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Department of Internal Medicine III, University of Leipzig (A.H., S.R.B., A.G., K.U., M.E.-B.), 04103 Leipzig; and Research Institute of Molecular Pharmacology (C.L.), 10315 Berlin, Germany; and the National Institute of Child Health and Human Development, National Institutes of Health (S.R.B.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: A. Haidan, Medizinische Klinik und Poliklinik III der Universität Leipzig, Philipp Rosenthal Strasse 27, 04103 Leipzig, Germany.
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Abstract |
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Cortisol release from cortical cells in coculture with chromaffin cells
was 10 times as high (mean ± SEM, 1035 ± 119%)
as that from the same number of isolated cortical cells (100 ±
11%). By a [3H]thymidine incorporation assay, it was
demonstrated that this effect was not due to a higher proliferation
rate. Northern analysis revealed an increasing expression of
P45017
mRNA in the coculture from days 1–5, whereas in
isolated cortical cells, P45017 mRNA decreased, leading
to a 6-fold difference on day 5. Inhibitors of protein (cycloheximide)
or RNA (actinomycin D) synthesis completely annulled the observed
increase in cortisol release, indicating that de novo
protein synthesis is required for this activation of adrenocortical
steroidogenesis. Addition of the cyclooxygenase inhibitor indomethacin
reduced the stimulatory effect, suggesting that this stimulation is in
part mediated by PGs. Locally produced ACTH, catecholamines, and
interleukin-1 accounted for 43% of the effect. Secretory products of
chromaffin cells that act in concert are believed to be responsible for
the stimulation of steroidogenesis in the coculture.
The coculture system is an in vitro model that corresponds to the in vivo situation in the intact adrenal gland, where both endocrine cell systems are in close contact. Our data demonstrate the requirement of intraadrenal cellular communication for the full strength of the adrenocortical hormonal response.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been known for many years from hypophysectomized patients (4, 5) and from animal models (6, 7, 8, 9) that adrenocortical function can be stimulated by several secretagogues, even in the absence of ACTH. Intraadrenal interactions could be responsible for this remaining adrenocortical activity.
Despite the knowledge of the complex intraadrenal regulatory mechanisms, the in vitro functions of chromaffin and cortical cells were studied in isolated primary cultures. However, results obtained from those cultures do not accurately reflect the situation in vivo. In addition, the investigation of effects of adrenomedullary secretory products on adrenocortical function has to take into account that chromaffin cells secrete a cocktail of transmitters and neuropeptides that may interact in the regulation of adrenocortical function. Therefore, the isolated investigation of adrenocortical cells and the isolated investigation of single adrenomedullary secretory products on adrenocortical function are certainly unreliable.
In the last few years the importance of coculturing cells that occur together in the intact organ or intact tissue was shown in different fields of research. For instance, embryo coculture systems are used to enhance embryo development (10, 11, 12). Various types of helper cells improve the rate of development, reduce the cell fragmentation rate, and, in some instances, increase pregnancy and implantation rates (10). The rapid loss of many cytochromes P450 (CYP) from hepatocytes grown in culture, comparable to the loss of CYP11B, CYP21, CYP17, and CYP11A from bovine adrenocortical cells in culture (13), can be prevented by adding liver epithelial cells (14). Another impressive example for cell to cell communication in vitro is that of isolated rat pituitary cells, which aggregate during gyratory shaking and become organized in a tissue-like configuration within a few days. Ultrastructural and functional investigations indicated that these reaggregates are viable and functional multicellular structures that have maintained in vivo characteristics (15).
Surprisingly, the effect of coculturing cortical and chromaffin cells
on the regulation of steroidogenesis in mammals has never been
analyzed. The objective of the present study was to establish a
coculture system of bovine adrenocortical and chromaffin cells that
comes close to the in vivo situation in the intact adrenal
gland. The regulation of basal steroidogenic activity was investigated
for the first time by comparing cortisol secretion in cocultures of
chromaffin and cortical cell with the steroidogenic activity in
isolated cortical cells. The effect was further characterized by
measuring P45017 messenger RNA (mRNA) expression and by
inhibiting transcription and ribosomal protein synthesis. The possible
involvement of catecholamines, medullary ACTH, and IL-1 was
investigated.
