Isolated guinea pig adrenocortical cells in vitro Morphology and steroidogenesis in control and ACTH-treated cultures.код для вставкиСкачать
THE AMERICAN JOURNAL OF ANATOMY 165225-248 (1982) Isolated Guinea Pig Adrenocortical Cells In Vitro: Morphology and Steroidogenesis in Control and ACTH-Treated Cultures V.H. BLACK, J. MIERLAK, T. KATZ, P. MIAO, T. HUIMA, AND N. MCNAMARA Department of Cell Biology, New York University School of Medicine, New York. New York 10012 ABSTRACT Isolated guinea pig adrenocortical cells were maintained in longterm culture in order to perform sequential experiments on the same cell populations. The cells produced fluorogenic steroids, shown by thin-layer chromatography to be at least aldosterone, cortisol, and corticosterone. In addition, they increased production of these steroids when treated with either ACTH or dibutyryl cyclic AMP. Of particular interest was the fact that cultures treated for the initial 24-hour culture period with ACTH maintained enhanced levels of secretion for several days in absence of hormone and had an enhanced response to ACTH later in the culture period. Such enhancement of secretion was not seen following early treatment with dibutyryl cyclic AMP. The fine structure of the ACTH-treated cells was consistent with increased steroidogenesis.They possessed more smoothsurfaced endoplasmic reticulum, larger mitochondria1cristal surfaces, and larger Golgi complexes than the cells in untreated cultures. While many studies have been done using isolated adrenocortical cells, most have used the cells immediately after isolation; only a few have maintained the cells in vitro for longer periods (Goodyer et al., 1976; Hornsby et al., 1973, 1974; Hornsby, 1980; O'Hare and Neville, 1973a,b,c;Neville and O'Hare, 1978; Ramachandran and Suyama, 1975; Simionian et al., 1979; Suyama et al., 19771, and fewer stillhave examined the fine structure of the cells during the culture period (Hornsby et al., 1974; O'Hare and Neville, 1973a;Suyama et al., 1977). Most of the long-term adrenocortical cultures have utilized cells of the rat adrenal, whose major secretory product is corticosterone. The guinea pig is of particular interest because, unlike most laboratory animals, cortisol is one of its major secretory products. In addition to cortisol it secretes corticosterone, aldosterone, cortisone, 11 deoxycortisol (Done et al., 1952), 11P-hydroxyandrostenedione,and androstenedione (Desphande et al., 19711, as well as dehydroepiandrosterone-sulfate(Jones and Griffiths, 1968; Lipsett and Hokfelt, 1961; "rout et al., 1967). Its steroid biosynthetic pathway thus has many similarities to the human adrenal, and, like the human adrenal, it has a requirement for ascorbic acid. Guinea pig adrenocortical cells may thus provide a n accessible alternative for in vitro analysis of cell types similar to those of the human adrenal cortex. 0002-9106/82/1653-0225$06.500 1982 ALAN R. LISS, INC. In this study, viable isolated guinea pig adrenocrotical cells, with morphological features similar to the cells in vivo, were obtained in high yield. The cells were maintained in culture for 3 weeks, and their functional and fine structural features were examined in control cultures and in cultures treated with ACTH and dibutyryl CAMP.Treatment with ACTH and dibutyryl cAMP a t early time periods resulted in similar levels of enhanced steroid secretion. However, ACTH had effects on steroidogenesis which persisted for several days in absence of further hormone treatment, whereas dibutyryl cAMP did not show such prolonged effects. When treated again a t later time points, cells pretreated with ACTH during the first 24 hours of culture showed a greater response than those pretreated with dibutyryl CAMP. MATERIALS AND METHODS Cell isolation Six male English short-hair guinea pigs (Camm Research Institute) ranging in weight from 600-800 gm per animal were used for Addreen reprint requesta to Dr. Virginia H. Black, D e p b e n t of Cell Biology, New York University School of Medicine, 560 First Avenue, New York, N.Y. 10016. Received December 22, 1981. Accepted July 7,1982. 226 V.H. BLACK ET AL. each experiment. The animals were anesthetized (0.1 m1/100 gm body weight) with Diabutal(60 mg/ml; Diamond Labs, IA) and their abdomens shaved. The adrenals were excised using sterile instruments, trimmed of adherent fat, and placed on ice in cold Hank’s Balanced Salt Solution (HBSS; Gibco, NY). They were rinsed three times in HBSS and then placed into capped, preweighed, conical tubes (50 ml; Falcon Plastics) which contained about 15 ml of ice-cold HBSS. By weighing again, the net weight of the adrenals was determined. The adrenals were kept in ice-cold HBSS until dissociation. The cells were isolated using an adaptation of the procedure of Hopkins and Farquhar (1973). Several different incubation media were tried. The procedure we found most suitable is described in detail below. The basic solution used in the dissociation procedure consisted of Krebs-Ringer bicarbonate buffer (KRB). It was used in its complete formula (complete KRB); as an “ion-free”buffer (IF-KRB), prepared by omitting Mg++ and Ca+ from the formula; and as a “low ion” buffer (LI-KRB), prepared by adding 0.5 ml each of 0.01M CaC12 and 0.12M MgCl2 to 50 ml of IF-KRB. Bovine serum albumin (Sigma, fraction V) was added to these KRB solutions as follows: 3 mg/ml to complete KRB and IFKRB, 5 mg/ml to LI-KRB. The pH of all solutions was adjusted to approximately 7.4 with 5% co2/95% 0 2 , using phenol red (1drop/ 50 ml) as an indicator. Solutions of enzymes and chemicals used in dissociation were prepared in complete KRB on the day of dissociation. All solutions were filtered through 0.45pm Millipore filters (Millipore Corp., Bedford, MA) into sterile Ehrlenmeyer flasks. The adrenals were minced by hand with scapel blades in a plastic petri dish containing a small volume of complete KRB. The pieces were placed in 20 ml of complete KRB in a conical test-tube (50 ml; Falcon Plastics) and allowed to settle. The supernatant was discarded and the tissue fragments were incubated sequentially in 25 ml each of trypsin (Gibco; 1 mg/ ml) and collagenase (Sigma; 2.5 mg/ml for 15 minutes), deoxyribonuclease (DNase) (Sigma; 2pg/ml for 1 minute), soy-bean trypsin inhibitor (Sigma; lmg/ml for 5 minutes), and IFKRB (15 minutes). All incubations were carried out in an Ehrlenmeyer flask (125 ml) at 37°C in a shaker bath (Precision Scientific, IL) oscillating at 65-70 strokes per minute. Between each incubation (except for the addition of DNase) the fragments and solution were + transferred back into a 50-ml conical tube and the pieces allowed to settle in order to facilitate supernatant removal. After the last step of incubation, the mixture was placed into another 50-ml conical test tube, the supernatant discarded, and the tissue fragments washed three times with 20 ml of IFKRB. The first two rinses were discarded after settling of the tissue fragments. In the third, the tissue fragments were sheared using a series of decreasing bore sterile Pasteur pipettes, which had been prepared by cutting the tips off at various levels and flame-polishing. Whenever the cell suspension became cloudy, larger pieces were allowed to settle, and the milky cell suspension was pipetted into another conical test tube containing 10 ml of LIKRB. Fresh IF-KRB was added to the remaining pieces of tissue and pipetting repeated until the majority of the tissue fragments were dispersed. The cells were pelleted by centrifugation at 200 x g for 5 minutes in a n International centrifuge (IEC PR-2). The supernatant was removed and the cells resuspended in 10 ml of LI-KRB. To separate isolated cells from cell debris, 10 ml of a 4% BSA solution in complete KRB was gently layered under the resuspended cells and this step gradient spun at 200 x g for 5 minutes in the International centrifuge. The supernatant fluid was discarded and the cell pellet was resuspended in 10 ml Eagles’ minimum essential medium (MEM), supplemented as described below. An aliquot of the suspension was mixed with trypan blue (0.04%)and the adrenocortical cells counted in a hemocytometer to determine cell yield and percentage viability. Cell culture The isolated