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.