atractyloside and Ischemia

Discrete morphologic, enzymatic and functional changes in skeletal muscle mitochondria have been demonstrated in patients with peripheral arterial disease (PAD). We examined mitochondrial respiration in the gastrocnemius muscle of nine patients (10 legs) with advanced PAD and in nine control patients (nine legs) without evidence of PAD.. Mitochondrial respiratory rates were determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Muscle samples were obtained from the anteromedial aspect of the gastrocnemius muscle, at a level 10 cm distal to the tibial tuberosity. Mitochondria respiratory rate, calculated as nanoatoms of oxygen consumed per minute per milligram of noncollagen protein, were measured at baseline (V(0)), after addition of substrates (malate and glutamate; (V(SUB)), after addition of adenosine diphosphate (ADP) (V(ADP)), and finally, after adenine nucleotide translocase inhibition with atractyloside (V(AT)). The acceptor control ratio, a sensitive indicator of overall mitochondrial function, was calculated as the ratio of the respiratory rate after the addition of ADP to the respiratory rate after adenine nucleotide translocase inhibition with atractyloside (V(ADP)/ V(AT)).. Respiratory rate in muscle mitochondria from patients with PAD were not significantly different from control values at baseline (0.31 +/- 0.06 vs 0.55 +/- 0.12; P =.09), but V(sub) was significantly lower in patients with PAD compared with control subjects (0.43 +/- 0.07 vs 0.89 +/- 0.20; P <.05), as was V(ADP) (0.69 +/- 0.13 vs 1.24 +/- 0.20; P <.05). Respiratory rates after atractyloside inhibition in patients with PAD were no different from those in control patients (0.47 +/- 0.07 vs 0.45 +/- P =.08). Compared with control values, mitochondria from patients with PAD had a significantly lower acceptor control ratio (1.41 +/- 0.10 vs 2.90 +/- 0.20; P <.001).. Mitochondrial respiratory activity is abnormal in lower extremity skeletal muscle in patients with PAD. When considered in concert with the ultrastructural and enzymatic abnormalities previously documented in mitochondria of chronically ischemic muscle, these data support the concept of defective mitochondrial function as a pathophysiologic component of PAD.

Topics: Adenosine Diphosphate; Aged; Atractyloside; Biopsy; Enzyme Inhibitors; Female; Glutamic Acid; Humans; In Vitro Techniques; Intermittent Claudication; Ischemia; Malates; Male; Middle Aged; Mitochondria, Muscle; Mitochondrial ADP, ATP Translocases; Muscle, Skeletal; Oxygen Consumption

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Cells, Cultured; Cold Temperature; Electric Stimulation; Energy Metabolism; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Membranes; Ischemia; Male; Mitochondria, Heart; Oligomycins; Ouabain; Proton-Translocating ATPases; Rats; Rats, Inbred Strains; Vanadium