A role for intracellular calcium in tight junction reassembly after atp depletion- repletion. Ye, Jiuming, Tatsuo Tsukamoto, Adam Sun and Sanjay K. Nigam. From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, and *the Renal Division, Rhode Island Hospital, Brown University School of Medicine, Providence, RI 02903.
APStracts 6:0107F, 1999.
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, using an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney (MDCK) cells, we have shown that TJ proteins such as zonula occludens-1 (ZO-1) reversibly form large complexes and associate with cytoskeletal proteins (J. Biol. Chem. 272,16133,1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator, 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, decreased solubility for ZO-1 and other TJ proteins by Triton-X-100 extraction assay, suggesting that this potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that this decreased solubility may in part result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) caused a dissociation of ZO-1 containing complexes from the cytoskeleton, sucrose gradient analyses of those solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Taken together, these results raise the possibility that intracellular calcium plays an important facilitory role in the reassembly of the TJ damaged by ischemic insults. 

Received 4 November 1998; accepted in final form 18 May 1999.
APS Manuscript Number F301-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1999 The American Physiological Society.
Published in APStracts on 14 June 1999