Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosisin bovine pulmonary artery endothelium

In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, re dox reactant and antioxidant pyruvate prevents programmed cell death. We te sted the hypothesis that such protection was mediated, at least in part, vi a pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM alpha -cyano-3-hydroxycinnamate, as a select ive inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 d ecreased cell viability by 30% within 24 h; this was associated with apopto sis-like bodies, nuclear condensation, and biochemical DNA damage consisten t with programmed cell death. Pyruvate (0.1-20 mM) enhanced cell viability in a dose-dependent manner, with greater than or equal to 85% viable cells at greater than or equal to 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining . In contrast, using greater than or equal to 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to approximate to 40%, which was associated with intense DNA laddering, posit ive TUNEL and Hoechst 33258 assays. alpha -Cyano-3-hydroxycinnamate alone d id not significantly decrease endothelial viability but reduced viability f rom 85 +/- 3 to 71 +/- 4% (p = 0.023) in presence of 3 mM pyruvate plus H2O 2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since alpha -cyan o-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consisten t with selective blockade of pyruvate uptake into the mitochondria, the fin dings support the hypothesis that pyruvate protection against H2O2 apoptosi s is mediated in part via the mitochondrial matrix compartment. Possible me diators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruv ate metabolism such as citrate that affect metabolic regulation and anti-ox idant status in the cytoplasm.