RESUMO
We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions.
Assuntos
Inflamação/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos/imunologia , Linfócitos/metabolismo , Óxido Nítrico/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Animais , Anticorpos Monoclonais , Células Cultivadas , Células Endoteliais/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Inflamação/imunologia , Interleucina-10/sangue , Interleucina-10/imunologia , NG-Nitroarginina Metil Éster/administração & dosagem , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: We have previously shown that melatonin inhibits bradykinin-induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin. EXPERIMENTAL APPROACH: RT-PCR assays were performed with rat cultured endothelial cells. The putative effect of melatonin upon arteriolar tone was investigated by intravital microscopy while NO production by endothelial cells in vitro was assayed by fluorimetry, and intracellular Ca(2+) measurements were assayed by confocal microscopy. KEY RESULTS: No expression of the mRNA for the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase, or for the melatonin MT(2) receptor was detected in microvascular endothelial cells. Melatonin fully inhibited L-NAME-sensitive bradykinin-induced vasodilation and also inhibited NO production induced by histamine, carbachol and 2-methylthio ATP, but did not inhibit NO production induced by ATP or alpha, beta-methylene ATP. None of its inhibitory effects was prevented by the melatonin receptor antagonist, luzindole. In nominally Ca(2+)-free solution, melatonin reduced intracellular Ca(2+) mobilization induced by bradykinin (40%) and 2-methylthio ATP (62%) but not Ca(2+) mobilization induced by ATP. CONCLUSIONS AND IMPLICATIONS: We have confirmed that melatonin inhibited NO production both in vivo and in vitro. In addition, the melatonin effect was selective for some G protein-coupled receptors and most probably reflects an inhibition of Ca(2+) mobilization from intracellular stores.
Assuntos
Células Endoteliais/efeitos dos fármacos , Melatonina/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Óxido Nítrico/biossíntese , Acetilserotonina O-Metiltransferasa/genética , Trifosfato de Adenosina/farmacologia , Animais , Arilalquilamina N-Acetiltransferase/genética , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Cálcio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fluorometria , Expressão Gênica/efeitos dos fármacos , Histamina/farmacologia , Masculino , Artérias Mesentéricas/fisiologia , Microscopia Confocal , Microscopia de Vídeo , NG-Nitroarginina Metil Éster/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor MT2 de Melatonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasodilatação/efeitos dos fármacosRESUMO
A new furnace, based on a halogen lamp, and a sample cell have been designed and constructed for in situ X-ray absorption spectroscopy experiments in conventional and dispersive mode (transmission and fluorescence geometries). The main application of the apparatus is thermal treatment studies under controlled conditions for dynamical processes. The sol-gel (gelatin) method has been utilized to synthesize NiO nanoparticles. During this heating process, in situ Ni K-edge X-ray absorption near-edge structural measurements provided evidence of the evolution of a Ni environment until complete NiO nanoparticle crystallization. This case is reported in order to show the furnace performance in dispersive mode.
Assuntos
Cristalização/métodos , Cristalografia por Raios X/instrumentação , Nanopartículas/química , Nanopartículas/ultraestrutura , Espectrometria por Raios X/instrumentação , Cristalografia por Raios X/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais/métodos , Espectrometria por Raios X/métodosRESUMO
The LNLS XAS beamline has been operating for external users since July 1997. Many facilities and improvements have been progressively added to it, extending the range of applications. Here, a technical description of the main beamline components is given, and results concerning important points, such as available flux at low and high energies, harmonic contamination, energy resolution and stability, are presented. Some key results are given to demonstrate the beamline performance and limitations. It is shown that the beamline can cover a large energy range, starting from the rather low energy of 2.3 keV up to 25 keV.
RESUMO
A multiwire proportional counter was used in fluorescence X ray absorption measurements and a comparison to a Si(Li) and NaI(Tl) detectors was done. The main features of the multiwire proportional counter are its high counting rate capability (10(7)) counts x s(-1)) and large active area (6 x 6 cm2). It was shown that the MWPC is suitable for fluorescence absorption. Although the maximum capability was not reached in the present experiments, it was found that as the counting rate increase the MWPC performance became better than Si(Li) detectors and shows a similar response to the scintillator counter at medium counting rates (up to 10(5) counts x s(-1)).
RESUMO
An X-ray absorption fine-structure spectroscopy beamline has been installed and commissioned at a bending-magnet source at LNLS. Three monochromators are available: a channel-cut, a double-crystal and a four-crystal set-up. They have been operated from 2500 up to 15000 eV, with a resolving power better than 5500 in the full range. Photon flux of the order of 10(8) photons s(-1) up to 10(10) photons s(-1) has been attained. The experimental station is equipped with a table that can withstand a weight of 300 kg and track the vertical position of the beam with a 2.5 micro m accuracy over a 120 mm stroke. The beamline has been fully characterized and the first results are presented.
RESUMO
Preliminary measurements in a proportional counter with two independently counting wires showed that counting rates up to 10(6) counts s(-1) wire(-1) can be reached without critical loss in the 'true versus measured' linearity relation. Results obtained with a detector containing 30 active wires (2 mm pitch) are presented. With each wire is associated a fast pre-amplifier and a discriminator channel. Global counting rates in excess of 10(7) events s(-1) are reported. Dead-time losses are corrected by use of simple mathematical-modelling functions. Data-acquisition systems are described for one-dimensional (real-time) and two-dimensional (off-line) position-sensitive detection systems.