RESUMO
We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.
Assuntos
Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Peróxido de Hidrogênio/metabolismo , Escopoletina/farmacologia , Trifosfato de Adenosina/metabolismo , Adesão Celular , Compostos Cromogênicos , Eosinófilos/citologia , Fibronectinas/metabolismo , Peroxidase do Rábano Silvestre , Humanos , Técnicas In Vitro , Oxazinas , Fator de Ativação de Plaquetas/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Ethanol can affect the regulation of liver hemodynamics through the release of vasoactive mediators such as nitric oxide and endothelins (ETs). The purpose of this study was to investigate the effects of ethanol on the changes in arterial and portal perfusion pressure induced by ET receptor activation. Ethanol significantly reduced portal, but not arterial perfusion pressure. ET-1 and norepinephrine (used as an ET receptor-independent vasoconstrictor) induced changes in hepatic arterial or portal inflow resistance that were not affected by ethanol treatment. However, an IRL 1620-induced increase in portal, but not arterial, inflow resistance was significantly reduced in ethanol-perfused preparations, an effect observed after either intra-arterial or intraportal administration of the agonist. These results suggest that ethanol can diminish the responsiveness of the portal vascular bed of the canine liver to ETB receptor activation.