The effect of phenothiazone on the metabolism of liver mitochondria

Abstract

Phenothiazone at a concentration of 10 μM stimulated oxygen uptake during the oxidation by liver mitochondria of l-glutamate, pyruvate, a-oxoglutarate and l-malate. This was due to the provision of a pathway of electron transfer from NADH to cytochrome c or oxygen, additional to the respiratory chain of electron carriers. Higher concentrations of phenothiazone inhibited NAD-dependent oxidations. Such inhibitions were relieved by additional NAD, or by treatment of mitochondria with EDTA. The mechanism of inhibition is considered to involve the formation of a complex between phenothiazone and NADH oxidase. Succinoxidase of mitochondria was inhibited by concentrations of phenothiazone from 10 μM upwards. The data suggest that inhibition of succinoxidase by low concentrations of phenothiazone is mediated through NAD inhibition of succinoxidase, or through a similar mechanism. At a higher concentration, 3.3 mM, phenothiazone inhibited cytochrome oxidase, thus explaining portion of the succinoxidase inhibition by this concentration. Phenothiazone apparently stimulated succinic dehydrogenase activity when it was isolated from the terminal respiratory chain by HCN and assayed by coupling succinate oxidation to the reduction of methylene blue. However, the apparent stimulation was due to phenothiazone itself acting as an auto-oxidizable electron donor additional to methylene blue. Phenothiazone at and above 10 μM lowered the efficiency of oxidative phosphorylation by mitochondria for both NAD-dependent substrate succinate oxidation. ATP-ase activity (Mg ++-activated) and the rate of swelling of mitochondria was unaffected by phenothiazone. The possible relationship of phenothiazone and phenothiazine in producing their effect on metabolism is discussed. It appears likely that phenothiazone or a precursor plays a role in the overall effects displayed by phenothiazine poisoning both in vivo and in vitro.