CONTRIBUTION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION TO LETHAL INJURY AFTER EXPOSURE OF HEPATOCYTES TO T-BUTYLHYDROPEROXIDE

We have developed a novel method for monitoring the mitochondrial perm eability transition in single intact hepatocytes during injury with t- butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with th e fluorescence probes, calcein and tetramethylrhodamine methyl ester ( TMRM), Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both c ytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged sim ultaneously using laser-scanning confocal microscopy. During normal in cubations, TMRM labelled mitochondria indefinitely (longer than 63 min ), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously, After addition of 100 mu M t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was ac companied by mitochondrial depolarization, as shown by loss of TMRM. S ubsequently, the concentration of ATP declined and cells lost viabilit y. Trifluoperazine, a phospholipase inhibitor that inhibits the permea bility transition in isolated mitochondria, prevented calcein redistri bution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mit ochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Triflu operazine did not prevent ATP depletion and cell death after the addit ion of CCCP. In conclusion, the permeability transition pore does not 'flicker' open during normal incubation of hepatocytes but remains con tinuously closed. Moreover, mitochondrial depolarization per se does n ot cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.