cells were plated as 1-ml aliquots a t concentrations of 1.25-7.0 x 105/ml in 35-mm plastic Petri dishes (Falcon Plastics). All culture media components were purchased from Gibco except as indicated: Eagles’ minimum essential medium (MEM) was supplemented with 12.67%(v/v)fetal calf serum, 29.2 mg/ml gutamine; 50 pg/ml ascorbic acid; 1% each of MEM vitamins and MEM nonessential amino acids; 2 pg/ml gentamycin (Schering, NJ); and 4 pg/ml fungizone. The pH was adjusted with sodium bicarbonate (7.5%)and aliquots were checked with a pH meter or with pHydrion paper to assure proper pH adjustment. The cells were maintained for periods of time up to 19 days either in the complete medium alone or in the complete medium supplemented GUINEA PIG ADRENOCORTICAL CELLS IN VITRO with either adrenocorticotrophic hormone (ACTH,Corticotrophin injection, Parke-Davis, 100 mU/ml) or dibutyryl CAMP, (N6, OZ1-dibutyryladenosine-3’:5‘-cyclicmonophosphoric acid, Sigma, 1 mM). In most cases the first medium change was not made until 3 days after plating, and thereafter the medium was changed every other day. The media were collected, unattached cells and debris pelleted at 200 x g for 10 minutes in the International centrifuge, and the supernatant stored in capped plastic tubes at -70°C for further assay. In some experiments the number of cells in the pellet was estimated using a hemocytometer and indicated a plating efficiency of 50-75% for cells plated a t 2.5-3.5 x 105/ml/plate.In a few experiments, 3H-pregnenolone (7-3H-pregnenolone, 1 pCi/ml, New England Nuclear, Boston, MA), purified to 88.8% by TLC, was added to the medium on days 7 or 9 in order to quantitate its incorporation into steroids. Medium controls consisted of dishes of media, without cells, incubated and changed in synchrony with the cultures. Microscopy Light micrographs of cultures in situ and of cultures fixed, as described below, were taken with a Leitz Diavert or a Wild Heerbrugg inverted phase-contrast microscope a t various time points throughout the experiments. For electron microscopy, some monolayers were fixed on day 7 of the experiment as zero-time controls, and some each of ACTH-treated and control cultures were fixed every 2-4 days thereafter. After removing the medium, the monolayers were rinsed three times in cold HBSS and fixed with 3%gluteraldehyde in 0.1 M sodium cacodylate for 30 minutes. Cultures were washed in 0.1 M sodium cacodylate for 5 minutes and then kept in the same buffer overnight. They were postfixed with 1%oso4 in 0.1 M sodium cacodylate buffer for 90 minutes, then stained with a solution of 1%uranyl acetate for 30 minutes. Monolayers were dehydrated with graded concentrations of ethanol and embedded in Epon. Silver to gold sections were cut parallel to the surface of the monolayers on an LKB-I11 ultramicrotome and examined with a Siemens Elmiskop IA electron microscope. Steroid assay To prepare for assay, 0.1 ml of sample medium was combined with 10 pl of 0.25M NaOH for 5 minutes and then extracted with 4 ml of methylene chloride (“spectranalyzed”,Fisher). 227 For qualitative assessment by thin-layer chromatography, aliquots of extracts of media from several similarly treated cultures were pooled and dried under nitrogen. The dried extracts were redissolved in methanol (Fisher) and spotted on precoated silica gel 60 TLC plates (20 x 20 cm, E. Merck). Chromatography was carried out in methylene ch1oride:methano1:water (150:9:0.5), benzene:acetone: water (75:50:0.2)or chloroform:methanol(95:5). The plates were air dried and treated with 2% HZSO4in ethanol and then heated at 100°C for 20 minutes. Fluorescent spots were visualized with UV light at 256 nm (mineralight lamp, Ultraviolet Products). Corticosteroids were identified using cochromatographed known standards on each plate. Standard solutions (cortisol, Sigma; corticosterone, Calbiochem;daldosterone, Sigma; deoxycorticosterone,Sigma; pregnenolone, Schwarz Mann; progesterone, Calbiochem) were prepared with absolute ethanol. In experiments where 3H-pregnenolone was added to facilitate identification of steroid products, media of individual culture plates were extracted and run on TLC plates in chloroform:methanol(95:5).The plates were cut into strips and counted in 4 ml ACS I1 (Amersham Co., Arlington, IL) in a Beckman Liquid Scintillation Counter. The percentage of total 3H counts extracted was determined for each strip. Steroids were identified by cochromatographed known standards. In initial experiments quantitative assessment of steroids secreted was done using the spectrophotometricassay of Cohen et al. (1957) which measures all A4-3-ketosteroids.Methylene chloride extracts were evaporated to dryness and the residue redissolved in 1ml of 95% ethanol. The ethanolic extracts were read a t 242 nm on a Zeiss spectrophotometer, and readings were expressed as pg equivalents of cortisol per ml & the standard error of the mean, using cortisol standards run with the assay. For more specific quantitative assessment of cortisol and corticosterone, the fluorometric method of Mejer and Blanchard (1973)was used. One milliliter of the methylene chloride extract was reacted with 0.5 ml of the fluorescence-inducing reagent (HzSOJethanol, 6535). Fluorescence was read 30 minutes later a t 520 nm using an excitation of 470 nm on an Aminco Bowman spectrofluorometer.Fluorescenceunits of culture media were determined from standards run with the assay and expressed as pg equivalents of cortisol per ml t the standard error of the mean. The acid-induced fluores- 228 V.H. BLACK ET AL. TABLE 1 . Yield of cells using various incubation media for cell isolation’ ~ Yield viable cell x lo5 Incubation medium lkypsin (1 mg/ml) lkypsin (1 mgiml) + 1 mM EDTA Trypsin (1 mg/ml) + neuraminidase (8 I*g/ml) lkypsin (1 mg/ml) + 1 mM EDTA + neuraminidase (8pg/ml) Trypsin (1 mg/ml) + collagenase (2.5 mg/ml) Collagenase (2.5mg/ml) ~ ZG ZF ZR 100 mg adrenal Viability as percentage of total yield 1.43 f 0.89 0.73 f 0.69 0.76 f 0.25 57.3 f 27.7 37.2 k 28.1 51.8 f 42.3 81 68 68 13 26 25 6 8 7 0.79 k 0.15 46.0 f 11.0 67 28 5 4.02 f 0.54 81.4 f 12.6 68 26 6 4.36 k 1.21 89.6 60 38 k Percentage of cell types 5.6 2 ~~ ~~ ‘Adrenocortical cells were prepared from adrenal fragments according to the general protocol outlined in Materials and Methods, with the following variations in composition of the media used for isolation: (1)Trypsin (1 mg/ml) followed by DNase, SBTI, and plain IF-KRB or IF-KRB containing 1 mM EDTA and/or neuraminidase (8p /mlJ (2)collagenase (2.5 mg/ml) followed by DNase and IF-KRB, and (3)trypsin (1 mg/ml) and collagenase (2.5m ml) kllowed by DNase, SBTI, and plain IF-KRB. The data are presented as the mean f the standard error of at least tfree determinations. cence of cortisol and corticosterone have identical maximum emission wavelengths, but cortisol has only 33%of the fluorescent capability of corticosterone(Silber et al., 1958;Carsia and Malamed, 1979). Therefore the values obtained represent their sum and the absolute levels of neither. Medium controls usually had a fluoresence similar to the assay blanks. If they were higher, the medium control, rather than the blank reading, was subtracted from that of the sample. RESULTS Cell isolation Several enzyme solutions were tested for their effectiveness in providing a good yield of viable and functionally responsive isolated guinea pig adrenocortical cells. The combination of trypsin and collagenase was the enzyme solution of choice for this study. The procedure yielded on the average 96.32 t 12.83 x lo5 single 1.07 x 105/adreadrenocortical cells (8.03 nal; 4.0 0.54 x 105/100mg adrenal) which were viable as judged by trypan blue exclusion (Tables 1,2).This represented 81%of the total yield of cortical cells. Trypsin (1mg/ml) alone gave a fair yield of cells but low viability. Inclusion of neuraminidase and/or EDTA in the IF-KRB incubation step decreased both yield and viability still further. Collagenase (2.5 mg/ ml) alone appeared to give better yields and viability; however, cells dispersed in this manner did not secrete fluorogenic steroids as well in vitro or respond as well to ACTH as those dispersed by both trypsin and collagenase. * * Morphology of isolated cells The population