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Materials and Methods |
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Cell preparation and culture
Bovine adrenal glands were obtained from freshly slaughtered 1-
to 3-yr-old steers, trimmed free of adipose tissue, and transported to
the laboratory in ice-cold PBS. The adrenals were put into 70% ethanol
for 10 s, and connective tissue was removed.
For the preparation of cortical cells, the glands were cut in half by longitudinal incision. The medulla was removed and the cortex was scraped off the capsule, cut into small pieces and washed three times for 15 min each time in washing medium [DMEM-Ham’s F-12 (Life Technologies, Eggenstein, Germany) containing 200 U/ml penicillin, 200 µg/ml streptomycin, and 50 µg/ml gentamicin]. Digestion was performed in washing medium with 2.5% trypsin (Life Technologies) at 37 C with shaking. The digestion medium was replaced every 20 min. The cells were pelleted by centrifugation and filtered through gauze.
Medullary cells were prepared by a modification of the method developed by Livett and co-workers (16, 17). For washing, the intact adrenal was infused several times through the vein with PBS using a syringe. For digestion, performed at 37 C, PBS containing 0.3% collagenase from Clostridium histolyticum (Serva, Heidelberg, Germany) and 0.01% deoxyribonuclease I was infused through the vein every 15 min. After a total time of 1 h, the adrenals were cut in half, and the digested medullary cells could be easily separated from the undigested cortex. Traces of adhering cortical tissue were carefully cut away. The medullary cells were filtered through gauze and washed six times. After this cell preparation, chromaffin cells were purified by differential plating, a method that exploits the different adhesiveness of chromaffin and nonchromaffin cells (18). Briefly, cells were suspended in culture medium (DMEM-Ham’s F-12 containing 10% FCS; Life Technologies) and plated on glass petri dishes. After 4 h, floating cells were transferred to plastic flasks. Floating cells were transferred to new plastic flasks every 90 min, three times. The cells that were not sessile 90 min after the third plating step were the purified chromaffin cells.
Erythrocytes were removed from cortical and medullary cell preparations by treatment with erythrocyte lysis buffer (0.15 M NH4Cl, 0.1 mM Na2EDTA, and 12 mM NaHCO3) for 2 min at 37 C. Lysis was stopped by adding ice-cold PBS and subsequently centrifuging the preparations.
The viability of isolated cells was checked by the trypan blue exclusion test and was found to be higher than 90%.
The cells were cultured in DMEM-Ham’s F-12 containing 10% (vol/vol) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin at 37 C under 5% CO2 in air. The day of cell preparation and cell seeding is subsequently described as day 0. Culture medium was replaced every 24 h. The serum-free medium that was used from days 4–5 contained 10-7 M ascorbic acid, 0.001% wt/vol transferrin, and 0.01% wt/vol bacitracin (19, 20).
Staining of chromaffin and cortical cells
Cells were grown on chamber slides (Nunc, Naperville, IL) for 4
days. For immunohistochemistry, the cells were fixed for 30 min in 4%
formaldehyde. Immunostaining was performed by the avidin-biotin
technique using the UniTect immunohistochemistry detection system
(Dianova, Hamburg, Germany) as previously described (21). Cortical
cells were stained by a 1-h incubation at 37 C with a 1:10 dilution of
the rabbit antiserum to 17-hydroxyprogesterone (Sigma). For specific
staining of chromaffin cells, cells were incubated for 1 h at room
temperature with a 1:1000 dilution of the rabbit antibovine
dopamine-ß-hydroxylase antibody (Incstar, Stillwater, MN).
Visualization was achieved by immersing the cells for 15 min in
3-amino-9-ethyl-carbazole (Dianova-Immunotech, Hamburg, Germany)
chromogen solution containing 0.05% H2O2.
Hematoxylin was used for counterstaining.
Day-dependent insertion of chromaffin cells
Cortical cells were seeded on 24-well plates at a density of
2 x 105 cells/well. Chromaffin cells were seeded in
inserts (2 x 105 cells/insert) with a 0.02-µm
anopore membrane (Nunc). The chromaffin cell-containing inserts
were transferred in wells with cortical cells in a day-dependent
manner. The first inserts were transferred 1 h after seeding the
cortical cells (day 0), resulting in 5 days of coculture. Inserts were
transferred to cortical cell-containing wells every 24 h until day
4, when the last inserts were transferred. On day 4, the FCS-containing
culture medium was replaced by serum-free medium, and on day 5, the
supernatants were collected for cortisol measurement.
