RESUMO
The genetic basis of many epilepsies is increasingly understood, giving rise to the possibility of precision treatments tailored to specific genetic etiologies. Despite this, current medical therapy for most epilepsies remains imprecise, aimed primarily at empirical seizure reduction rather than targeting specific disease processes. Intellectual and technological leaps in diagnosis over the past 10 years have not yet translated to routine changes in clinical practice. However, the epilepsy community is poised to make impressive gains in precision therapy, with continued innovation in gene discovery, diagnostic ability, and bioinformatics; increased access to genetic testing and counseling; fuller understanding of natural histories; agility and rigor in preclinical research, including strategic use of emerging model systems; and engagement of an evolving group of stakeholders (including patient advocates, governmental resources, and clinicians and scientists in academia and industry). In each of these areas, we highlight notable examples of recent progress, new or persistent challenges, and future directions. The future of precision medicine for genetic epilepsy looks bright if key opportunities on the horizon can be pursued with strategic and coordinated effort.
Assuntos
Epilepsia , Medicina de Precisão , Epilepsia/diagnóstico , Epilepsia/genética , Epilepsia/terapia , Testes Genéticos , Humanos , Convulsões/genética , SugestãoRESUMO
The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.
Assuntos
Catepsina K/antagonistas & inibidores , Osteoporose/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos Radiofarmacêuticos/farmacocinética , Animais , Osso e Ossos/diagnóstico por imagem , Radioisótopos de Carbono/química , Inibidores de Cisteína Proteinase/química , Avaliação Pré-Clínica de Medicamentos , Feminino , Ligantes , Macaca mulatta , Ligação Proteica , Coelhos , Compostos Radiofarmacêuticos/efeitos adversos , Compostos Radiofarmacêuticos/química , Distribuição TecidualRESUMO
During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.
Assuntos
Catepsina K/antagonistas & inibidores , Catepsina K/metabolismo , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Animais , Osso e Ossos/enzimologia , Avaliação Pré-Clínica de Medicamentos , Cinética , CoelhosRESUMO
Cannabinoid receptors (CBRs) play an important role in a variety of physiological functions and have been considered drug targets for obesity and psychiatric disorders. In particular, the CB1R is highly expressed in brain regions crucial to learning and memory processes, and several lines of evidence indicate that pharmacological blockade of this receptor could have therapeutic applications in the treatment of cognitive disorders. In this study, we investigated whether MK-7128 (0.1, 0.3, and 1 mg/kg, orally), a novel and selective CB1R inverse agonist, could improve learning and memory deficits induced by scopolamine (1 mg/kg, subcutaneously) in mice. The investigators also assessed CB1R occupancy in the brain to ensure target engagement of MK-7128, and showed that MK-7128 significantly improved both Y-maze spontaneous alternation and object habituation performance in scopolamine-treated mice and inhibits the binding of radioiodinated AM251 in murine cortex and hippocampus. These data indicate that MK-7128 improves cognitive performance in a model of cholinergic hypofunction and suggest that efficacy is achieved at relatively low levels of CB1R occupancy in the brain. Our results extend earlier findings suggesting a role of CB1Rs in the modulation of memory processes and a potential therapeutic application for CB1R inverse agonists in cognitive disorders.
Assuntos
Azetidinas/farmacologia , Agonismo Inverso de Drogas , Transtornos da Memória/tratamento farmacológico , Oxidiazóis/farmacologia , Receptor CB1 de Canabinoide/agonistas , Animais , Azetidinas/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Oxidiazóis/administração & dosagem , Piperidinas/metabolismo , Ligação Proteica , Pirazóis/metabolismo , EscopolaminaRESUMO
Effects of estradiol benzoate (EB), ERα-selective agonist, propyl pyrazole triol (PPT) and ERß-selective agonists, diarylpropionitrile (DPN) and Compound 19 (C-19) on memory were investigated in OVX rats using object recognition (OR) and placement (OP) memory tasks. Treatments were acute (behavior 4h later) or sub chronic (daily injections for 2 days with behavior 48 h later). Objects were explored in sample trials (T1), and discrimination between sample (old) and new object/location in recognition trials (T2) was examined after 2-4h inter-trial delays. Subjects treated sub chronically with EB, DPN, and C-19, but not PPT, discriminated between old and new objects and objects in old and new locations, suggesting that, at these doses and duration of treatments, estrogenic interactions with ERß contribute to enhancements in recognition memory. Acute injections of DPN, but not PPT, immediately after T1, also enhanced discrimination for both tasks (C19 was not investigated). Effects of EB, DPN and PPT on anxiety and locomotion, measured on elevated plus maze and open field, did not appear to account for the mnemonic enhancements. Monoamines and metabolites were measured following DPN treatment in subjects that did not receive behavioral testing. DPN was associated with alterations in monoamines in several brain areas: indexed by the metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), or the MHPG/norepinephrine (NE) ratio, NE activity was increased by 60-130% in the prefrontal cortex (PFC) and ventral hippocampus, and NE activity was decreased by 40-80% in the v. diagonal bands and CA1. Levels of the dopamine (DA) metabolite, homovanillic acid (HVA), increased 100% in the PFC and decreased by 50% in the dentate gyrus following DPN treatment. The metabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was increased in the PFC and CA3, by approximately 20%. No monoaminergic changes were noted in striatum or medial septum. Results suggest that ERß mediates sub chronic and acute effects of estrogens on recognition memory and that memory enhancements by DPN may occur, in part, through alterations in monoaminergic containing systems primarily in PFC and hippocampus.
