oligomycins and acetoacetic-acid

2-Oxoglutarate-supported rat liver mitochondria were loaded with moderate amounts of calcium and submitted to O2 deprivation and reoxygenation. In the presence of acetoacetate, anaerobic energy production maintained Ca2+ retention by mitochondria during the anoxia period unless the Pi concentration of the incubation solution was raised to 4-6 mM. Acetoacetate prompted Ca2+ release from O2-deprived mitochondria at elevated Pi levels, presumably due to occurrence of a permeability transition of the inner membrane. Providing 3-hydroxybutyrate and malate, together with acetoacetate, was found to delay the permeability transition until O2 was reintroduced, i.e., O2 triggered a paradoxical release of Ca2+ from mitochondria under these conditions. Whether initiated by O2 in the presence of Pi or by Pi under aerobic conditions, Ca2+ release was initially activated and subsequently inhibited or reversed in the presence of alpha-tocopherol (10-90 mumol.g protein-1). Similar effects were exerted by alpha-tocopherol during Pi-induced Ca2+ release from oligomycin-treated mitochondria supported by succinate (+ rotenone). In addition, the permeability transition was delayed by retinol (3-30 mumol.g protein-1) while beta-carotene, ubiquinone, and water-soluble antioxidants, including Trolox C, were ineffective. Other observations suggest that the Ca(2+)-releasing and/or -retaining effects of alpha-tocopherol and retinol may be independent from pro- and/or antioxidant activities. Effects resembling those of alpha-tocopherol were exerted by alpha-tocopherol succinate, which is devoid of antioxidant activity. Our data indicate that the permeability transition of Ca(2+)-loaded liver mitochondria may be triggered by O2, in the presence of ketone bodies, and affected by lipid-soluble antioxidants through mechanisms seemingly unrelated to free-radical generation or scavenging.

Topics: Acetoacetates; Anaerobiosis; Animals; Antioxidants; beta Carotene; Calcium; Carotenoids; Chromans; Hypoxia; Kinetics; Mitochondria, Liver; Oligomycins; Oxygen; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Ubiquinone; Vitamin A; Vitamin E

Acetoacetate provision to Ca(2+)-loaded liver mitochondria (less than 40 micrograms-ion Ca2+ x g protein-1), supplied with 2 mM Pi and 2-oxoglutarate as substrate, was found to prevent the mitochondrial deenergization and Ca2+ release induced by either rotenone during aerobic incubations or by O2 deprivation. Under the latter condition, the acetoacetate-promoted Ca2+ retention was entirely supported by ATP produced anaerobically at the succinylthiokinase step of the tricarboxylic acid cycle and was therefore abolished by addition of oligomycin. Surprisingly, oligomycin was also found to trigger Ca2+ release in rotenone-inhibited mitochondria in the presence of acetoacetate under aerobic conditions, unless a Pi acceptor was supplied. ADP deprivation at the succinylthiokinase step is likely to be involved. As estimated from rates of succinate production in O2-deprived mitochondria or from respiration rates in rotenone-inhibited mitochondria at supramaximal acetoacetate concentrations (above 1.2 mM) in the presence of a Pi acceptor, ATP production by substrate-level phosphorylation was close to 10 mumol.g protein-1.min-1 and appeared to be limited by rates of ketone body transport across the inner membrane. The rates of anaerobic energy production obtained by coupling 2-oxoglutarate oxidation to acetoacetate reduction were markedly higher than those obtained by reactions involved in the anaerobic metabolism of amino acids, simulated by providing 2-oxoglutarate and malate to mitochondria. Energy production was limited by rates of oxidant equivalent generation under the latter condition. Our data suggest that acetoacetate could effectively contribute to sustaining anaerobic energy production from endogenous substrates in liver tissue.

Topics: Acetoacetates; Adenosine Diphosphate; Adenosine Triphosphate; Anaerobiosis; Animals; Calcium; Citric Acid Cycle; Energy Metabolism; Ketoglutaric Acids; Malates; Mitochondria, Liver; NAD; Oligomycins; Oxygen; Phosphates; Rats; Rats, Inbred Strains; Rotenone; Succinates; Succinic Acid