates the mammalian response to stress. In the amygdala, the CRF system appears to be responsible, at least in part, for the behavioral responses resulting from stress. Associated with amygdalar CRF is a 37 kDa binding protein (CRF-BP) which may also play a role in regulating stressful stimuli. Aging has been shown to be associated with abnormal neuroendocrine stress systems and little is known with regards to how amygdalar stress systems change with aging. In our study, we have assessed levels of amygdalar CRF and CRF-BP mRNA in Fischer 344 rats of 4, 12 or 24 months of age following 14 days of hourly restraint. Prior to sacrifice, rats were also tested for anxiety-like behaviors on the elevated plus maze. After behavioral testing, rats were perfused with 4% paraformaldehyde and the brains were processed for in situ hybridization. Twenty micron sections were hybridized with a CRF as well as a CRF-BP riboprobe. Following hybridization, tissue sections were oppossed to X-ray film and relative amounts of mRNA in the amygdala were quantitated. Levels of CRF mRNA in the amygdala of 12 and 24 month-old rats following chronic restraint were significantly lower relative to rats which were handled for 14 days. There were no significant differences in amygdalar CRF gene expression between stressed and handled 4 month-old rats. At 12 and 24 months of age but not 4 months, there were also significant effects of restraint associated with decreases in amygdalar CRF-BP gene expression. Furthermore, there were reciprocal decreases in anxiety-like behaviors in the 12 and 24 month-old rats which were significant; the changes in anxiety-like behaviors between restrained vs. handled 4 month-old rats were not significantly different. The decreased gene expression of CRF in the amygdala in concert with decreased anxiety-like behaviors following restraint is consistent with the known behavioral effects of exogenously applied intra-amygdalar CRF. The changes in amygdalar CRF-BP observed may be secondary to the known regulatory effects that CRF exhibits on its binding-protein. These studies have relevance to better understanding the molecular basis of aging related changes in neuroendocrine stress systems.
Assuntos
Envelhecimento/metabolismo , Tonsila do Cerebelo/metabolismo , Comportamento Animal/fisiologia , Hormônio Liberador da Corticotropina/genética , Emoções/fisiologia , Receptores de Hormônio Liberador da Corticotropina/genética , Tonsila do Cerebelo/citologia , Animais , Peso Corporal/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Restrição Física/efeitos adversos , Estresse Psicológico/fisiopatologiaRESUMO
Corticotropin-releasing factor-binding protein (CRF-BP) is known to regulate the bioavailability of CRF and may also play a role in stress behaviours. CRF-BP has been localized in the pituitary as well as central nervous system (CNS) limbic and cortical areas, including the amygdala. The signal transduction pathways which regulate amygdalar CRF-BP are not well understood. In this report, we have examined the effect of protein kinase A and C activators, CRF, dexamethasone and interleukin-6 (IL6) on CRF-BP mRNA and protein expression in dissociated fetal amygdalar cultures. CRF-BP mRNA levels were determined by Northern analysis following 12 h treatment with the following agents: forskolin (1-30 microM), CRF (1-1000 nM), phorbol-12-myristate-13-acetate (TPA; 1-50 nM), dexamethasone (1-100 nM) and IL6 (10-500 pM). Significant increases in CRF-BP mRNA were observed in response to forskolin (30 mM), CRF (100, 1000 nM), IL6 (100, 500 pM), TPA (50 nM) and dexamethasone (100 nM; P<0.05 for all; n=3-6 for all). We extended our observations of CRF-BP expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with the same agents. Twenty-four hour treatment with 30 microM forskolin, 1000 nM CRF, 50 nM TPA, 100 pM IL6 or 100 nM dexamethasone significantly increased CRF-BP expression (P<0.05, n=3 for each treatment). The primary cultures were then transfected with a rat CRF-BP-reporter construct containing 3500 base pairs of CRF-BP 5' flanking DNA. Treatment with all five agents produced statistically significant increases above control (P<0.05; n=3 for each). The results suggest that CRF-BP in the amygdala is stimulated by numerous pathways which may play a significant role in promoting behavioural changes.
Assuntos
Tonsila do Cerebelo/citologia , Proteínas de Transporte/genética , Neurônios/fisiologia , Regiões 5' não Traduzidas/genética , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dexametasona/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glucocorticoides/farmacologia , Interleucina-6/fisiologia , Neurônios/química , Neurônios/citologia , Gravidez , Proteína Quinase C/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiopatologiaRESUMO
The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis. a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium potassium dependent adenosine triphosphatase (Na+ K+ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome.
Assuntos
Inibidores Enzimáticos/metabolismo , Ouabaína/metabolismo , Peptídeos/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Glicosídeos Digitálicos/isolamento & purificação , Dados de Sequência Molecular , Neurofisinas/metabolismo , Neuro-Hipófise/metabolismo , Neuro-Hipófise/patologia , Precursores de Proteínas/metabolismo , Coloração e Rotulagem/métodos , Suínos , Vasopressinas/metabolismoRESUMO
Adaptation in aging may become impaired from abnormal expression of corticotropin-releasing factor (CRF) and altered CRF receptor function. In this study, we measured CRF mRNA levels in Fischer 344 rats at various ages. The brains of these rats were processed for in situ hybridization. Relative to 3-month-old rats, levels of CRF mRNA were significantly decreased in the following brain areas at the following ages: at 24 months in the paraventricular hypothalamus, at 11, 17, and 24 months in the amygdala and at 17 and 24 months in the bed nucleus of the stria terminalis. These changes may contribute to impaired adaptations to stress, cognitive decline and other pathophysiological processes during aging.
