Optimizing ACTH-stimulated steroid secretion by cultured adrenocortical tumor cells

If 1 mM supplemental Ca++, or 1.6 mg hemoglobin (Hb)/ml medium (23.5 uM, a concentration comparable to that in 1 ml mouse blood), was added to Eagle's minimum essential medium (MEM) (which contains 1 mM Ca++), basal and maximally stimulated 20-dihydroprogesterone (20-DHP) secretion by cultured Y-1 mouse adrenal tumor cells [49-65th passages] could be measured by radioimmunoassay after a 0.5 hr incubation. ACTH-stimulated, but not basal, 20-DHP secretion increased after 1 mM Ca++ treatment. 5 mM EGTA significantly reduced basal and stimulated 20-DHP secretion, although significant ACTH stimulation still remained. Basal and stimulated secretion significantly increased when Hb was present. Although O2 involvement in the Hb effect was tested by bubbling medium with 100% O2 for 10 minutes, basal and stimulated secretion was unaffected. Since proteins, such as Hb, non-specifically bind free steroids, enhancing secretion, albumin (Al) was compared to Hb. Al enhanced unstimulated, but not ACTH-stimulated, 20-DHP secretion. The Hb effect may not be due to non-specific protein-steroid binding. Ca++ supplementation and chelation studies suggest the necessity to optimize co-factors in steroidogenic tissue incubation media to maximize basal and stimulated steroid synthesis and secretion. Since physiologically relevant Hb levels enhanced basal and stimulated Y-1 cell steroid secretion by mechanisms other than protein-steroid binding and soluble O2 had little effect, Hb may more efficiently transport O2 to cultured cells than soluble O2 diffusion through medium does.