To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11β-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11β-hydroxylase activity declined with a half-time of about 40 h. When 50 pM cortisol, which acts as a pseudosubstrate for 11β-hydroxylase, was added to such cultures, 11β-hydroxylase activity declined with a halftime of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11β-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11β-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 μM to 5 mM did not inhibit 11β-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11β-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11β-hydroxylase activity in the presence of 50 μM cortisol. 11β-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 fiM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11β-hydroxylase is specific; neither α-tocopherol nor selenium prevented loss of 11β-hydroxylase in the presence of cortisol or enhanced reinduction of 11β-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or α-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome P-450s.
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