Assuntos
Envelhecimento/fisiologia , Química Encefálica/fisiologia , Hormônio Liberador da Corticotropina/genética , Tonsila do Cerebelo/química , Tonsila do Cerebelo/fisiologia , Animais , Expressão Gênica/fisiologia , Masculino , Núcleo Hipotalâmico Paraventricular/química , Núcleo Hipotalâmico Paraventricular/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Estresse Fisiológico/fisiopatologiaRESUMO
Levels of hypophysial portal arginine-vasopressin (AVP), plasma ACTH and plasma corticosterone (B) were measured in four and 24 month old Fischer 344 rats. Portal levels of AVP were lower in older rats whereas plasma ACTH levels were elevated. Older rats given adrenalectomies with physiologic replacement doses of B exhibited levels of AVP, but not ACTH, which were comparable to that observable in younger rats. The findings suggest that altered AVP, but not ACTH, can be accounted for by higher B levels which are commonly demonstrable in aging rats.
Assuntos
Hormônio Adrenocorticotrópico/sangue , Envelhecimento/fisiologia , Arginina Vasopressina/metabolismo , Química Encefálica/fisiologia , Corticosterona/sangue , Animais , Hormônio Liberador da Corticotropina/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Estresse Fisiológico/metabolismoRESUMO
CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.
Assuntos
Tonsila do Cerebelo/metabolismo , Proteínas de Transporte/biossíntese , Hormônio Liberador da Corticotropina/biossíntese , Regulação da Expressão Gênica , Neurônios/metabolismo , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/embriologia , Animais , Proteínas de Transporte/genética , Linhagem Celular Transformada , Colforsina/farmacologia , Hormônio Liberador da Corticotropina/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Interleucina-6/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The BE(2)-M17 and BE(2)-C human neuroblastoma cell lines have been shown to synthesize and secrete corticotropin-releasing factor (CRF) following retinoic acid treatment. It has been demonstrated that CRF secretion and intracellular synthesis increases in response to forskolin treatment. In this report, we have further characterized these cells in response to protein kinase C activators, dexamethasone, interleukin-1 alpha, as well as various neurotransmitters and peptides. Nanomolar concentrations of the phorbol ester--phorbol 12 myristate 13--acetate (TPA), increased intracellular CRF content in both cell lines while increasing secretion only in the BE(2)-M17 cell. Nanomolar concentrations of dexamethasone were not able to alter basal levels of secretion and content in either cell type. However, in the BE(2)-M17 cell but not the BE(2)-C cell, the same concentrations of dexamethasone added to 30 microM forskolin augmented levels of CRF secretion and content. Likewise, the same augmented response in CRF secretion and content was seen only in the BE(2)-M17 cell line when nanomolar concentrations of dexamethasone were added to 20 nM TPA. Furthermore, only in the BE(2)-M17 cell line were micromolar levels of the biogenic amine serotonin able to increase levels of CRF secretion and content. No effects on CRF in both cell lines were demonstrable with picomolar levels of interleukin-1 alpha as well as micromolar levels of acetylcholine, norepinephrine, arginine-vasopressin, oxytocin, and angiotensin-II. The potential usefulness of these cells as models of central nervous system or placental CRF-containing neurons is discussed.
Assuntos
Hormônio Liberador da Corticotropina/biossíntese , Hormônio Liberador da Corticotropina/metabolismo , Neuroblastoma/metabolismo , Colforsina/farmacologia , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Proteína Quinase C/metabolismo , Serotonina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Corticotropin-releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of "antisense knockdown" strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15-mer antisense (5' CTG-CGG-GCG-CCG-TCC 3') and "scrambled" control (5' CGT-CCG-CGC-GCT-GCG 3') oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 mumol/L of antisense oligonucleotide for 40-67 h resulted in significant (17-36%) decreases in 125I-ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 mumol/L of "scrambled" oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 mumol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30-min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Adeno-Hipófise/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Hormônio Liberador da Corticotropina/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Cinética , Masculino , Dados de Sequência Molecular , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
LH-RH analogs cause some inhibition of growth of pancreatic cancers. Syrian golden hamsters bearing chemically induced pancreatic cancers were treated with [D-Trp6]LH-RH for 3 d before sacrifice. LH-RH receptors were localized by electron-microscopic immunohistochemistry in the tumor cells of both treated and untreated hamsters. [D-Trp6]LH-RH treatment resulted in a marked increase in the concentration of LH-RH receptors in the nuclei. The dissociation constants (Kd) and the maximal binding capacity of the LH-RH receptors (Bmax), measured by radioreceptor assay, were higher in the nuclei of the pancreatic tumor cells of hamsters treated with [D-Trp6]LH-RH than in the untreated controls. Pancreatic cells of tumor-free hamsters did not show immunostaining for LH-RH receptors. A possible correlation between the increase in the concentration of the LH-RH receptors in the nuclei and the tumor growth-inhibiting activity of [D-Trp6]LH-RH is suggested.