Effects of different culture conditions
Isolated cortical cells were cultured in wells (2 x
105 cells/well; Fig. 1A
). For
the coculture, cortical cells were seeded in wells (2 x
105 cells/well), and medullary cells were seeded at the
same density in inserts (Fig. 1B). In mixed cultures, cortical and
chromaffin cells were mixed 1:1 and seeded at a density of 4 x
105 cells/well (Fig. 1C). Chromaffin-cell conditioned
medium was prepared by replacing the culture medium from the medullary
cells every 24 h. The conditioned medium was either immediately
transferred to cortical cells or kept frozen at -20 C. In all
experiments the same volume of medium per cells (0.4 ml medium/2
x 105 cells) was used. On day 4, the FCS-containing
culture medium was replaced by serum-free medium, and on day 5, the
supernatants were collected for cortisol measurement.
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[3H]Thymidine incorporation assay
Cortical cells were seeded on 96-well plates at a density of
5 x 104 cells/well and cultured either in
unconditioned FCS-containing medium or in chromaffin cell-conditioned
FCS containing medium. The media were changed every 24 h. To
assess cell growth, cells were incubated with 2.5 µCi/ml
[3H]thymidine (Peninsula Laboratories, Belmont, CA)
for 24 h (days 4–5). All equipment was purchased from Packard
(Meriden, CT). Cells were removed from the culture plate by
trypsinization and harvested with Filtermate 196. After drying the
filter with the harvested cells at 60 C for 1 h, a plastic
scintillator sheet (FlexiScint) was placed over the filter, placed in
an oven at 70 C for approximately 30 min, and counted in a microplate
scintillation and luminescence counter (TopCount).
Northern analysis
Cortical cells were seeded on six-well plates at a density of
106 cells/well and maintained either in culture alone or in
coculture with chromaffin cells (106 cells/insert). Total
RNA was isolated from 2 x 106 cortical
cells/experiment using the RNAzol B RNA isolation kit from AGS
(Heidelberg, Germany). Equal amounts of total RNA (5 µg) were loaded
onto the gel based on ethidium bromide staining of the 28S and 18S
ribosomal RNA bands. RNA was fractionated by electrophoresis through a
1.2% agarose gel containing 0.61 mol/liter formaldehyde under
denaturing conditions and transferred to uncharged nylon membranes
(Quiagen, Hilden, Germany).
Bovine cytochrome P45017 complementary DNA was labeled
with digoxigenin (Dig)-UTP using the Dig RNA labeling kit (SP6/T7) from
Boehringer Mannheim (Mannheim, Germany) by in vitro
transcription. The P45017 complementary DNA was provided
by Prof. M. R. Waterman, Department of Biochemistry, Vanderbilt
University School of Medicine (Nashville, TN).
The filters were prehybridized for 1 h and hybridized overnight
with Dig-UTP-labeled RNA probe at 68 C. Filters were washed twice for 5
min each time in 2 x SSC (standard saline citrate)-1% SDS at
room temperature, followed by 0,1 x SSC-1% SDS at 68 C twice for
15 min each time. For detection of chemiluminesence, CDP-Star was used
according to the manufacturer’s protocol for the Dig luminescence
detection kit (Boehringer Mannheim). The resultant blots were exposed
to Hyperfilm-ECL (Amersham, Braunschweig, Germany) for 2–5 min.
Hybridization signals in the blots were analyzed quantitatively by
densitometric scanning. A sense probe were used to control the
specificity of the antisense P45017 probe.
Effects of inhibitors
Cortical cells were incubated with an inhibitor of transcription
[actinomycin D (Act D); 10 µg/ml] or an inhibitor of protein
synthesis [cycloheximide (CHX); 10 µg/ml] as described by Yuhi
et al. (22). From days 2–3, isolated cortical cells were
pretreated for 24 h with or without the addition of the
inhibitors. After this pretreatment, dishes were divided into two
groups and incubated for an additional 24 h (days 3–4) with
culture medium or chromaffin cell-conditioned medium in the continued
absence or presence of Act D or CHX.
