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 t o Eagle's mimimum essential medium (MEM) (which contains 1 mM Ca++), b asal and maximally stimulated 20-dihydroprogesterone (20-DHP) secretio n by cultured Y-1 mouse adrenal tumor cells [49 - 65th passages] could be measured by radioimmunoassay after a 0.5 hr incubation. ACTH-stimu lated, but not basal, 20-DHP secretion increased after 1 mM Ca++ treat ment. 5 mM EGTA significantly reduced basal and stimulated 20-DHP secr etion, although significant ACTH stimulation still remained. Basal and stimulated secretion significantly increased when Hb was present. Alt hough O-2 involvement in the Hb effect was tested by bubbling medium w ith 100% O-2 for 10 minutes, basal and stimulated secretion was unaffe cted. Since proteins, such as Hb, non-specifically bind free steroids, enhancing secretion, albumin (Al) was compared to Hb. Al enhanced uns timulated, but not ACTH-stimulated, 20-DHP secretion. The Hb effect ma y not be due to non-specific protein-steroid binding. Ca++ supplementa tion and chelation studies suggest the necessity to optimize co-factor s in steroidogenic tissue incubation media to maximize basal and stimu lated 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 O-2 had litt le effect, Hb may more efficiently transport O-2 to cultured cells tha n soluble O-2 diffusion through medium does.