oligomycins and Acidosis--Lactic

Mitochondrial encephalomyopathy and lactic acidosis with strokelike episodes (MELAS) is a severe young onset stroke disorder without effective treatment. We have identified a MELAS patient harboring a 13528A-->G mitochondrial DNA (mtDNA) mutation in the Complex I ND5 gene. This mutation was homoplasmic in mtDNA from patient muscle and nearly homoplasmic (99.9%) in blood. Fibroblasts from the patient exhibited decreased mitochondrial membrane potential (Deltapsim) and increased lactate production, consistent with impaired mitochondrial function. Transfer of patient mtDNA to a new nuclear background using transmitochondrial cybrid fusions confirmed the pathogenicity of the 13528A-->G mutation; Complex I-linked respiration and Deltapsim were both significantly reduced in patient mtDNA cybrids compared with controls. Inhibition of the adenine nucleotide translocase or the F1F0-ATPase with bongkrekic acid or oligomycin caused a loss of potential in patient mtDNA cybrid mitochondria, indicating a requirement for glycolytically generated ATP to maintain Deltapsim. This was confirmed by inhibition of glycolysis with 2-deoxy-D-glucose, which caused depletion of ATP and mitochondrial depolarization in patient mtDNA cybrids. These data suggest that in response to impaired respiration due to the mtDNA mutation, mitochondria consume ATP to maintain Deltapsim, representing a potential pathophysiological mechanism in human mitochondrial disease.

Topics: Acidosis, Lactic; Adenosine Triphosphate; Adult; Anti-Bacterial Agents; Antimetabolites; Bongkrekic Acid; Cell Line, Tumor; Deoxyglucose; DNA, Mitochondrial; Electron Transport Complex I; Female; Fibroblasts; Glycolysis; Humans; Membrane Potential, Mitochondrial; Mitochondria, Muscle; Mitochondrial Encephalomyopathies; Mitochondrial Proteins; Oligomycins; Oxygen Consumption; Point Mutation; Proton-Translocating ATPases; Stroke

Oxidative phosphorylation of isolated rat skeletal muscle mitochondria after exposure to lactic acidosis in either phosphorylating or nonphosphorylating states has been evaluated. Mitochondrial respiration and transmembrane potential (DeltaPsi(m)) were measured with pyruvate and malate as the substrates. The addition of lactic acid decreased the pH of the reaction medium from 7.5 to 6.4. When lactic acid was added to nonphosphorylating mitochondria, the subsequent maximal ADP-stimulated respiration decreased by 27% compared with that under control conditions (P < 0.05), and the apparent Michaelis-Menten constant (K(m)) for ADP decreased to 10 microM vs. 20 microM (P < 0.05) in controls. In contrast, maximal respiration and ADP sensitivity were not affected when mitochondria were exposed to acidosis during active phosphorylation in state 3. Acidosis significantly increased mitochondrial oxygen consumption in state 4 (post-state 3), irrespective of when acidosis was induced. This effect of acidosis was attenuated in the presence of oligomycin. The addition of lactic acid during state 4 respiration decreased DeltaPsi(m) by 19%. The ratio between added ADP and consumed oxygen (P/O) was close to the theoretical value of 3 in all conditions. The addition of potassium lactate during state 3 (i.e., medium pH unchanged) had no effect on the parameters measured. It is concluded that lactic acidosis has different effects when induced on nonphosphorylating vs. actively phosphorylating mitochondria. On the basis of these results, we suggest that the influence of lactic acidosis on muscle aerobic energy production depends on the physiological conditions at the onset of acidity.

Topics: Acidosis, Lactic; Adenosine Diphosphate; Animals; Disease Susceptibility; Hydrogen-Ion Concentration; Lactic Acid; Male; Membrane Potentials; Mitochondria; Oligomycins; Oxygen Consumption; Phosphorylation; Rats; Rats, Sprague-Dawley