To inhibit PG synthesis, cortical cells were pretreated for 1 h with indomethacin. After the pretreatment, cells were incubated for 24 h (days 3–4) with culture medium or chromaffin cell-conditioned medium in the continued absence or presence of indomethacin.
Incubation with ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), epinephrine, norepinephrine,
propranolol, and IL-1ß
ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (Synacthen, Ciba Geigy, Wehr, Germany),
norepinephrine, and/or human recombinant IL-1ß (Endogen, Woburn, MA)
were added to isolated cortical cells every 24 h during the entire
time of culture (days 0–5). Epinephrine (Jenapharm, Jena, Germany) was
added to isolated cortical cells every 24 h from days 0–5 or from
days 4–5. Propranolol
(1-(isopropylamino)-3-(1-naphthyloxy)-2-propanol, Obsidan, Isis Pharma,
Zwickau, Germany) was added to isolated cortical cells and to mixed
cultures every 24 h from days 0–5. On day 4, the FCS-containing
culture medium was replaced by serum-free medium, and on day 5, the
supernatants were collected for cortisol measurement.
Hormone measurements
For cortisol measurements, the supernatants were kept frozen at
-20 C; for ACTH and catecholamine measurements, the supernatants were
stored at -80 C. ACTH was stabilized by addition of 4 mM
Na2EDTA as demanded by the manufacturer of the ACTH RIA
kit. To prevent oxidation of catecholamines, 0.027 mM
Na2EDTA and 0.57 mM ascorbic acid were added
(23).
Hormone concentrations in the incubation media were measured by RIA, using the following kits. The cortisol RIA was purchased from Biermann (Bad Nauheim, Germany; sensitivity, 5.5 nmol/liter; cross-reactivity: cortisol, 100%; prednisolone, 76%; 11-deoxycortisol, 11.4%; prednisone, 2.3%; other steroids, <1%; intra- and interassay variations, 5.1% and 6.4%, respectively). The DYNOtest for ACTH was obtained from Brahms Diagnostica (Berlin, Germany; sensitivity, 0.44 pmol/liter; intra- and interassay variations, <10 and <20%, respectively). Norepinephrine and epinephrine were measured by HPLC according to the manufacturer’s protocol for plasma levels (Chromsystems, Munich, Germany). Catecholamines were detected with the Waters 460 electrochemical detector (Waters Associates, Milford, MA), and the evaluation of the chromatogram was performed using Millennium 2000 software (Millipore, Milford, MA).
Statistical analysis
Results are expressed as the mean ± SEM, and
statistical significance was determined by ANOVA using the software
package SPSS for Windows, version 6. Differences were considered
significant at P < 0.05, and very significant at
P < 0.01. All experiments for cortisol measurements
were repeated for a minimum of three different cell preparations (n)
using four wells per experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyprogesterone. (Fig. 2A). Besides
17-hydroxyprogesterone-positive cells, these primary cultures
contained 6.8 ± 1.7% (mean ± SEM; n = 4)
other cell types, such as fibroblasts, macrophages, lymphocytes, and
endothelial cells. In contrast to cortical cells, chromaffin cells did
not grow to confluent monolayers, but in cell groups. They were
characterized with an antibody against dopamine-ß- hydroxylase (Fig. 2B). Chromaffin cells obtained by differential plating were highly
purified and contained, in accordance with the report by Unsicker and
Mueller (18), only 2.5 ± 0.6% (mean ± SEM;
n = 4) other cell types. When adrenocortical and adrenomedullary
cells were cultured together in mixed cultures, both cell types formed
close cellular contacts (Fig. 2C).
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). No cortisol was detectable in the
supernatants from chromaffin cells (days 0–5).
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), indicating that the increased
cortisol release in mixed cultures and cocultures was not due to the
doubled cell number.
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). The
cortisol secretion from isolated cortical cells was on a near-constant
low level, ranging from 4.0–7.9 pmol/well·24 h. In contrast, the
cortisol secretion from chromaffin cell-conditioned medium-treated
cortical cells was on a much higher level and increased during culture,
reaching a value of 139.3 ± 10.3 pmol/well·24 h on days
4–5.
