Metyrapone and antioxidants are required to maintain aldosterone synthesis by cultured bovine adrenocortical zona glomerulosa cells

Bovine adrenal zona glomerulosa cell cultures were prepared by microdissection and collagenase digestion, followed by unit gravity sedimentation, to give populations of glomerulosa cells which were more than 95% pure. Glomerulosa cell-specific function was assayed by measuring the formation of [³H]aldosterone from saturating concentrations of precursor [³H] deoxycorticosterone and by measuring aldosterone production with a specific RIA. The purity of glomerulosa cell cultures was documented by demonstrating low levels of steroid 17α-hydroxylase activity, which is specific to the zona fasciculata-reticularis. [³H]Aldosterone production, expressed as a percentage of total ³H-labeled steroid products of [³H]deoxycorticosterone metabolism, decreased to 50% of the initial value within 12 h when cells were placed in primary culture. The following factors prolonged relative [³H]aldosterone synthesis: increasing cell density (<2 × 104 cells/cm2), lowering the oxygen concentration from 19% to 2% and including the radioprotectant dimethyl sulfoxide (100 mM) in the culture medium. Metyrapone (2-methyl-l, 2-di- 3-pyridyl-l-propanone) was the most effective agent identified, maintaining the relative aldosterone synthesis constant at initial levels for 3 days. In the presence of metyrapone, butylated hydroxyanisole (2- and 3-tert-butyl-4-methoxyphenol; 100 μM), H₂SeO₃ (50 nM), and dimethyl sulfoxide, K⁺ (21 mM) increased relative aldosterone synthesis to 150% of the initial value. There was an absolute requirement for metyrapone and antioxidants for the K⁺-mediated increase in relative aldosterone synthesis. Whereas in untreated cultures, the ability to respond to angiotensin II and ACTH was lost, in metyrapone-pretreated cultures, angiotensin II and ACTH acutely increased aldosterone production as measured by specific RIA (15- and 7-fold, respectively). Under defined conditions, maximal angiotensin II-stimulated aldosterone production per cell remained constant through more than three population doublings. Prolonged treatment with ACTH or angiotensin II decreased relative aldosterone synthesis, suggesting that conditions were not sufficient to protect against the loss of activity of aldosterone-synthesizing enzymes due to elevated steroidogenesis. High antioxidant conditions coupled with rapid dissipation of steroid products in vivo appear to be essential for the maintenance of differentiated function of the zona glomerulosa.