Assuntos
Encéfalo/metabolismo , Estradiol/fisiologia , Receptor beta de Estrogênio/agonistas , Memória de Curto Prazo/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Animais , Monoaminas Biogênicas/metabolismo , Encéfalo/efeitos dos fármacos , Aprendizagem por Discriminação/efeitos dos fármacos , Estradiol/administração & dosagem , Estrogênios/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória de Curto Prazo/fisiologia , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Nitrilas/farmacologia , Ovariectomia , Fenóis , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Propionatos/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Reconhecimento Psicológico/efeitos dos fármacos , Comportamento Espacial/efeitos dos fármacos , Comportamento Espacial/fisiologia , Estatísticas não ParamétricasRESUMO
Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited (125)I-NPS binding in the CNS when administered to rats.
Assuntos
Receptores de Neuropeptídeos/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/metabolismo , Humanos , Radioisótopos do Iodo/química , Ligação Proteica , Quinolonas/síntese química , Quinolonas/química , Quinolonas/farmacologia , Ratos , Receptores de Neuropeptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Cathepsin K (CatK) inhibitors exhibited chondroprotective and pain-reducing effects in animal models, however, improvements were relatively modest at dose levels achieving maximal suppression of CatK biomarkers in urine. In this report, a previously characterized CatK inhibitor (MK-1256) is utilized to explore the potential of reduced target engagement and/or suboptimal exposure (free drug) as limiting factors to the pharmacological potential of CatK inhibitors in the knee joint. Following oral administration of MK-1256 at a dose level achieving maximal inhibition of urinary biomarker (helical peptide) in dogs, full suppression of the biomarker in synovial fluid was observed. Subsequent tissue distribution studies conducted in dogs and rabbits revealed that MK-1256 levels in synovial fluid and cartilage were consistent with the free-drug hypothesis. Reasonable projection (within twofold) of drug levels in these tissues can be made based on plasma drug concentration with adjustments for binding factors. These results indicate that the previously observed efficacies in the animal models were not limited by compound distribution or target engagement in the knee tissues.
RESUMO
Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.
Assuntos
Estrogênios/metabolismo , Prosencéfalo/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Sítios de Ligação , Estradiol/metabolismo , Feminino , Imuno-Histoquímica , Radioisótopos do Iodo , Microscopia Eletrônica , Modelos Biológicos , Prosencéfalo/ultraestrutura , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.
Assuntos
Sítio Alostérico/genética , Aminoácidos/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Regulação Alostérica/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Linhagem Celular , Células Cultivadas , Humanos , Masculino , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica/genética , Ratos , Ratos Sprague-DawleyRESUMO
The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.
Assuntos
Catepsina K/sangue , Inibidores de Cisteína Proteinase/sangue , Adolescente , Adulto , Idoso , Animais , Biomarcadores/urina , Compostos de Bifenilo/sangue , Compostos de Bifenilo/farmacocinética , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/urina , Proteínas Sanguíneas/metabolismo , Catepsina K/antagonistas & inibidores , Colágeno Tipo I/urina , Inibidores de Cisteína Proteinase/farmacocinética , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/urina , Cães , Feminino , Humanos , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Peptídeos/urina , Ligação Proteica , Pirazóis/sangue , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirazóis/urina , Coelhos , Sulfonas/sangue , Sulfonas/farmacocinética , Sulfonas/farmacologia , Sulfonas/urina , Adulto JovemRESUMO
Estrogen receptor (ER)beta is present in hypothalamic and limbic neurons of female rat brains, but little is known about its regulation under physiological conditions. To determine whether ER beta expression varies during physiological conditions in which sex steroid hormone profiles are significantly different, we used in situ hybridization to assess ER beta mRNA expression in the periventricular preoptic area, bed nucleus of stria terminalis, paraventricular nucleus, supraoptic nucleus, and the posterodorsal medial amygdala of female rats on proestrus, on d 22 of pregnancy, or on d 10 of lactation (L10). In the periventricular preoptic area, d-22 pregnant females had fewer ER beta-mRNA-expressing cells than did females at proestrus, but the level of ER beta mRNA expression per cell in pregnant females was higher than in the two other groups. In the paraventricular nucleus, no changes in ER beta mRNA expression were observed; whereas in the supraoptic nucleus, proestrous females had fewer ER beta-mRNA-expressing cells than L10 females. In the posterodorsal medial amygdala, proestrous females had a greater number of ER beta-mRNA-expressing cells than did L10 females. These results demonstrate that ER beta mRNA expression is differentially regulated in a brain-region-specific and temporal manner under physiological conditions and suggest that ER beta may participate in the regulation of estrogen-sensitive reproductive functions in female rats.
