Previous work suggested that autoregulation of adrenocortical function by steroids may be important in the development of different functional zones of the adrenal cortex, possibly through the generation of oxygen-derived free radicals after an interaction with cytochrome P-450s. The present experiments investigate the effects of pretreatment with various steroids on subsequent conversion of deoxycorticosterone (DOC) to aldosterone by primary cultures of bovine adrenocortical zona glomerulosa cells. The products of DOC metabolism, quantitated by high performance liquid chromatography, were aldosterone, 18-hydroxycorticosterone (18-OH B), 18-hydroxydeoxycorticos-terone (18-OH DOC), and corticosterone. Corticosterone was the major product formed (approximately 80%), whereas 18-OH B and 18-OH DOC were formed in equal amounts (approximately 10%) and aldosterone was formed in lesser amounts (1%). The maximal rate of production of aldosterone using DOC as precursor was twice the maximal rate using corticosterone as precursor. After plating of glomerulosa cells in culture, conversion of DOC to aldosterone and 18-OH corticosterone decreased with apparent first order kinetics (t½ = 10 and 17 h, respectively)over a time period in which DOC conversion to corticosterone declined only slightly (t½ = 50 h). Addition of 100 μM butylated hydroxyanisole, 50 nM H2SeO3, and 100 mM dimethyl sulfoxide to the culture medium slowed the loss of formation of aldosterone, 18-OH B, and corticosterone but did not halt it (t½ = 23, 26, and 65 h, respectively). In order of potency, pretreatment with androstenedione, cortisol, 18-OH DOC, and 11β3-hydrox yandrostenedione accelerated the loss of the subsequent conver sion of DOC to aldosterone and 18-OH B. Aldosterone production decreased at a faster rate than production of corticosterone or 18-OH B. Pretreatment with cortisol also depressed the subsequent conversion of corticosterone to aldosterone. Analysis of effective steroid structures suggested interaction with glo merulosa cytochrome P-45011β as a requirement for activity Antioxidants prevented or reduced the effects of steroids on DOC metabolism. The most potent agent wasthe OH -scavenger dimethyl sulfoxide, butylated hydroxyanisole was also effective, whereas the natural antioxidant α-tocopherol was not. The suppression of corticosterone conversion to aldosterone by cortisol makes it unlikely that the reduction in glomerulosa cytochrome P-45O11β activity (i.e. reduction in the production of the precursor corticosterone) is responsible for the reduction in DOC conversion to aldosterone. It is more likely that glomeru losa aldosterone production is reduced by the interaction of cortisol with glomerulosa cytochrome P-45O11β with the concomitant production of oxygen-derived free radicals resulting in the initiation of lipid peroxidation and formation of lipid hydrope roxides. Lipid hydroperoxides then cause loss of the terminal cytochrome P-450 (P-450CMO, corticosterone methyl oxidase) in the aldosterone biosynthetic pathway at a faster rate than that of glomerulosa cytochrome P-45011β. (Endocrinology 113: 235, 1983).
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