of intact cells obtained was assessed for the percentage of each adrenocor- TABLE 2. Recovery of viable isolated cells from adrenals treated with trypsin and collagenase’ Total ZG ZF ZR ____ Cells per adrenal ( x lo6) 120.41 30.54 46.67 43.20 Viable cells ( x lo6)in total 96.32 65.43 25.21 5.54 isolate f12.83 k3.59 kl.ll f0.86 Viable cells ( x lo5)per 8.03 5.45 2.10 0.46 adrenal in total isolate -t 1.07 10.29 fO.09 fO.07 8 viable cells recovered per 6.67 17.84 4.50 1.06 adrenal after isolation ~ _ _ _ _ The number of cells in the intact adrenal was approximated using data derived from stereological analysis as reported by Black and Russo (1980).The formula used was N = V,z. V. . V+ellsz/V, where N = cell number, V,z = volume density (percentage) of zone in adrenal, V. = volume of adrenal, V,cells’ = percentage of zone occupied by adrenocortical cells, v = volume of adrenoeorticalcell. The data for Vsellsz and t were taken from our prior paper, after checking cell size and density on Epon thick sections of adrenals from animals weighing 750-800 gm.V, was calculated by dividing the average weight of adrenals from animals of this size by the specific gravity, 1.02,previously reported for the guinea pig adrenal. Vvz of each zone was obtained by stereological analysis of adrene cortical radial strips embedded in Epon and cut into sections 1.5 pm thick. The data presented for the isoted cells are the means the standard errors of seven determinations. * tical cell type by phase-contrast light microscopy. The different cell types could be easily identified by their distinctive morphological characteristics (Fig. 1).Zona glomerulosa cells were small, with indented nuclei and a few lipid droplets. Zona fasciculata cells were larger and filled with refractile lipid droplets, which were of more uniform size and more numerous in zona fasciculata externa cells than in zona fasciculata interna cells. Zona reticularis cells were the largest cells. They possessed few lipid droplets and had a characteristic rim of clear periplieral cytoplasm.Although most of the cells occurred as single cells, zona reticularis cells were occasionally found in pairs or clusters (Fig. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO Fig. 1. The total isolate is heterogeneousin composition; but, as shown in these phase-contrast light micrographs, the adrenocortical cell types can be easily identified in the cell suspension. Zona glomerulosa (ZG) cells are of small diameter, with few refractile lipid droplets. In some cells their characteristically indented nuclei can be distinguished. Zona fasciculata (ZF) cells are filled with lipid drop- a) ZA), and some zona reticularis cells had small anucleate fragments adhering to them (Fig. 1B-D). Among the viable cells, about 68%were zona glomerulosa cells, 26% were zona fasciculata externa and interna cells, and 5.5%were zona reticularis cells (Table 1). To assess whether morphological changes occurred during cell isolation, the cells were examined by electron microscopy. Such examination revealed that the characteristic fine structural features of each cell type (Black et al., 1979) were retained during the isolation procedure (Fig. 2). Zona glomerulosa cells had a large nuclear-to-cytoplasmicratio, with deeply indented nucleus, a few lipid droplets, rodshaped mitochondria, a prominent Golgi complex, and a moderate amount of endoplasmic reticulum which was either tubular or cisternal in form and had patches of ribosomes scattered on its surface (Fig. 2A). Zona fasciculata 229 lets which are of fairly uniform size in extema cells (ZFe). Interna cells (ZFi) contain some very large droplets. Zona reticularis (ZR) cells are the largest cells and contain virtually no lipid droplets. These cells are sometimes seen in clusters of two or more (A). Occasionally smaller fragments appear to be adherent to single reticularis cells (B-D). A$, x 320; B-D, x 360. cells possessed a greater amount of cytoplasm filled with lipid droplets, more abundant smooth endoplasmic reticulum, a fairly prominant Golgi complex, and many mitochondria of larger diameter than those of glomerulosa cells (Fig. 2B,C). A single layer of fenestrated cisternal smooth reticulum could be seen around many of the lipid droplets. Zona fasciculata interna cells had larger lipid droplets and a more prominent smooth reticulum than the externa cells (Fig. ZC). Zona reticularis cells were characterized by very abundant smooth endoplasmic reticulum which filled the peripheral region of the cells (Fig. 2D). Other organelles were clustered in the juxtanuclear area. Many mitochondria of various shapes, large lysosomes, small peroxisomes, and the Golgi complex were all seen in this region. Lipid droplets were rarely seen in the zona reticularis cells. Two general changes in organelles were noted in almost all Fig. 2. The adrenocortical cells retain their characteristic fine structural features during the isolation procedure, as shown in these electron micrographs of freshly isolated cells. A, Zona glomerulosa cells possess a few lipid droplets (l), elongated mitochondria (m) with straight cristae and endoplasmic reticulum (er) which has scattered patches of ribosomes on its surface. The Golgi complex (G can be seen near the indented nucleus (N). x 5,800. B, Zona fasciculata externa cells have more abundant lipid droplets (11, more prominent tubular smooth-surfacedendoplasmic reticulum (ser), and mitochondria (m) of larger diameter. X 5,800. C, Zona fasciculata interna cells have some very large lipid droplets (11,more polymorphous mitochondria (m) with convoluted cristae, and abundant smooth-surfacedendoplasmic reticulum (ser). x 6,000. D, Zona reticularis cells have a juxtanuclear region filled with polymorphous mitochondria (m), lysosomes (ly), and the Golgi complex (G).The perimeter of the cell contains tightly packed elements of smooth reticulum (ser). x 5,900. Two general changes can be observed in all the cells: increased mitochondria1 density and vesicularization of tubular smooth reticulum. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO freshly isolated cells: increased density of mitochondrial matrices and vesicularization of some smooth endoplasmic reticulum elements. Morphology of cell cultures The cells were monitored throughout the culture period by phase-contrast light microscopy to assess cell attachment and to obtain a total view of the cell population. In settling on the culture dish, the zona glomerulosa and zona fasciculata cells usually formed aggregates of two to 20 or more cells, with varying proportions of each cell type. These cell types could be easily distinguished from one another by the small size of the former and the large amount of lipid in the latter (Figs. 3A, 4A). Zona glomerulosa cells spread out on the surface of the culture dish very quickly, zona fasciculata cells more slowly. However, zona reticularis cells adhered less well and many did not attach. When seen, they occurred adjacent to the glomerulosa-fasciculata clusters or as single cells. They could be distinguished by their larger size, large amount of relatively clear cytoplasm, paucity of lipid droplets, and juxtanuclear granular content (mitochondria,etc.) (Fig. 3A). They often spread out uniformly around their perimeter, taking on a disk shape rather than the polyhedral shape assumed by the glomerulosa and fasciculata cells (Fig. 3A, inset). Other cell types were also seen in the cultures, as illustrated in Figure 3. Fibroblasts were rare in the early days in vitro (days 1-31, but they divided rapidly, filling in the areas between cortical cells by 7-11 days (Fig. 4).This monolayer appeared almost glandular. The cultures retained this appearance for periods up to 3 weeks or until the fibroblasts formed multilayers and retracted. Retraction was either focal, producing clear areas on the dish, or total, whereupon the whole monolayer lifted off the dish. Fibroblasts filled in such clear areas; cortical cells were never found there. There was little difference in general morphology between control and ACTH-treated cultures (Fig. 4). Fine structure of cultured cells-effects of ACTH The moderate changes seen in mitochondria and endoplasmic reticulum after isolation were reversed