Assuntos
Lactação/metabolismo , Prenhez/metabolismo , Proestro/metabolismo , Prosencéfalo/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Receptor beta de Estrogênio , Feminino , Hibridização In Situ , Neurônios/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Ratos , Receptores de Estrogênio/genética , Distribuição TecidualRESUMO
Genome-wide transcriptional profiling was used to characterize the molecular underpinnings of neocortical organization in rhesus macaque, including cortical areal specialization and laminar cell-type diversity. Microarray analysis of individual cortical layers across sensorimotor and association cortices identified robust and specific molecular signatures for individual cortical layers and areas, prominently involving genes associated with specialized neuronal function. Overall, transcriptome-based relationships were related to spatial proximity, being strongest between neighboring cortical areas and between proximal layers. Primary visual cortex (V1) displayed the most distinctive gene expression compared to other cortical regions in rhesus and human, both in the specialized layer 4 as well as other layers. Laminar patterns were more similar between macaque and human compared to mouse, as was the unique V1 profile that was not observed in mouse. These data provide a unique resource detailing neocortical transcription patterns in a nonhuman primate with great similarity in gene expression to human.
Assuntos
Macaca mulatta/anatomia & histologia , Neocórtex/citologia , Neocórtex/metabolismo , Transcriptoma/fisiologia , Análise de Variância , Animais , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Análise em Microsséries , Microdissecção , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/fisiologia , Neurônios , Análise de Componente Principal , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
We assessed the effects of subtype-selective ER agonists on monoamine levels in discrete regions of the female rat brain. Ovariectomized (ovx) rats were treated for 4 days with vehicle, 17ß-estradiol (E; 0.05mg/kg), an ERß agonist (C19; 3mg/kg) or an ERα agonist (PPT; 3mg/kg) and samples from brain regions were assessed for monoamines and metabolites. We also assessed effects of ERß modulation on baseline and fenfluramine-induced release of monoamines in hippocampus using microdialysis. In the first study, E and the ERα agonist increased norepinephrine in cortex and all three ER ligands increased it in the ventral hippocampus. Changes in levels of the noradrenergic metabolite, MHPG and the dopaminergic metabolite, DOPAC were noted in brain areas of ER ligand-treated animals. E also increased levels of 5HIAA in three brain areas. In the microdialysis study, there were no differences among groups in baseline levels of monoamines. However, E and the ERß agonist increased levels of the dopaminergic metabolite, HVA following fenfluramine. In summary, activation of the two nuclear ERs with selective agonists affects monoamine and metabolite levels in discrete brain areas, a number of which are known to play key roles in cognitive and affective function.
Assuntos
Monoaminas Biogênicas/metabolismo , Córtex Cerebral/efeitos dos fármacos , Estradiol/farmacologia , Fluorenos/farmacologia , Hipocampo/efeitos dos fármacos , Fenóis/farmacologia , Pirazóis/farmacologia , Receptores de Estrogênio/agonistas , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Córtex Cerebral/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Feminino , Fenfluramina/farmacologia , Hipocampo/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Metoxi-Hidroxifenilglicol/metabolismo , Norepinefrina/metabolismo , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.
Assuntos
Proteínas Sanguíneas/metabolismo , Estradiol/metabolismo , Espectrometria de Massas/métodos , Fenóis/metabolismo , Plasma/química , Pirazóis/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Dados de Sequência Molecular , Tamanho do Órgão , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/metabolismo , Útero/anatomia & histologiaRESUMO
The frequency of pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is high in the breeding season and low in the nonbreeding season. These alterations in the patterns of GnRH and LH release are due to an interaction of daylength and gonadal steroid negative feedback. A vast amount of data indicates that steroid-responsive neural systems may play a role in regulating seasonal changes in GnRH release. One candidate system is neuropeptide Y (NPY). To determine the independent and interactive influences of photoperiod and steroid exposure on NPY mRNA levels, we used hypothalamic tissue from four groups (n = 4 per group) of castrated male sheep that were simultaneously housed in photochambers and exposed to: (1) a 16L:8D photoperiod (LD); (2) LD and implanted with testosterone (LD + T); (3) a 10L:14D photoperiod (SD), and (4) SD + T. Circulating levels of T averaged 2.8 +/- 0.2 ng/ml in implanted animals, but were undetectable in nonimplanted males. Mean LH levels were significantly reduced (p < 0.01) in the LD + T group as compared with the other groups which did not differ from each other. The silver grain area per NPY neuron in the arcuate nucleus, as assessed by in situ hybridization, was inversely related to mean LH values, with the grain area per cell being significantly greater (p < 0.05) for LD + T males than for all other groups which did not differ from each other. NPY cell numbers were not significantly different (p > 0.10) among the treatment groups. These results show that NPY mRNA expression is increased in male sheep during a LD photoperiod in a T-dependent manner. Our data are consistent with the idea that NPY is involved in the seasonal regulation of GnRH and LH release in the male sheep.