Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes

Abstract

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to <1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained >90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na+-free buffer at pH 7.4, pHi did not rise and cell viability remained >80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5–1 μM) at pH 7.4, the MPT was prevented and cell viability remained >80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30–40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.

Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes

“…17,30 -32 Glycine is a membrane-stabilizing amino acid that prevents plasma membrane failure and onset of necrotic cell killing. 22,33,34 Fructose plus glycine prevented acetaminophen-induced necrotic cell killing almost completely 16 hours after acetaminophen administration ( Fig. 2A).…”

“…Hepatocytes were isolated from 25-to 30-g overnight-fasted male C3Heb/FeJ mice (Jackson Laboratory, Bar Harbor, ME) via collagenase perfusion through the inferior vena cava, as described previously. 22 Hepatocytes were resuspended in Waymouth's medium MB-752/1 containing 2 mmol/L L-glutamine, 10% fetal calf serum, 100 nmol/L insulin, 100 nmol/L dexamethasone, 100 U/mL penicillin, and 100 g/mL streptomycin. Cell viability was greater than 90%, as determined via trypan blue exclusion.…”

“…Confocal microscopy after loading of hepatocytes with calcein and tetramethylrhodamine methylester (TMRM) was performed essentially as described previously. 22,24 Briefly, hepatocytes plated on cover glasses were incubated in HDM with 50 mmol/L hydroxyethylpiperazine-N-2 ethanesulfonic acid (pH 7.4) to stabilize pH. After 2.5 hours of treatment with acetaminophen or no addition, cells were loaded with 100 nmol/L TMRM, 1 mol/L acetoxymethyl ester of calcein, and 3 mol/L PI in HDM.…”

“…After centrifugation at 9,000g for 1 minute, the supernatant was diluted 200-fold in H 2 O, and ATP was determined with a luciferin-luciferase kit (Promega Enliten, Madison, WI), as described previously. 22 Nuclear Morphology. Hepatocytes plated on cover glasses were fixed with cold 1% paraformaldehyde in PBS.…”

Inhibitory effects of Schisandra chinensis on acetaminophen-induced hepatotoxicity

“…According to the methods of Qian et al (18), hepatocytes were isolated from overnight-fasted wild-type C57BL/6 male mice by collagenase digestion and plated on type 1 collagen-coated 24-well microtiter plates, 6-cm culture dishes or glass bottom Petri dishes in Waymouth's medium MB-752/1 (HiMedia Laboratories, Mumbai, India) supplemented with 2 mM L-glutamine, 10% fetal calf serum, 100 nM insulin, 100 nM dexamethasone, 100 U/ml penicillin and 100 µg/ml streptomycin. Cell viability was identified as >90% by trypan blue exclusion, according to the manufacturer's instructions (Beyotime Institute of Biotechnology).…”