after the cortical cells attached and spread on the culture dish, and their fine structural appearance was very similar to that of the corresponding cell types in situ (Black et al., 1979).The endoplasmic reticulum assumed its characteristic form in each cell type (Figs. 5-11). The Golgi complex was prominent in all 231 of the cells. Gap junctions, some quite long, some annular, could be seen between the cultured cells a t early time points and throughout the culture period, in both control and ACTHtreated cultures (Figs. 5,8). However, in other respects, the fine structure of cells maintained without ACTH became progressively different from that of cells treated with ACTH. Zona fasciculata cells treated with ACTH were larger in size and developed more abundant tubular smooth endoplasmic reticulum, convoluted mitochondria1 cristae, and larger Golgi complexes (Figs. 5-7). Cells maintained in medium without ACTH gradually lost their abundant tubular smooth-surfaced endoplasmic reticulum (Figs. 8, 9). The number of mitochondria and the size of the Golgi complex became reduced as the overall size of the cells became smaller in older cultures (Fig. 9). Large residual bodies were found in the cytoplasm of many cells after 11 days. The larger diameter of the adrenocortical mitochondria, paucity of rough endoplasmic reticulum, and presence of lipid droplets became the main characteristics distinguishing these cells from surrounding fibroblasts. Zona glomerulosa cells did not show such dramatic changes; however, they became somewhat difficult to distinguish from zona fasciculata cells in thin section as time in culture progressed. This was apparently due to changes in their mitochondria. A population of cells was seen, in both control and ACTHtreated cultures, which lacked the abundant lipid of fasciculata cells, yet had mitochondria of large diameter, similar to those of fasciculata cells (Figs. 10, 11).These cells did not possess abundant smooth reticulum, even in ACTH-treated cultures (Fig. lo), but in some sections had the long cisternae of endoplasmic reticulum bearing patches of ribosomes and the small peripheral lysosome-like granules characteristic of glomerulosa cells in situ (Black et al., 1979) (Fig. 11). Zona reticularis cells, as noted above, were rarely seen in long-term cultures, but cells with very polymorphous mitochondria, very abundant smooth reticulum, and few lipid droplets were occasionally encountered. Their occurrence was not frequent enough, however, to allow comparisons to be made between control and ACTH-treated cells. Steroid production-basal and ACTHstimulated Spectrophotometricassay indicatedthat there were A4-3-ketosteroids being secreted by the cultured cells, and these were shown by thin- 232 V.H. BLACK ET AL. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO layer chromatography to be predominantly corticosterone, cortisol, and aldosterone (Table 3). The fluorometric assay allowed quantitation of the fluorogenic steroids being produced over time. The overall pattern indicated that fluorogenic steroids were produced throughout the culture period with a basal level that remained fairly stable until 5-7 days of culture and then gradually declined (Fig. 12). ACTH treatment enhanced secretion by the cultures and delayed, but did not prevent, the gradual decline of fluorogenic steroid production by the cells (Fig. 12). Levels of fluorogenic steroid secreted by cells isolated with collagenase alone were one-third (on days 3-7) to one-sixth (on days 9-11, with and without ACTH) those of cells isolated with trypsin and collagenase. In experiments where both spectrophotometricand fluorogenic assays were performed, the spectrophotometric assay gave values similar to those of the fluorogenic assay up until day 9. After this point, however, the assays gave different pictures of steroid production. While the fluorogenic analysis, which measures chiefly corticosterone and cortisol, showed a steady decline of secretion between days 9 and 19, the spectrophotometric assay, which measure A43-ketosteroids, showed a fairly stable basal secretion in control cultures and a sustained, enhanced level in ACTH-treated cultrues (Fig. 12). The data obtained from duplicate or triplicate plates in any one experiment were remarkably consistent. However, there was variation between experiments in the amount of fluorogenic steroid produced, the day at which Fig. 3. Adrenocortical cells are the predominant cell types seen during the early days of culture, but some other cell types do occur. A, The cortical cells are easily recognized by characteristic features, shown in this culture of 3 days. Zona glomerulsoa (ZG) cells are of small size with few lipid droplets. Zona fasciculata (ZF) cells possess abundant refractile lipid droplets which sometimes obscure the nucleus of the cells. Interna cells (ZFi) have some large droplets, while in externa cells (ZFe) the droplets are of more uniform size. Zona reticularis (ZR) cells, when they attach, occupy larger areas of the culture dish surface, taking a polyhedral shape when among other cortical cells or spreading radially when occumng as single cells (inset). Sparse fibroblasts occur between the cortical cell clusters at this early time point. B, Another cell type, perhaps of endothelial origin, divides, forming “cobblestone”-appearingareas on the dish, shown here at 4 days. C, Vacuolatedcells (V), such as the one shown here, are occasionally seen. D,Some cells at the periphery of a cortical cell cluster assume a dendritic shape (D), as shown in this culture maintained for 4 days without ACTH. E, Neuronal-appearing cells (N),such as this, are rarely seen. F, Single cells in control and ACTH-treated cultures can also assume a dendritic form (D), as shown in this 4day control culture A X , x 320;D-F, x 380. 233 steroid production peaked, and the degree of responsivenessto ACTH. A few cultures (three out of 24 experiments) showed no response to ACTH, although their steroid production a t basal levels was similar to that of the other cultures. Conditions affecting ACTH response Variation in the time of introduction of ACTH into the cultures influenced the level of response and the length of responsiveness. Treatment with ACTH beginning a t day 7 resulted in secretion a t significantly higher than basal levels. After 4 days of exposure to ACTH, a total of 11days in culture, however, the levels of fluorogenic steroid in these ACTH-treated cultures gradually decreased. By day 17 of culture, they were only slightly higher than those in untreated cultures (Fig. 12).Treatment with ACTH a t earlier time points, on days 1, 2, or 3, resulted in higher secretory levels compared to basal levels; and these cultures maintained significantly stimulated levels for longer periods than those where treatment was begun on day 7 (Fig. 13). Analysis of other culture conditions such as pH and cell density indicated that variations in these could also influence the secretory capabilities of the cells and their level of response to ACTH. The basal levels of fluorogenic steroids found in media of cultures plated in media of pH 8.5 were two to three times lower than those plated in media of pH 7.5. However, response to ACTH, i.e., the ratio of ACTHstimulated to control levels, beginning on day 7, was greater in cultures exposed to pH 8.5 (pH 7.5, ratio = 2-3; pH 8.5, ratio = 4-5). Cultures plated more densely had higher basal levels of secretion throughout and secreted higher levels of fluorogenic steroids in response to ACTH from day 7 to 9 (Figs. 1, 14); but the responsiveness, as measured by the ratio of ACTH-stimulated to control levels, was not as great as that of lower density cultures (Fig. 15). The decrease in response to ACTH with increased cell density, particularly from day 9 to 11, was gradual across the density range used (1.25-7.0 x lo5 cells/ml) (Fig. 15). Steroid production-comparison of dibutyryl CAMP and ACTH effects To compare the effects of dibutyryl CAMP with those of ACTH and to compare the secretory capabilities of freshly isolated cells with those cultured for longer periods, cells were incubated for 2 and 22 or for 24 hours after initial plating in plain medium or medium supplemented with ACTH (100 mU/ml) or dibu- Fig. 4. The adrenocortical cells retain their characteristic features at the light-microscopic level throughout the culture period as shown here in cultures from 3 days (A), 7 days (B,),8 days ( C P )and 11 days (EJi?. Zona glomerulosa (ZG), zona fasciculata externa (ZFe), and zona fasciculata interna (ZFi) cells are easily recognized. Zona reticularis (ZR) cells, rarely seen, are not shown here. Fibroblasts (F), which are rare at 3 days, quickly fill in the areas between the cortical cells until by 7-11 days the cultures take on a glandular appearance. Little difference can be seen, in this general morphology, between those cultures treated with ACTH ( D f l and those maintained without ACTH (C,E). x 280. