Endotoxin pretreatment in vivo increases the mitochondrial respiratory capacity in rat hepatocytes

Administration of sublethal doses of endotoxin produces tolerance to subsequent oxidative stress in diverse animal models. Although endotoxin induces antioxidant enzymes, particularly manganous superoxide dismutase (Mn-SOD), the phenomenon of tolerance remains incompletely understood. Previously I determined that endotoxin treatment in rats increased lung mitochondrial respiration-dependent (i.e., independent of Mn-SOD) scavenging of superoxide anion. Because nonenzymatic scavenging of superoxide anion correlates with the mitochondrial membrane energy gradient, I hypothesized that endotoxin increases the mitochondrial transmembrane potential. Endotoxin treatment (500 micrograms/kg intraperitoneally 48 h earlier) increased the hepatocyte mitochondrial transmembrane potential as determined by two separate methods: the intramitochondrial sequestration of triphenylmethylphosphonium (electrical potential or delta psi) and the fluorescence intensity of the hepatocyte mitochondria when stained with rhodamine-123 and examined by confocal microscopy. These findings suggest that endotoxin treatment increased the total mitochondrial membrane potential per hepatocyte. In parallel, endotoxin treatment increased the fluorescence intensity of hepatocyte mitochondria after staining with 10-N-nonyl-acridine orange, a dye that binds to the mitochondrial inner membrane independently of the transmembrane potential. This suggests that an increase in mitochondrial inner membrane mass is responsible for the net increase in inner membrane potential per cell following endotoxin pretreatment. These findings complement previous studies in which endotoxin treatment increased the mitochondrial-specific antioxidant Mn-SOD and support the more recent finding that endotoxin treatment also increased nonenzymatic scavenging of superoxide by lung mitochondria. Taken, together, these observations suggest that mitochondrial biogenesis, and the subsequent increase in both enzymatic and nonenzymatic scavenging of superoxide anion, is a central feature of endotoxin-mediated tolerance to oxidative stress.