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Abstract: Transforming growth factor-β (TGFβ) is synthesized as a precursor protein, pro-TGFβ, that must be cleaved by a furin-like proteinase before it becomes biologically active. We hypothesized that alkalinization of [...]

Epithelial mucous membranes are repeatedly exposed to oxidants and xenobiotics. CFTR plays a role in glutathione transepithelial flux and in defining the hydration and viscoelasticity of protective mucus. We therefore hypothesized that CFTR expression and function may be modulated by oxidant stress. A sublethal oxidant stress (tert-butylhydroquinone, BHQ) in CFTR-expressing epithelial cells (T84) induced a significant increase in cellular glutathione that was associated with an increase in expression of the gene encoding the heavy subunit of the rate-limiting enzyme for glutathione synthesis, [gamma]-glutamylcysteine synthetase ([gamma]-GCShs). CFTR gene expression was markedly decreased according to a time course that mirrored the changes in [gamma]-GCShs. Western blot analysis confirmed that the decrease in CFTR gene expression was associated with a decrease in CFTR protein. cAMP-dependent iodide efflux was also decreased by the oxidant stress. Nuclear run-on assays indicated that the oxidant stress had no effect on CFTR gene transcription, but the mRNA stability in the oxidant-stressed cells was markedly reduced. Furthermore, BHQ increased [gamma]-GCShs mRNA while decreasing CFTR mRNA in Calu-3 cells, and taurine chloramine induced similar effects in T84 cells. We conclude that suppression of CFTR expression may represent an adaptive response of mucosal epithelium to an exogenous oxidant stress. antioxidants: chloride channels: epithelial cells: cystic fibrosis: reactive oxygen species

Asthma is characterized by an increased production of eosinophil-active C-C chemokines by the airway epithelium. Recent studies have identified the presence of important quantities of labile zinc in the conducting airways. We hypothesized that modulation of this labile zinc could influence the production of proinflammatory chemokines in respiratory epithelial cells. The zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the heavy metal chelator 2,3-dimercapto-l-propanesulfonic acid (DMPS) were used to reduce the labile zinc content of A549, BEAS-2B, and HFL-1 cells. Northern blot analysis and RNase protection assay were used to study the effects of the zinc chelators on mRNA expression. DMPS and TPEN specifically inhibited the production of eotaxin, regulated on activation, normal T-cell expressed, and presumably secreted, and monocyte chemotactic protein-1 in TNF-[alpha]-stimulated respiratory epithelial cells and fibroblasts through labile zinc chelation. The inhibitory effects of DMPS and TPEN were associated with the decreased binding of the zinc-finger transcription factor GATA-1, whereas no change in NF-[kappa]B activation was observed. Together these results demonstrate that modulation of the labile pool of zinc can regulate gene expression and protein synthesis of C-C chemokines in lung epithelial cells and fibroblasts. CC chemokines; inflammation; asthma; regulated on activation, normal T-cell expressed, and presumably secreted; monocyte chemotactic protein

A series of [beta]-lactam derivatives were synthesized and tested to determine the structure-activity relationship for inhibition of human leukocyte elastase (HLE), a serine protease involved in several degenerative lung and tissue diseases. The most potent [IC.sub.50] values were obtained with neutral hydrophobic 7[alpha]-methoxy cephalosporanic acid derivatives. Tryptophanyl-9-fluorenylmethyl ester and N-benzhydryl piperazine derivatives of 7[alpha]-methoxy cephalosporanic acid represent two novel HLE inhibitors, with length of action persisting beyond 24 h. Key words: [beta]-lactams, neutrophil elastase, protease inhibitors, peptides.

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, [Beta]-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases. leukocyte; inflammation; adherens junctions; adhesion molecules; epithelium; inflammatory mediators; tight junctions