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO Fig. 5. Zona fasciculata cells treated with ACTH beginning on day 7 retain their abundant smooth reticulum (ser) and numerous mitochondria (m), a s shown in these cells of ll days, 4 days with ACTH. Lipid droplets (1)are abundant but of somewhat smaller size in ACTH-treated cells. In this 235 micrograph peroxisomes (p) are easily identified. Annulate lamellae (al), seen infrequently in the cells in situ, are seen here in one cell at the lower right. Gap junctions occur between cells, and may be quite long (arrows). x 13,200.Inset: a gap junction shown a t higher magnification. x 62,100. 236 V.H. BLACK ET AL. Fig. 6. The Golgi complex (G) becomes large in ACTHtreated zona fasciculata cells, such as this cell from an 11day culture, 4 days in ACTH. The abundance of smooth surfaced reticulum (ser) in ACTH-treated cells can also be appreciated in this micrograph. x 12,200. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO Fig. 7. Zona fasciculata cells maintained for up to 3 weeks in vitro, 2 of these being with ACTH in the medium, remain larger in size, and develop abundant smooth reticulum (ser), mitochondria with convoluted cristae, a large Golgi complex 237 (G),and many small lipid droplets (1). Lysosomes (ly), some quite large, occur in these cells, as well as those of control cultures at this time. X 9,500. 238 V.H. BLACK ET AL Fig. 8. Zona fasciculata cells maintained in vitro without ACTH show progressive changes at the fine-structural level. In this cell of a 7-day culture, although lipid droplets (1) remain numerous, there is a diminution in the amount of smooth-surfaced endoplasmic reticulum (ser) and mitochondrial cristae (m) have become less convoluted. A portion of the Golgi complex (G), still quite large in control cells at this fairly early time point, is seen in the upper leR. Changes in these organelles become more pronounced with increasing time in culture. Lysosomes (ly) and peroxisomes (p) can also be seen in the cytoplasm. Gap junctions ( Q ) here seen as annular junctions (arrow and inset) continue to Occur between adjacent cells. x 13,500; inset, x 62,100. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO Fig. 9. In older culture, the fine-structural differences between control and ACTH-treated cultures become greater. Zona fasciculata cells maintained in vitro without ACTH eventually become smaller, a s shown here in cells from an 18-day culture. The loss of smooth endoplasmic reticulum (ser) is more marked and the Golgi complex (GI becomes 239 reduced in size. Lipid droplets (1) remain a feature of the cells as do mitochondria (m) of fairly large diameter. The number of mitochondria, however, must decrease as the cell size becomes smaller, because they do not appear to increase in number per unit area of cytoplasm. X 11,610. 240 V.H. BLACK ET AL. Fig. 10. Zona glomerulosa cells become somewhat difficult to distinguish from zona fasciculata cells, particularly in ACTH-treated cultures such as this one of 14 days, 7 days with ACTH. Their mitochondria (m) become larger in dimeter, although they retain their characteristically straight cristae. Smooth reticulum (ser)does not become as abundant in these cells as in zona fasciculata cells, nor does the Gold complex ( G )enlarge. One distinguishing feature of the glomerulosa cells is the clusters of small lysosome-like membrane-bounded granules (gr) located at the periphery of the cells. Large lysosomes (ly) are also seen in the cells. x 12,400. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO 241 Fig. 11. Occasionally some cells are seen, such as these from a 14-day culture, which retain more of the features of zona glomerulosa cells in situ: indented nuclei (N); long cisternaeof endoplasmic reticulum (er)with scattered patches of ribosomes; elongate mitochondria (m) with straight cristae; and small, peripherally located, lysosome-like granules (gr). Large lysosomes (ly) also occur in the cells, as they do in many cells of these older cultures. x 9,500. tyryl cAMP (1mM). After the 24-hour period the medium was changed to fresh plain medium, and the cells were maintained in that medium until day 7 with changes on day 3 and 5. On day 7 they received medium containing the same supplements they had been exposed to during the first 24 hours of culture. 3H-pregnenolone was added to some of the cultures on day 7 or 9 in order to facilitate identification of the steroids produced and quantitate their ratio of production. The response to both dibutyryl cAMP and ACTH was similar within the early 2- and 24hour periods, but the cells treated with ACTH during the initial 24 hours of culture continued to secrete at higher levels than controls for the next 4 days, in the absence of further hormone (Fig. 16). Early exposure to dibutyryl cAMP had no such effect (Fig. 16). When measured 1 week later, over a 2-day period, response to dibutyryl cAMP was less than that of ACTH (Fig. 16). The effects of dibutyryl cAMP and ACTH on the ratio of steroids produced also differed in some respects (Table 3). Over days 7-9, incorporation of 3H-pregnenoloneinto cortisol and aldosterone in control cultures was approximately equal (21.7% vs. 25.1%), that into corticosterone was much less (4.4%). A considerable degree of incorportation into polar compounds (41%),which remained a t the origin, occurred. ACTH effected a large increase in incorporation into cortisol (45.6%)and a corresponding decrease in incorporation into aldosterone (13.5%). This change in ratios was much less prominent following dibutyryl CAMP treatment. In both cases, however, incorporation into polar compounds decreased by about 10%. Over days 9-11, incorporation into cortisol and aldosterone decreased in control cul- 242 V.H. BLACK ET AL. tures (7.2 and 18%), and aldosterone became the primary labeled product separated. Incorporation into polar compounds remained the same (41%).Following treatment with ACTH, but not with dibtyryl CAMP,incorporation into cortisol was enhanced twofold. Incorporation into polar compounds over this period increased, rather than decreased, following both ACTH and dibutyryl CAMP. DISCUSSION Isolation protocol Several methods have been reported in the literature for isolating adrenocortical cells. Collagenase (Kloppenborg et al., 1968) or trypsin (Swallow and Sayers, 1969) have been used to derive cell suspensions of rat adrenocortical cells. Use of trypsin has the advantages of gentle dispersion, of control over enzymatic digestion with soybean trypsin inhibitor, and of less interference by additional proteases which are known to be contaminants of many collagenase I4 preparations. However, in a detailed analysis of the effect of isolation conditions (Barofsky et al., 1973), it was found that both trypsin and collagenase were necessary to obtain a maximum yield of responsive cells. The combined enzyme treatment employed in this study followed by incubation with soybean inhibitor and then IF-KRB seems to have provided effective control of enzymatic digestion by inhibiting trypsin and diluting the collagenase and any contaminating proteases. Although neuraminidase and EDTA have been used with success in preparing anterior pituitary cells (Hopkins and Farquhar, 1973), their inclusion in our incubation medium for the adrenal resulted in poor yield and low viability. The sensitivity of adrenocortical cells to lysis by neuraminidase solutions of greater than 50 mM has been noted previously, and lower concentrations, while not resulting in lysis, were reported to result in decreased responsiveness of cells to ACTH (Haksar et al., 1973). T --E 12 ..5 .... .. . - ‘E 10- s .2 & 0- W - 53 .c b 0 6- 0 v # U .