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Isolated guinea pig adrenocortical cells in vitro Morphology and steroidogenesis in control and ACTH-treated cultures.

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Isolated Guinea Pig Adrenocortical Cells In Vitro:
Morphology and Steroidogenesis in Control and
ACTH-Treated Cultures
Department of Cell Biology, New York University School of Medicine, New
York. New York 10012
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
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.
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
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
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.
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
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).
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
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-
TABLE 1 . Yield of cells using various incubation media for cell isolation’
viable cell x lo5
Incubation medium
lkypsin (1 mg/ml)
lkypsin (1 mgiml) + 1 mM EDTA
Trypsin (1 mg/ml) + neuraminidase (8
lkypsin (1 mg/ml) + 1 mM EDTA +
neuraminidase (8pg/ml)
Trypsin (1 mg/ml) + collagenase (2.5
Collagenase (2.5mg/ml)
100 mg adrenal
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
0.79 k 0.15
46.0 f 11.0
4.02 f 0.54
81.4 f 12.6
4.36 k 1.21
Percentage of cell types
‘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.
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’
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
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
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.
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-
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
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.
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.
Fine structure of cultured cells-effects of
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
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-
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.
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.
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
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
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
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.
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.
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
(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.
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.
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
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.
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.
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-
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.
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
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.,
..5 ....
‘E 10-
& 0-
5 23
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
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
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.
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.
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).
H D i b u t y r y l cAMP
2 hr
22 hr
Hormone present
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
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.
TABLE 3. Percentage incorporation of 3H-pregnenolone into secreted steroids1
Percent conversion of 3H-pregnenolone
(% of extracted 3H)
(without hormone)
Dibutyryl cAMP
(without cells)
7-9 days
11 deoxycorticosterone
Polar compounds
9-11 days
11 deoxycorticosterone
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
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
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
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
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.
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.
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guinea, morphology, isolated, pig, culture, acth, adrenocortical, steroidogenesis, vitro, treated, control, cells
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