Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells.
To further understand and investigate how ischemia affects the tight junction we have developed a 2-h model of rapidly reversible ATP depletion and cellular injury in confluent LLC-PK1 monolayers. ATP depletion was achieved utilizing substrate-free medium containing 0.1 microM antimycin A (AA). Cellular ATP levels dropped rapidly to less than 5% of control values, but recovery of ATP and cell morphology was possible even after 2 h of exposure to AA. Ruthenium red, an electron-dense marker of tight-junction integrity, was excluded from the tight junctions of control monolayers but penetrated cellular tight junctions during ATP depletion in a duration-dependent manner. Electrical resistance across the monolayers remained unchanged in control monolayers but decreased linearly during ATP depletion to 59% of control values. Transmonolayer movement of [3H]mannitol increased from a control level of 7 to 13.5% during ATP depletion. Recovery of tight-junction integrity was demonstrated by a slowing of [3H]mannitol transfer from the basolateral to the apical medium. The transfer rate in control monolayers was 0.0126%/min. During the initial 120 min of cellular recovery from 2 h of ATP depletion, the transfer rate was 0.0789%/min, but this decreased to 0.0045%/min between 2 and 4 h of recovery. In summary, physiology, biochemical, and morphological evidence indicates that reversible ATP depletion results in rapid opening of cellular tight junctions. After ATP-repletion physiological studies indicate a recovery of tight-junction integrity.
Canfield
P E
PE
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
Geerdes
A M
AM
Molitoris
B A
BA
D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000502:physiology*; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D004553:Electric Conductivity; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007687:Kidney Tubules, Proximal / Q000648:ultrastructure*; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008854:Microscopy, Electron; D012430:Ruthenium Red / Q000378:metabolism
D012430:Ruthenium Red; D008353:Mannitol; D004533:Egtazic Acid; D000255:Adenosine Triphosphate