- 4- 2 al 5 23 5 7 9 Fig. 12. Steroid secretion by adrenocortical cell cultures. Cells isolated with trypsin and collagenase were plated a t 1.25-7 x lo5cells/ml, an average of4.29 ? 0.72 x lo5cells/ ml (lines), and 2 4 . 9 x lo5 cells/ml, a n average of 4.29 ? 0.72 x lo5 cells/ml (dots). They were maintained in vitro for periods up to 19 days. All cultures were kept in media lacking ACTH ( 0 ) for the first 7 days. Beginning on day 7 some of the cultures were changed to media containing ACTH (A), and were kept in such media for the remainder of the culture period. Steroid content of the media was assayed II 13 15 17 19 using either the fluorometric method (lines) or the spectrophotometric method (dots) outlined in Materials and Methods. Data points for fluorometric data represent the mean values for data from 17-20 experiments for days 1-11 and 4-10 experiments for days 13-19. Spectrophotometric assay data points represent mean values of two to four experiments for days 1-15 and 1 experiment for days 17 and 19. The bars indicate the SEM, where it is greater than the symbol. GUINEA PIG ADRENOCORTICAL CELLS IN VITRO X I .. 5 7 9 II 13 Days in Culture Fig. 13. Difference in responsiveness to ACTH treatment begun on day 3 (open bars) and day 7 (shaded bars). Cultures with 3-5 x lo5cells/ml were kept in medium lacking ACTH for 3 or 7 days. At those time points some cultures received media containing ACTH and were maintained in ACTH-containing media for the rest of the culture period, a total of 13 days. Fluorogenic steroids were measured in the media and the mean values from seven ACTH-treated cultures were divided by those from seven control cultures to obtain the response to ACTH. 243 Cell yield The yield of cells from our protocol is higher (8 x lo5 celldadrenal) than the yield from rat adrenal, reflecting the larger size of the guinea pig adrenal (guinea pig, 27.3 mg/100 gm body weight, Black and Russo, 1980; rat, 12-14 mg/ 100 gm body weight, Nussdorfer and Mazzocchi, 1972). If the yields are expressed per mg adrenalllO0 gm body weight or per 100 mg adrenal', the results are more comparable. In most other protocols, the yield of cells cannot be compared with the number of cells in the adrenal in situ. However, by comparing our yield with available stereological data from the guinea pig adrenal (Black and Russo, 1980)we calculated that the recovery in terms of total cortical cells is 7%and of each cell type is about 17.8%for zona glomerulosa, 4.5% for zona fasciculata externa and interna combined, and 1.1%for zona reticularis (Table 2). Although 'Sayers et al. (1971) used 1 6 2 0 rats weighing 300450 g m each. Thus the maximumamount of adrenal tissue used, assuming a weight of 55 mgiadrenal, would have been about 2.5 gm with a total of 160 x lo5 cells or 4 x lo5 cellsilO0 mg adrenal. We used six guinea pigs per experiment, weighing 750-850 g m each, giving about 2.4 gm of adrenal tissue and a total of about 96 x lo5 cells or 4 x lo5 cells/ 100 mg adrenal. Days in Culture Fig. 14. Difference in fluorogenic steroid production by cultures plated a t 3 4 X lo5 celldm1 (dashes) and those plated at 5-7 x lo5 cells/ml (lines). Cultures were maintained without ACTH ( 0 ) until day 7, a t which time some began to receive media containing ACTH (A).The mean -t SEM of data from seven experiments for 3-4 x lo5 cells/ml and from 5 experiments for 5-7 X lo5 celldm1 is represented. 244 V.H. BLACK ET AL. nil Fig. 15. Responsiveness to ACTH of cells plated a t increasing cell density. Cultures were plated at 1.25-7.0 X lo5cells/ml and maintained without ACTH until day 7 when some cultures began to be maintained in media containing ACTH. Fluorogenic steroids in the media were assayed, and the average value of steroids produced by duplicate cultures maintained with ACTH was divided by the values from duplicate control cultures to obtain the response to ACTH from days 7-9 (stippled bars) and days 9-11 (open bars). 0Control H D i b u t y r y l cAMP ACTH T T - 2 hr 22 hr Hormone present 24hr. 3d 5d 1-ime in Culture Fig. 16. Responsiveness to dibutyryl CAMPand ACTH during the first day of culture and after 1 week. Cells were plated a t 1.1-1.5 x lo5cellslml in complete medium without (open bars) or with the addition of dibutyryl CAMP(1mM) (cross-hatched bars) or ACTH (100 mU/ml) (stippled bars). They were kept in this medium for 24 hours. Media were collected from some culture dishes at 2 hours and 22 hours and from other culture dishes a t 24 hours. After the 24 hours they were given complete medium, lacking dibutyryl cAMP 7d - 9d Ild Hormone pcesent or ACTH, and changed a t 3, 5, and 7 days of culture. On day 7 each culture received the same type of medium it had during the first 24 hours. These media were changed a t 9 days and 11 days of culture. Fluorogenic steroids were assayed and the values, expressed as p,g cortisol equivalent/ ml, are shown in the histogram above. Each value represents the mean -C SEM of eight to 12 individual culture dishes, four plated a t 1.1 x lo5 cells/ml and four to eight plated a t 1.5 x lo5 cells/ml. 245 GUINEA PIG ADRENOCORTICAL CELLS IN VITRO TABLE 3. Percentage incorporation of 3H-pregnenolone into secreted steroids1 Percent conversion of 3H-pregnenolone (% of extracted 3H) Control (without hormone) (100mU/ml) ACTH Dibutyryl cAMP (1mM) Control (without cells) 21.7 25.1 4.4 0.5 0.3 0.4 41.1 45.6 13.5 5.2 0.6 0.3 0.3 28.0 27.1 21.0 9.6 1.5 0.3 0.9 30.3 1.5 3.5 1.4 1.0 1.3 77.7 2.2 7.2 18.1 7.0 0.9 0.7 1.7 40.9 15.9 17.1 3.3 1.0 1.0 0.2 53.1 7.5 23.2 4.8 0.8 0.8 1.1 47.3 7-9 days Cortisol Aldosterone Corticosterone 11 deoxycorticosterone Progesterone Pregnenolone Polar compounds 9-11 days Cortisol Aldosterone Corticosterone 11 deoxycorticosterone Progesterone Pregnenolone Polar compounds ~ 3H-pregnenolone(1 Ci, 7-3H pregnenolone, New England Nuclear) was added to the medium of some culture dishes on days 7 and 9 of culture. Some of the dishes’ media contained ACTH (100mU/ml) or dibutyryl CAMP(1 mM). Cultures treated on day 7 were plated at 1.1 x lo5 cells/ml. Those treated on day 9 were plated at 1.5 X lo5 cells/ml. Medium was collected 48 hours later, extracted, and run on TLC plates in ch1oroform:methanol(955)as described in Materials and Methods. The lates were cut into strips and counted in 4 ml ACS 11. The percentage of total 3H counts extracted with the medium was &ermined for each strip, and the corresponding steroids were identified by cochromatographed standards. Polar compounds were not separated on this system. Each value represents the average of determinations from duplicate disappointingly low at first glance, this level of recovery is similar on a per-gram basis to that obtained with large organs such as t4e liver (Howard et al., 1973; LaBrecque and Howard, 1976), and as noted above, is comparable with other protocols for the rat adrenal. The yield of each cell type was, however, not in proportion to its occurrence in the gland (Table 2). This may be due to the presence of larger gap junctions between the deeper cortical cells (Black et al., 1979), which were not uncoupled during isolation. Mechanical shearing of such connected cells would result in damage to one of the two adjacent cells. In cells having the largest gap junctions, this damage may be so severe that reannealing of the plasma membrane cannot take place. In the total isolate, whole zona reticularis cells were often seen closely apposed to one another, perhaps still attached by junctions. Single zona reticularis cells often had small fragments adherent to them, probably representing remnants of an adjacent cell. Similar observations were made by Amsterdam and Jamieson (1974)during the isolation of pancreatic acinar cells. that these cells were morphologically similar to their in situ counterparts. As in rat adrenocortical cell cultures (O’Hare and Neville, 1973a), guinea pig zona fasciculata cells retained their lipid-filled cortical cell morphology throughout the culture period, even in the absence of ACTH, and even after fibroblast overgrowth. This is in contrast