Mitochondrial Iron and Sphingosine Synergize Initiation of Hepatocyte Death by Augmenting Oxidative Stress

Hepatocytes exposed to ischemia/reperfusion (I/R) succumb to cell death after reperfusion. Sphingosine and ceramide profiles revealed substantial accumulation of sphingosine after 4h of ischemia to rat hepatocytes, whereas other sphingoid bases did not change. A lysosomotropic inhibitor of acid ceramidase suppressed I/R-induced death, indicating a lysosomal origin of sphingosine. Addition of exogenous sphingosine to hepatocytes increased cell death, which was insensitive to the ceramide synthase inhibitor, fumonisin B1. This finding indicates that accumulation of sphingosine, not ceramide formed from sphingosine, promoted cell death. Exogenous sphingosine also inhibited complex IV (cytochrome oxidase), the terminal component of the respiratory chain, in isolated mitochondria. Accordingly, we hypothesized that downstream respiratory inhibition by sphingosine leads to increased formation of O2•- radicals after reperfusion, which by themselves have only a moderately harmful effect. However when Fe2+redistributes from lysosomes into mitochondria during ischemia, Fenton chemistry occurs after reperfusion, leading to formation of highly reactive OH• radicals, potent inducers of the mitochondrial permeability transition pore and cell death. This hypothesis was directly tested using bafilomycin, which induces the release of Fe2+ from lysosomes with subsequent uptake into mitochondria. Indeed, bafilomycin potentiated sphingosine-induced cell death. The data highlight a novel mechanism mediating I/R injury, which involves sphingosine accumulation and uptake of lysosomal iron into mitochondria during ischemia, leading to respiratory chain inhibition, iron-dependent oxidative stress, mitochondrial permeability transition and cell death after reperfusion. DK073336, DK037034 and 14.Z50.31.0028 (JJL) and NS083544 (TIG).