Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. Many disease states of the kidney such as ischemia/reperfusion, inflammation, or toxic injury to the kidney and gut lead to loss of the epithelial barrier. Reactive oxygen species are directly involved in the pathophysiology of some of these diseases. Targets for hydrogen peroxide (H 2 O 2 ) 1 include DNA, proteins, and lipids. Reported effects of hydrogen peroxide on renal epithelial cell lines include DNA damage with induction of apoptosis or necrosis (1-3), decreased activity of membrane transporters (4), and membrane lipid peroxidation (5). Hydrogen peroxide decreases transepithelial resistance (TER) and increases transcellular permeability of MDCK cell monolayers (6, 7), which is also well documented in non-kidney epithelial cell lines (8 -11) and endothelial cell lines (12-15). However, the biochemical and subcellular effects of hydrogen peroxide on tight junction (TJ) proteins have not been studied, nor have they been distinguished from effects due to partial ATP depletion. Short-term depletion and repletion of intracellular ATP in cultured cells has been used to study the disassembly and/or reassembly of junctions as a model of organ ischemia and reperfusion (16 -20). However, little is known about the reassembly of the tight junction after exposure to reactive oxygen species due to the lack of a reproducible model system. We have now developed and analyzed a model of reversible hydrogen peroxide-induced disassembly and reassembly of the TJ in vitro and show that the reassembly pathway, as monitored by physiological, biochemical, and immunocytochemical parameters, depends upon tyrosine kinase activity. Furthermore, the cellular and biochemical consequences of H 2 O 2 exposure are distinct from those caused by ATP depletion-repletion. EXPERIMENTAL PROCEDURESCell Culture and Materials-Madin-Darby canine kidney cells (MDCK, type II) were obtained from American Type Tissue Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 50 IU/ml penicillin, and 50 g/ml streptomycin. Cells were incubated at 37°C in 5% CO 2 and were passaged weekly. DMEM was from Cellgro (Herndon, VA), streptomycin, penicillin, and fetal calf serum from Sigma. Plasticware was from ...

The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133-16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.