The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia-reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe(2+), ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe(2+). Desferal and starch-desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch-desferal, and the inhibitor of the mitochondrial Ca(2+) uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch-desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe(2+) during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.
Keywords: Cell death; Free radicals; IR; Iron; Ischemia; KRH; Krebs–Ringer–Hepes; LTR; LysoTracker Red; Lysosome; MCU; MPT; Mitochondria; OH(•); PI; ROS; RhDex; TMRM; V-ATPase; chloromethyldichlorofluorescein; chloromethyldihydrodichlorofluorescein diacetate; cmDCF; cmH(2)DCF-DA; hydroxyl radical; ischemia–reperfusion; mitochondrial Ca(2+) uniporter; mitochondrial membrane potential; mitochondrial permeability transition; propidium iodide; reactive oxygen species; rhodamine–dextran; tetramethylrhodamine methyl ester; vacuolar proton-pumping ATPase; ΔΨ.
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