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Northern analysis
Day-dependent cytochrome P45017 mRNA expression in
isolated cortical cells compared with that in cortical cells in
coculture with chromaffin cells was investigated by Northern analysis
(Fig. 6). The amount of
P45017 mRNA in the coculture increased from days 1–5,
whereas it decreased in isolated cortical cells. This led to a sixfold
increased P45017 mRNA expression (605 ± 61% of
the hybridization signal for isolated cortical cells; n = 3) on
day 5, as revealed by densitometric scanning.
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).
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). It has been shown previously that in
bovine adrenals, the stimulatory effect of IL-1 on adrenocortical
steroidogenesis is mediated by the local release of PGs and could be
totally blocked by indomethacin (24). Therefore, PG synthesis was
inhibited by adding the cyclooxygenase inhibitor indomethacin to the
culture medium. Indomethacin partly inhibited the stimulatory effect of
chromaffin cell-conditioned medium. Cortisol secretion decreased from
133 ± 10 to 99 ± 9 pmol/well·24 h by the addition of 10
µM indomethacin and to 72 ± 7 pmol/well·24 h by
the addition of 100 µM indomethacin (Fig. 9).
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Epinephrine and norepinephrine concentrations in mixed cultures ranged
from 6.5 x 10-8 to 4 x 10-7
M and from 2.3 x 10-8 to 3.6 x
10-7 M, respectively, on day 3. On day 5, the
epinephrine concentration ranged from 9.2 x 10-10 to
4.3 x 10-9 M, and the norepinephrine
concentration ranged from nondetectable to 2.8 x
10-9 M (n = 3; Table 1).
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).
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).
The coincubation of ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), epinephrine, norepinephrine, and IL-1
during the entire 5-day culture resulted in an additive effect of these
substances. On day 5, cortisol secretion was increased 4.3-fold over
basal secretion (Fig. 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies, we have shown that adrenal medulla and adrenal cortex are highly interwoven in the mammalian adrenal (25), and it has been supposed that this interrelationship is the prerequisite for a paracrine regulation of the adrenal cortex by the adrenal medulla (1, 3). Chromaffin cells secrete a wide variety of transmitters and neuropeptides, and it was concluded that under basal conditions, stimulatory and inhibitory effects annul each other (3). However, our data show that stimulatory influences predominate, leading to a pronounced enhancement of steroidogenesis. This stimulatory effect was independent from direct cellular contacts, as cortisol release reached the same values in cultures where both cell types were mixed and therefore were in direct contact as in the coculture where both cell types were separated by a membrane. Chromaffin cell-conditioned medium added every 24 h had the same effect as coculturing, indicating that the factor(s) involved are not subject to fast degradation. The stimulatory effect was clearly time dependent, and the activity of cortical cells increased with the time the two cell types were cultured together or with the time the cortical cells were treated with chromaffin cell-conditioned medium. The increased activity does not reflect an increased proliferation rate of cortical cells. In contrast, the proliferation rate of cortical cells decreased as measured by [3H]thymidine incorporation. This agrees with the effects of other stimulators of adrenocortical function, i.e. ACTH that is a potent stimulator of steroidogenesis, while at the same time inhibiting cell replication (26).
On the transcriptional level, the large differences in cortisol release
were reflected by an increased expression of P45017 mRNA
in the coculture compared with a decrease in isolated cortical cells,
leading to a 6-fold difference on day 5. Adrenocortical steroid
17-hydroxylase is a strongly inducible enzyme. In vitro
and in vivo 17-hydroxylase is induced and maintained by
corticotropin or other stimulators of cAMP production. Enzyme levels
correlate with mRNA levels after induction. Although the rate-limiting
step in steroid hormone biosynthesis is the side-chain cleavage of
cholesterol (27), it is P45017 mRNA expression that is
regulated most rapidly, and a decrease in adrenocortical activity is
primarily reflected by a decrease in P45017 mRNA
expression (28, 29). The maintenance and stimulation of
P45017 expression and cortisol production suggest that
in the coculture, the environment of cortical cells in vivo
is more accurately mimicked than that in primary cultures of isolated
adrenocortical cells. In addition, the observed steroidogenic effect
may be responsible for the remaining activity of the adrenal cortex
after hypophysectomy.
