The role of ascorbic acid in the function of the adrenal cortex: Studies in adrenocortical cells in culture

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.