Steroid hydroxylations in rat adrenal mitochondria: IV. An inhibition of NADH oxidase and succinate-supported deoxycorticosterone hydroxylation by steroid and rotenone

Abstract

The effects of steroid and rotenone on succinate-supported deoxycortieosterone hydroxylation were compared. In intact rat adrenal mitochondria, the rate of deoxycortieosterone hydroxylation decreased as the concentration of the steroid substrate or the major steroid product (corticosterone) or rotenone was increased. Steroid had no effect on either succinate-supported oxidative phosphorylation or on deoxycorticosterone hydroxylation supported by NADPH plus calcium ions. In submitochondrial particles, NADPH formation by the energy-linked transhydrogenase was only slightly inhibited by steroid. NADH oxidase, however, was markedly inhibited by deoxycortieosterone, corticosterone or rotenone suggesting that steroid (like rotenone) decreased the rate of succinate-supported steroid hydroxylation by specifically inhibiting reversed electron transport. Further support for this interpretation was found in the inhibition by either steroid or rotenone of the ATP-dependent, succinate-supported hydroxylation of deoxycortieosterone in KCN-inhibited mitochondria. It is concluded that the deleterious effect of steroid on succinate-supported steroid hydroxylation is due to an inhibition of reversed electron transport in the region of the first phosphorylation site. The steroid, rotenone and antimycin sensitivity of the NADH oxidase of rat adrenal submitochondrial particles differed depending on the method of assay. When measured by NADH disappearance, NADH oxidase activity was sensitive to all three inhibitors and KCN. When measured by oxygen consumption, NADH oxidase activity was sensitive to KCN but only slightly sensitive to steroid, rotenone or antimycin. Rat adrenal submitochondrial preparations were found to contain an active rotenone-and antimycin-insensitive NADH-cytochrome c reductase in addition to the typical inhibitor-sensitive NADH oxidase. Exogenous cytochrome c converted the rotenone or antimycin-sensitive NADH oxidase to an inhibitor-insensitive NADH oxidase. However, sensitivity to KCN remained. The results indicate that a shuttle of cytochrome c between the inhibitor-insensitive NADH cytochrome c reductase and the inhibitor-sensitive NADH oxidase may explain the rotenone- and antimycin-insensitive NADH oxidase activity of adrenal cortex submitochondrial preparations.