Adrenocorticotropic hormone (ACTH) 160-180 g) were anesthetized with Nembutal, and the adrenal glands were aseptically removed from surrounding fat, trimmed, bisected, and decapsulated. The tissue was then minced and collected in a sterile 50-ml capped conical polypropylene tube containing 15 ml of phosphate-buffered saline at room temperature. The phosphate-buffered saline solution was removed and replaced with 15 ml of a sterile solution of medium 199 with Earle's salts containing collagenase (4 mg/ml) and DNase (0.1 mg/ml). The tube was gassed with 95% 02-5%CO2, capped, and placed in a gyratory water bath at 370 for 1 hr. The tissue fragments were allowed to settle after this incubation. The medium was removed and replaced with 5 ml of a sterile solution of medium 199 containing no enzymes, and the tissue was dispersed mechanically by drawing it through a sterile siliconized pasteur pipet with a flamepolished tip. The fragments were allowed to settle, and the supernatant was removed and filtered through two layers of cheese cloth. Five milliliters of medium 199 were again added to the tissue fragments, and the procedure was repeated until the tissue was completely dissociated. The cells were then collected by centrifugation, and the pellet was washed twice with medium. Finally, the cells were suspended in medium 199 with 10% fetal calf serum, 100 IU/ml of penicillin, and 100 ug/ml of streptomycin.The cells were plated at a density of about 2.5 to 3.0 X 105 cells per cm2 on 10-cm2 plastic petri dishes. Staining with trypan blue showed that 80-90% of the cells were viable. The medium was removed and replaced with fresh medium after 48 hr of culture. At this time, about 30-50% of the cells had firmly attached and, thereafter, the medium was replaced every 2 days. These cultures were maintained for periods of up to 1 month.Adrenocortical cells from 17-to 20-day-old male SpragueDawley rats were also prepared in a similar manner. In addition, the capsular tissue of the adult rats (160-180 g) was also digested with collagenase and cells were prepared for culture. 113Abbreviations: ACTH, adrenocorticotropin; NPS-ACTH, o-nitrophenyl sulfenyl derivative of ACTH.

Adrenocorticotropin (ACTH) produces dramatic changes in the ultrastructural morphology of ado renocortical cells in hypophysectomized animals (for review, see reference 8). Similar changes have been observed in adrenocortical cells maintained in primary culture. found that fetal rat adrenal cells have ultrastructural characteristics typical of zona glomerulosa cells of the adult rat and that ACTH induced a transformation of the mitochondrial architecture to that of adult zona fasciculata cells. The reorganization of the tubulolamellar cristae of the mitochondria to vesicular cristae and the abundance of smooth-surfaced endoplasmic reticulum are the most widely recorded changes caused by ACTH (1,9-11, 13). Milner (13) compared the effects of ACTH and cyclic AMP (cAMP) on the ultrastructure of fetal rat adrenal cells in tissue culture and concluded that cAMP induced some, but not all, of the changes normally induced by ACTH.We have investigated the effects of ACTH on the growth, function, and morphology of normal rat adrenocortical cells in primary culture (18). In the course of these studies it was found that both ACTH and its o-nitrophenyl sulfenyl derivative (NPS-ACTH), which produces virtually no cAMP, were able to inhibit the replication of these cells with a concomitant stimulation of steroidogenesis and characteristic change in morphology. We have now examined the ultrastructural changes induced by ACTH and NPS-ACTH and compared these with the effects of dibutyryl cAMP (dbcAMP). During these studies we have also investigated the nature of the electron-opaque granules which are found in the mitochondria of adrenocortical cells and which disappear upon stimulation with ACTH. MATERIALS AND METHODSHighly purified ovine ACTH and NPS-ACTH were prepared as described (5). dbcAMP was purchased from Calbiochem (San Diego, Calif.). Sera, antibiotics, and medium 199 were purchased from Grand Island Biological Co. (Grand Island, N.Y.). CoUagenase (209 U/rag) and DNase (2,000 U/rag) were obtained from Worthington Biochemical Corp. (Freehold, N. J.). Cell CultureAdrenocortical cells were isolated from the adrenal glands of adult male Sprague-Dawley rats (350-450 g) by digestion with collagenase and DNase as previously described (18). The cells were suspended in medium 199 with 10% fetal serum, 100 IU/ml of penicillin, and 100 p.g/ml of streptomycin, and plated at a density of 4 x 105 cells/cm 2 on 10 cm ~ plastic Petri dishes. The cells were maintained at 37~ in a humidified environment of 5% COz in air. The medium was replaced every 2 days. For ultrastructural studies the cells were plated on plastic cover slips in plastic Petri dishes. Steroid production was measured by sulfuric acid fluGrescence as described (19). Electron MicroscopyPlastic cover slips bearing cultured adrenal cells were fixed for 1 h at room temperature in 3.0% glutaraldehyde (Polysciences, Inc., Warrington, Pa.) buffered with 0.1 M sodium cacodylate (pH 7.4). After several rinses in the buffer, the cells were posffixed in chilled 2.0% osmium tetroxi...

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