Oxidants increase paracellular permeability in a cultured epithelial cell line

Abstract

Inflammation of epithelia is an important step in the pathophysiology of a wide variety of diseases. Because reactive oxygen metabolites are important effector molecules of acute inflammation, we examined the effect of oxidants on the barrier function of a cultured epithelium, Madin Darby Canine Kidney cells, by measuring the transepithelial electrical conductance, G(t), of monolayers grown on permeable supports. We found that H2O2, added directly or generated with glucose oxidase, increased G(t). Similar effects were observed with addition of xanthine and xanthine oxidase, a system which enzymatically generates superoxide radical O-2. The oxidant-induced increase in G(t) was reversible if the exposure to oxidants was not prolonged (<20 min), and if the concentration of H2O2 was <5 x 10-3 M. The increase in G(t) suggested that oxidants increase the permeability of the paracellular pathway, a suggestion supported by an oxidant-induced increase in the permeability to 14C-mannitol, which primarily crosses epithelia via the extracellular route. In addition to functional changes in the epithelial monolayer, oxidants changed the cell morphology; after H2O2 exposure, the cells tended to pull apart, most prominently at their basolateral surfaces. These changes were heterogeneous with most areas showing no changes. Some of the morphologic changes could be reversed if the exposure to H2O2 was limited. We also observed a disruption of the normal pattern of the actin-cytoskeleton, particularly in the area of cell to cell junctions, as demonstrated by fluorescent staining of f-actin with rhodamine phallicidin. These functional and structural findings indicate that oxidants increase the permeability of the paracellular pathway in a cultured epithelium. The changes can be reversible, and are accompanied by alterations in organization of the cell cytoskeleton. These studies demonstrate the dynamic nature of the interaction between epithelial cells and oxygen metabolites.