to the changes which have been reported by several individuals in general morphology of adrenocortical cell cultures in the absence of ACTH and their reversal following ACTH treatment. Some human adrenal cells also lose their corticallike appearance in the absence of ACTH, but retain it in the presence of ACTH (Neville and OHare, 1978).Slavinsky-Turley and Auersberg (1978) reported the isolation from rat adrenal of fibroblastic and epithelial cells; both responded to ACTH with increased steroid production and a more rounded shape. Retraction of cortical cells following ACTH treatment has been reported by many individuals (Goodyeret al., 1976;Hornsby et al., 1974; O’Hare and Neville, 1973a; Neville and OHare, 1978;Ramachandran and Suyama, 1975; Simionian et al., 1979; Suyama et al., 1977). However, in our cultures the line of separation beGeneral morphology of isolated cells and tween clusters of cortical cells and fibroblasts cultures was not increased by ACTH or dibutyryl cyclic Microscopic analysis of the population of cells AMP, and no retraction of the cells within the obtained with trypsin and collagenase showed cluster to the dendritic form reported by some 246 V.H. BLACK ET AL. investigators occurred under the culture conditions used. Such retraction could be induced only if the cells were treated with ACTH or dibutyryl cyclic AMP in the absence of serum (Black, unpublished observation). In both control and ACTH-treated cultures, single cortical cells were occasionally seen to take on a very ruffled, almost dendritic appearance. The ability to acquire dendritic form, or a lack thereof, may therefore be a reflection of the absence or presence of junctional contacts between adjacent cortical cells. In our cultures, gap junctions could be seen between both control and ACTH-treated cells. Fine structure of cultured cells-effectsof ACTH Although few changes in the morphology of the cultures were visible with the light microscope, cortical-cell fine structure did vary with the presence or absence of ACTH. The appearance of zona fasciculata cells maintained in the absence of ACTH eventually resembled that of the relatively undifferentiated cells of early fetal life (Black, 1972). There was little endoplasmic reticulum, rough or smooth; the Golgi complex was reduced in size; and the mitochondria, while retaining a large diameter, showed a decrease in inner membrane surface, reflected in their less convoluted cristae. Abundant lipid droplets remained in the cells. With ACTH treatment, many of the cells showed increases in smooth endoplasmic reticulum, mitochondrial cristae and the Golgi complex. Similar changes occur in these cells in vivo &r ACTH treatment (Black and Russo, 1980). Zona glomerulosa cells underwent less dramatic changes. Their mitochondria became larger in diameter, but changes in smooth reticulum and Golgi were not as great. The morphological response to ACTH by the guinea pig adrenocortical cells at the fine structural level agrees in general with that described by others for long-term cultures of adult adrenocortical cells (Armato and Nussdorfer, 1972; Hornsby et al., 1974; O’Hare and Neville, 1973a; Slavinsky-Turley and Auersberg, 1978; Suyama et al., 1977). All have observed an increase in smooth reticulum and Golgi complex coincident with enhanced secretion. In some cases, mitochondria were reported to increase in size (OHare and Neville, 1973a),and their cristae to increase in surface area (Armato and Nussdorfer, 1972)and change in form (Armato and Nussdorfer, 1972;Armato et al., 1978; Hornsby et al., 1974; O’Hare and Neville, 1973a; Suyama et al., 1977). Relationship of fine structure to steroid production-A CTH The morphological appearance of the guinea pig cortical cells did not, however, entirely coincide with the functional capacity of the cells. While the cells could be maintained in vitro for up to 3 weeks, their production of fluorogenic steroids (cortisol/corticosterone) peaked at day 5 and then gradually declined, in the absence of ACTH. This was consistent with the decrease in smooth endoplasmic reticulum, mitochondria, and Golgi complexes. ACTH caused increased secretion by the cells a t the time of administration, and in its continued presence higher levels of secretion were maintained for 4-6 days. This was consistent with the retention of abundant smooth reticulum, convoluted mitochondrial cristae and large Golgi complexes. However, after this period, fluorogenic steroid production declined to a level barely distinguishable from that in controls cultured for the same period, despite the fact that the cells retained their characteristic fine structural features. One explanation for this may be that the cells in the presence of ACTH, continued to produce steroids which were not fluorogenic. In support of this was our observation that spectrophotometricassays showed that A43-ketosteroids continued to be secreted through day 19 of culture and that their production was enhanced by ACTH. This may reflect continued production of polar compounds, into which pregnenolone incorporation was enhanced following ACTH beginning on day 9. Effects of culture conditions on ACTHstimulated steroid production Basal secretion by guinea pig adrenocortical cells maintained in MEM was significant: onehalf to one-eighth that of the maximum stimulated level achieved with 100 mU/ml ACTH, with the precise level depending on the individual culture conditions, including cell density, as discussed below. This is much greater than the baseline secretion reported for rat cortical cells (1/1OOth the maximum level achieved with 100 mU ACTH/ml) (Hornsby et al., 1973; OHare and Neville, 1973c)but is more similar to that of human cortical cells (6.7-10% the maximum level achieved with 100 mU ACTW ml) (Neville and O’Hare, 1978). More densely plated cultures showed higher basal and ACTH-stimulated output of steroids than less densely plated cultures and longer maintenance of fluorogenic steroid production. However, the response to ACTH (ACTHmasal) was less. The higher basal levels of secretion GUINEA PIG ADRENOCORTICAL CELLS IN VITRO could have been due to increased cell contact, as suggested by Neville and O’Hare (1978) for human cortical cell cultures, or to a protective effect of lowered POz in cultures of higher density, as suggested by Hornby (1980). The decreased responsiveness to ACTH could have been due to relatively fewer available nutrients or other factors per cortical cell, especially in the face of increasing numbers of fibroblasts. In addition, there may have been faster breakdown of ACTH by the larger number of cells. ACTH has been shown to be rapidly degraded in vitro by both plasma (Purvis and Sirett, 1968) and adrenocortical cells (Voigt et al., 1974). Its degradation is enhanced by the presence of both (Voigt et al., 1974), is more rapid with denser cell populations, and may be due to proteases released into the medium from damaged (dying) cells (Bennett et al., 1974). Thus it is possible that even though we used maximal dosages of ACTH, significant degradation occurred in our cultures over 2 days, particularly at the high cell densities. Another possibility is that the higher basal levels partially suppressed the ACTH response. Kowal and Fiedler (1968),in their studies of Y-1 cells, felt that the eventual decline in response to ACTH which they observed in small culture volumes was due to feedback inhibition by steroids secreted in the medium. Carsia and Malamed (1979) recently tested this hypothesis and found that addition of exogenous steroids at levels similar to those achieved by the cells in vitro can suppress secretion by rat adrenocortical cells. Comparison of effects of ACTH and CAMP Levels of steroid equivalent to those achieved with ACTH were produced upon exposure to dibutyryl CAMP during the first 2-24 hours of culture. However, results with these two agents differed in the long term. Initial exposure of cultures to ACTH enhanced steroid production in absence of ACTH for a t least the next 4 days, but no such effect was observed with dibutyryl CAMP. In addition, cultures initially exposed to ACTH had a greater response to ACTH later in the culture period, in terms of both the levels of steroid produced