SOURCES OF REDUCING EQUIVALENTS FOR CYTOCHROME P-450 MITOCHONDRIAL STEROID HYDROXYLATIONS IN RAT ADRENAL CORTEX CELLS

Abstract

Pyruvate-supported 11β-hydroxylation of 11-deoxycorticosterone (DOC) via cytochrome P-450 reductase chain in incubated rat adrenal mitochondria was maximal when either traces of oxaloacetate (OAA) or 2 mM ATP were added to the incubation medium. This showed that reducing equivalents formed at the pyruvate dehydrogenase level as well as those derived from Krebs-cycle activity were needed for maximal corticosterone (B) formation from DOC. These findings were confirmed in experiments with whole cells isolated from rat adrenals designed to show the possible pathways of intramitochondrial NADPH generation for steroid hydroxylations. Whereas citrate, isocitrate, succinate and malate were not metabolized because of their impermeability to the plasma membranes of the cells, both [1-14C] and [2-14C]-pyruvate were efficiently utilized by the mitochondria of the cells for B formation from DOC. Whereas arsenite completely inhibited the pyruvate- 14C supported B formation, comparison of the data obtained with two inhibitors, 2,4-dinitrophenol (2,4-DNP) and fluorocitrate, showed that for the same per cent inhibition of 14CO2 production the inhibition of 11β-hydroxylation of DOC was greater with 2,4-DNP than with fluorocitrate. It is concluded that operation of the Krebs-cycle is essential for optimizing the production of reducing equivalents needed for 11β-hydroxylation to occur. It is also concluded that the rate of oxidation of pyruvate in the mitochondria of the cells and the activity of the Krebs-cycle is dependent on availability of OAA. When ATP levels were reduced by the uncoupling effect of 2,4-DNP resulting in a decreased production of OAA via ATP-requiring pyruvate carboxylase, a concomitant inhibition in production of reducing equivalent occurred which led to a lack of B production from DOC.