Optimizing acth-stimulated steroid secretion by cultured adrenocortical tumor cells

Abstract

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 mimimum 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 (A1) was compared to Hb. A1 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.