RESUMO
ASCT2 is an ASC (alanine-, serine-, cysteine-preferring) neutral amino acid exchanger that may regulate CNS function by transporting amino acid substrates including L-serine, L-cysteine, L-glutamine, L-glutamate and D-serine. Despite the potentially important role of ASCT2 in influencing metabolic and signaling functions of these amino acids in brain, there has been little description of its distribution in brain tissue. We employed a commercially available human ASCT2 antibody in immunohistochemistry studies in adult mouse brain and found a wide regional distribution for ASCT2 that was limited to dendrites labeled by anti-microtubule-associated protein-2 in cortex, hippocampus and striatum. No ASCT2 immunoreactivity was observed in areas labeled by antibodies against a neuronal cell body marker (NeuN), or either of the astrocyte markers, glial fibrillary acidic protein or S100beta. In cerebellum both Purkinje cell bodies and dendrites were positive for ASCT2 immunoreactivity. In support of a dendritic localization for ASCT2 in cortex, low affinity (K(T) > 1 mM), Na(+)-dependent D-serine and L-glutamine uptake characteristic of ASCT2-mediated transport was observed in P2 synaptosomal preparations. These results suggest that ASCT2 may be an important neuronal neutral amino acid transporter and highlight a discrepancy between findings of astrocyte ASCT2 function in tissue culture and brain in situ.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Neurônios/metabolismo , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Células Cultivadas , Dendritos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Antígenos de Histocompatibilidade Menor , Fatores de Crescimento Neural/metabolismo , Neurônios/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
BACKGROUND: The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob Disease (vCJD) have led to profound changes in the production and trade of agricultural goods. The rapid tests currently approved for BSE monitoring in slaughtered cattle are all based on the detection of the disease related isoform of the prion protein, PrPd, in brain tissue and consequently are only suitable for post-mortem diagnosis. OBJECTIVES: In instances such as assessing the health of breeding stock for export purposes where post-mortem testing is not an option, there is a demand for an ante-mortem test based on a matrix or body fluid that would permit easy access and repeated sampling. Urine and urine based analyses would meet these requirements. RESULTS: Two dimensional differential gel eletrophoresis (2D-DIGE) and mass spectrometry analyses were used to identify proteins exhibiting differential abundance in the urine of BSE infected cattle and age matched controls over the course of the disease. Multivariate analyses of protein expression data identified a single protein able to discriminate, with 100% accuracy, control from infected samples. In addition, a subset of proteins were able to predict with 85% +/- 13.2 accuracy the time post infection that the samples were collected. CONCLUSION: These results suggest that in principle it is possible to identify biomarkers in urine useful in the diagnosis, prognosis and monitoring of disease progression of transmissible spongiform encephalopathy diseases (TSEs).
RESUMO
N-methyl-D-aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, D-serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl-D-aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of D-serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, D-2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and D-serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and D-serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Artéria Cerebral Média/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Denervação , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos , Artéria Cerebral Média/inervação , Artéria Cerebral Média/metabolismo , Miografia , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Vasodilatação/fisiologiaRESUMO
BACKGROUND: We previously reported that resveratrol, a polyphenol found in red grapes, attenuated changes in small artery geometry and stiffness, as well as cardiac hypertrophy and cardiac function in the spontaneously hypertensive rat (SHR). However, in addition to resveratrol, grapes contain a variety of bioactive polyphenols such as catechins, anthocyanins, and flavonoids. Therefore, we investigated the effects of grape consumption in SHR. METHODS: Wistar-Kyoto (WKY) rats and SHR were treated with freeze-dried grape powder for 10 weeks. Dilatory, geometry, and stiffness properties of mesenteric small arteries were assessed by pressurized myography. Left ventricular mass index and cardiac function were assessed by two-dimensional guided M-mode and pulse-wave Doppler echocardiography. RESULTS: Elevated blood pressure in SHR was associated with remodeling and impaired endothelium-dependent relaxation of small arteries. Augmented left ventricular mass index (reflecting hypertrophy) and diminished cardiac function were also evident in SHR. Although grape treatment failed to affect cardiac dysfunction, it elicited a significant reduction in blood pressure, improved arterial relaxation, increased vascular compliance, and attenuated cardiac hypertrophy. CONCLUSIONS: Treatment with whole grape powder conferred mild vascular and cardiac benefits in SHR. Therefore, dietary grape consumption may be a feasible and salutary adjunct to pharmacological treatment of human hypertension.
Assuntos
Hipertensão/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Artérias Mesentéricas/efeitos dos fármacos , Fitoterapia , Preparações de Plantas/uso terapêutico , Vitis , Animais , Pressão Sanguínea/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Astrocytes mediate either constriction or dilation of local brain arterioles in response to synaptic activity. Recent work indicates that the directionality of this response may be dictated by ambient oxygen levels.
Assuntos
Astrócitos/fisiologia , Encéfalo/irrigação sanguínea , Sistema Vasomotor/fisiologia , Arteríolas/fisiologia , Encéfalo/citologia , Metabolismo Energético , Oxigênio/análise , Oxigênio/fisiologia , Vasoconstrição , VasodilataçãoRESUMO
BACKGROUND: The pathological hallmarks of transmissible spongiform encephalopathy (TSE) diseases are the deposition of a misfolded form of a host-encoded protein (PrPres), marked astrocytosis, microglial activation and spongiosis. The development of powerful gene based technologies has permitted increased levels of pro-inflammatory cytokines to be demonstrated. However, due to the use of assays of differing sensitivities and typically the analysis of a single model system it remained unclear whether this was a general feature of these diseases or to what extent different model systems and routes of infection influenced the relative levels of expression. Similarly, it was not clear whether the elevated levels of cytokines observed in the brain were accompanied by similar increases in other tissues that accumulate PrPres, such as the spleen. RESULTS: The level of expression of the three interferon responsive genes, Eif2ak2, 2'5'-OAS, and Mx2, was measured in the brains of Syrian hamsters infected with scrapie 263K, VM mice infected with bovine spongiform encephalopathy and C57BL/6 mice infected with the scrapie strain ME7. Glial fibrillary acidic expression confirmed the occurrence of astrocytosis in all models. When infected intracranially all three models showed a similar pattern of increased expression of the interferon responsive genes at the onset of clinical symptoms. At the terminal stage of the disease the level and pattern of expression of the three genes was mostly unchanged in the mouse models. In contrast, in hamsters infected by either the intracranial or intraperitoneal routes, both the level of expression and the expression of the three genes relative to one another was altered. Increased interferon responsive gene expression was not observed in a transgenic mouse model of Alzheimer's disease or the spleens of C57BL/6 mice infected with ME7. Concurrent increases in TNFalpha, TNFR1, Fas/ApoI receptor, and caspase 8 expression in ME7 infected C57BL/6 mice were observed. CONCLUSION: The identification of increased interferon responsive gene expression in the brains of three rodent models of TSE disease at two different stages of disease progression suggest that this may be a general feature of the disease in rodents. In addition, it was determined that the increased interferon responsive gene expression was confined to the CNS and that the TSE model system and the route of infection influenced the pattern and extent of the increased expression. The concurrent increase in initiators of Eif2ak2 mediated apoptotic pathways in C57BL/6 mice infected with ME7 suggested one mechanism by which increased interferon responsive gene expression may enhance disease progression.