and the ratio of cortisol to aldosterone, than did cultures reexposed to dibutyryl CAMP.This is the first time that such a discrepancy has been seen. It may indicate that the action of ACTH involves intermediaries other than CAMP.It also points out one advantage of long-term cultures where cells can be repeatedly analyzed on successive days. 247 ACKNOWLEDGMENTS The authors wish to thank Dr. Kumiko Martin for her help with thin-layer chromatography and Dr. Mortimer Levitz for helpful discussions. A preliminary report of a portion of this work was included in a presentation before the American Society for Cell Biology (Miao, P., and V.H. Black, J. Cell Biol., 79:251a, 1978). This research was supported by NIH research grant HD-04005. LITERATURE CITED Amsterdam, A,, and J.D. Jamieson 1974 Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristic of separated cells. J. Cell Biol., 63t1037-1056. Armato, U., and G.G. Nussdorfer 1972 Tissue culture of rat adult decapsulated adrenal glands. Z. Zellforsch., 135:245-273. Armato, U., G.G. Nussdorfer, G. Neri, E. Draghi, P.G. Andreis, G. Mazzocchi, and F. Mantero 1978 Effects of ACTH and 3’, 5’-cyclic purine nucleotides on the morphology and metabolism of normal adult human adrenocortical cells in primary tissue cluture. Cell Tissue Res., 190t187-205. Barofsky, A.-L., M. Feinstein, and I.D.K. Halkerston 1973 Enzymatic and mechanical requirements for dissociation of cortical cells from rat adrenal glands. Exp. Cell Res., 79t263-274. Bennett, H.P.J., G. Bullock, P.J. Lowry, C. McMartin, and J. Peters 1974 Fate of corticotrophin in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification. Biochem. J., 138t185-194. Black, V.H. 1972 The development of smooth-surfaced endoplasmic reticulum in adrenal cortical cells of fetal guinea pigs. Am. J. Anat., 135:381418. Black, V.H., E. Robbins, N. McNamara, and T. Huima 1979 A correlated thin-section and freeze fracture analysis of guinea pig adrenocortical cells. Am. J. Anat., 156t453-504. Black, V.H., and JJ. Russo 1980 Stereological analysis of the guinea pig adrenal: Effects of dexamethasone and ACTH treatment, with emphasis on the inner cortex. Am. J. Anat., 159t85-120. Carsia, R.V., and S. Malamed 1979 Acute self-suppression of corticosteroidogenesis in isolated adrenocortical cells. Endrocrinology, 105r911-914. Cohen, A.I., E. Bloch, and E. Celozzi 1957 In uitro response of functional experimental adrenal tumors to corticotropin (ACTH). Proc. Soc. Exp. Biol. Med., 95t304309. Deshpande, N., P. Carson, and S. Harley 1971 Adrenal biogenesis zn uiuo of androgens and cortisol in the guinea pig using a continuous infusion technique. J. Endocrinol., 50t467484. Done, A.K., R.S. Ely, R.B. Raile, and V.C. Kelley 1952 Species differences in circulating 17-hydroxycorticosteroid concentrations. Proc. Soc. Exp. Biol. Med., 81t667-669. Goodyer, C.G., J.S. Torday, B.T. Smith, and C.J.P. Giroud 1976 Preliminary observations of bovine adrenal fasciculata-reticularis cells in monolayer culture: Steroidogenesis, effect of ACTH and CAMP. Acta Endocrinol., 83:373385. Haksar, A., S. Baniukiewicz, and F.G. Peron 1973 Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase. Biochem. Biophys. Res. Commun., 52:959-966. Hopkins, C.R., and M.G. Farquhar 1973 Hormone secretion by cells dissociated from rat anterior pituitaries. J. Cell Biol., 59t27G303. Hornsby, P.J. 1980 Regulation of cytochrome P-450-sup- 248 V.H. BLACK ET AL. ported llp-hydroxylation of deoxycortiaol by steroids, oxygen and antioxidents in adrenocortical cell cultures. J. Biol. Chem., 255t4020-4027. Hornsby, P.J., M.J. O’Hare, and A.M. Neville 1973 The effect of ACTH on biosynthesis of aldosterone and corticosterone by monolayer cultures of rat adrenal zona glomerulosa cells. Biochem. Biophys. Res. Commun., 54rl 1561559. Hornsby, P.J., M.J. O’Hare, and A.M. Neville 1974 Functional and morphological observations on r a t adrenal zona glomerulosa cells in monolayer cultures. Endocrinology, 95,1240-1251. Howard, R.B., J.C. Lee, and L.A. Pesch 1973 The fine structure, potassium content and respiratory activity of isolated rat liver parenchymal cells prepared by improved enzymatic techniques. J. Cell Biol., 57t642-658. Jones, T., and K. Griffiths 1968 Ultramicrochemical studies on the site of formation of dehydroepiandrosterone sulphate in the adrenal cortex of the guinea pig. J. Endocrinol., 42559-565. Kloppenborg, P.W.C., D.P. Island, G.W. Liddle, A.M. Mikelakis, and W.E. Nicholson 1968 A method of preparing adrenal cell suspensions and its applicability to the in uitro study of adrenal metabolism. Endocrinology, 82r1053-1058. Kowal, J., and R. Fiedler 1968 Adrenal cells in tissue culture. I. Assay of steroid products; steroidogenic responses to peptide hormones. Arch. Biochem. Biophys., 128r406-42 1. LaBreque, D.R., and R.B. Howard 1976The preparation and characterization of intact isolated parenchymal cells from rat liver. In: Methods of Cell Biology. D.M. Prescott, ed. Academic Press, New York, vol. 1.4, pp. 327339. Lipsett, M., and B. Hokfelt 1961 Conversion of 17a-hydroxypregnenolone to cortisol. Experientia, 17r449450. Mejer, L.E., and R.C. Blanchard 1973 Fluorometric determination of plasma 11-hydroxycorticosteroids.I. Rapid procedure for clinical screening. Clin. Chem., 19t710-717. Miao, P., T. Huima, and V.H. Black 1978 Characterization of guinea pig adrenocortical cells separated by equilibrium density centrifugation. J. Cell Biol., 79r251a. Neville, A.M., and M.J. O’Hare 1978 Cell culture and histopathology of the human adrenal cortex in relation to hypercorticalism. In: Adrenal Cortex. V.H.T. James, ed. Academic Press, New York, pp, 229-249. Nussdorfer, G.G., and G. Mazzocchi 1972 A stereologic study of the effects of ACTH and cyclic 3‘ 5’-AMP on adrenocortical cells of intact and hypophysectomized rats. Lab Invest., 26,4542. O’Hare, M.J., and A.M. Neville 1973a Morphological re- sponses to corticotrophin and CAMPby adult rat adrenocortical cells in monolayer cultures. J. Endocrinol., 56r529-536. O’Hare, M.J., and A.M. Neville 1973b Steroid metabolism by adult adrenocortical cells in monolayer culture. J. Endocrinol., 58r447462. O’Hare, M.J., and A.M. Neville 1973c The steroidogenic response of adult adrenocortical cells in monolayer culture. J. Endocrinol., 56r537-549. Purvis, H.D., and N.E. Sirett 1968 The stability of endogenous and exogenous corticotrophin in rat plasma as established by a bioassay in intact rats. J. Endocrinol., 41r491497. Ramachandran, J., and A.T. Suyama 1975 Inhibition ofreplication of normal adrenocortical cells in culture by adrenocorticotmpin. Proc. Natl. Acad. Sci., USA 72:11%117. Sayers, G., R. Portanova, R.J. Beall, S. Seelig, and S. Malamed 1971 Techniques for the isolation of cells of the adrenal cortex, the anterior pituitary and the corpus luteum: Morphological and functional evaluation of the isolated cells. In: In Vitro Methods in Reproductive Cell Biology. E.D. Diczfalusy, ed. Bogtrykkeriet Forum, Cophenhagen, pp. 11-22. Silber, R.H., R.D. Busch, and R. Oslapas 1958 Practical procedure for estimation of corticosterone or hydrocortisone. Clin. Chem., 4;27%285. Simionian, M.H., P.J. Hornsby, C.R. Ill, M.J. O’Hare, and G.N. Gill 1979 Characterization of cultured bovine adrenocortical cells and derived clonal lines: Regulation of steroidogenesis and culture life span. Endocrinology, 105r99-108. Slavinsky-Turley, E.A., and N. Auersberg 1978 Cultured adrenocortical cells in various states of differentiation: Electron microscopic characterization and ultrastructural response to adrenocorticotrophin. J. Endocrinol., 78:427-434. Suyama, A.T., J.A. Long, and J. Ramachandran 1977 U1trastructural changes induced by ACTH in normal adrenocortical cells in culture. J. Cell Biol., 72r757-763. Swallow, R.L., and G. Sayers 1969 A technic for the prepExp. Biol. aration of isolated rat adrenal cells. Proc. SOC. Med., 131rl-4. Trout, E.C., K.Y.T. Kao, and T.H. McGovak 1967 Conversion of 17 a-hydroxypregnenolone to dehydroepiandrosterone by adrenal homogenates of immature and adult guinea pigs. Steroids, 9r57-70. Voigt, K.H., H.L. Fehm, R. Lang, and E.F. Pfeiffer 1974 Degradation of ACTH by isolated adrenal cells. Influence of plasma. Acta Endocrinol. ISuppl.1 (Copenh.) 18:165.