Effect of reversible ATP depletion on tight-junction integrity in LLC-PK1 cells

P. E. Canfield, A. M. Geerdes, B. A. Molitoris

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Abstract

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 μM antimycin A (AA). Cellular ATP levels dropped rapidly to <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.

Original languageEnglish (US)
Pages (from-to)F1038-F1045
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume261
Issue number6 30-6
StatePublished - Dec 1 1991

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Keywords

  • Electrical resistance across monolayers
  • Paracellular permeability
  • Ruthenium red

ASJC Scopus subject areas

  • Physiology

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