A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion

The integrity of the tight junction (TJ), which is responsible for the perm eability 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) w ere shown to reversibly form large complexes and associate with cytoskeleta l 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 r eassembly after ATP depletion-repletion by employing the cell-permeant calc ium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (B APTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability bar rier following ATP repletion as measured by transepithelial electrical resi stance and mannitol flux, marked alterations in the subcellular localizatio n 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 w ith the cytoskeleton. Coimmunoprecipitation studies indicated that decrease d solubility may partly result from the stabilization of large TJ protein-c ontaining complexes with fodrin. Although ionic detergents (SDS and deoxych olate) appeared to cause a dissociation of ZO-1-containing complexes from t he cytoskeleton, sucrose gradient analyses of the solubilized proteins sugg ested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium pl ays an important facilitatory role in the reassembly of the TJ damaged by i schemic insults. Calcium appears to be necessary for the dissociation of TJ -cytoskeletal complexes, thus permitting functional TJ reassembly and parac ellular permeability barrier recovery.