To further determine whether protein synthesis is involved in the stimulation of adrenocortical steroidogenesis by adrenomedullary cells, the effects of inhibitors of protein (CHX) or RNA (Act D) synthesis on steroidogenesis were examined. CHX and Act D both blocked the stimulatory effect of chromaffin cell-conditioned medium on cortisol release from adrenocortical cells, indicating that de novo RNA and protein syntheses are required for this stimulation by adrenomedullary secretagogues.
How can adrenomedullary secretory products in vivo reach the adrenal cortex? The blood flow within the adrenal is directed centripetally from the cortex to the medulla (30), thus making an effect via the vascular system unlikely. However, the observed interweaving of both cell systems to various degrees in mammals (25, 31, 32, 33, 34, 35) may be the prerequisite for this local regulation of adrenocortical function. The close anatomical colocalization may form the basis for a paracrine interaction of the two endocrine systems. Interestingly, such a stimulatory effect of chromaffin cells on corticosteroid secretion has also been demonstrated in nonmammalian species such as the frog, in which the adrenal is composed of highly intermingled adrenocortical and chromaffin cells (36, 37). In addition to a direct paracrine action, some adrenomedullary secretory products, especially larger molecules such as proteins and neuropeptides, may reach the adrenal cortex via interstitial fluid and lymphatics (38).
Adrenomedullary chromaffin cells produce, store, and secrete a whole series of neuropeptides and transmitters in addition to catecholamines. Which factors are responsible for the stimulation of steroidogenesis in the coculture?
The most dominant secretory products of the adrenal medulla are the
catecholamines epinephrine and norepinephrine, which are both able to
stimulate corticosteroidogenesis (39, 40, 41) and to up-regulate
17-hydroxylase (41, 42). The most effective intraadrenal stimulator
of adrenal steroidogenesis is most likely to be adrenomedullary ACTH
(1, 43). In the present study, the concentration of ACTH in the
supernatant of mixed cultures of chromaffin and cortical cells was at
the detection limit of the RIA of 0.44 pmol/liter or even below.
Incubation of isolated adrenocortical cells with 10-12
M ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) for 5 days led to a doubling of cortisol
secretion on day 5 compared with a 10-fold increase in cocultures or by
incubation of cortical cells with chromaffin cell-conditioned medium.
Therefore, the high up-regulation of basal cortisol release in
cocultures is only partly caused by adrenomedullary ACTH. POMC is
produced within the medulla, but has been found not to stimulate
steroidogenesis itself (44).
In our coculture system, epinephrine concentrations in the supernatant
ranged from 6.5 x 10-8 to 4 x
10-7 M on day 3. Therefore, 10-7
and 10-6 M epinephrine were added to isolated
adrenocortical cells for 5 days, resulting in an increased cortisol
release to 223% and 162% of basal secretion on day 5, respectively
(compared with 1080% in cocultures). The observation that
10-6 M epinephrine had a more pronounced
stimulatory effect on cortisol release when added for only
24 h than after incubation of the cells for 5 days may be
due to a homologous desensitization of the cells (39). The
epinephrine-stimulated cortisol release was completely blocked by
addition of the ß-blocker propranolol. Propranolol at
10-6 M has been demonstrated to arrest the
effect of 10-5 M epinephrine completely,
whereas it has no effect on ACTH-induced cortisol production (45, 46).
In addition to epinephrine, chromaffin cell-conditioned medium, in
lower concentrations, contained norepinephrine (Table 1). It has been
demonstrated that propranolol blocks not only the effect of epinephrine
but also the effect of norepinephrine (39). Only 20% of the cortisol
release in mixed cultures of chromaffin and cortical cells was
inhibited by propranolol, suggesting the involvement of other factors
besides catecholamines.
The partial inhibition of steroidogenesis by indomethacin reflects the involvement of PGs in the stimulation of cortical function in the coculture. It has been shown that different cytokines stimulate adrenal steroidogenesis via the local release of PGs (24, 47, 48). Cytokines are very likely to be involved in such a local paracrine interaction. IL-1, the only cytokine shown to be produced by adrenomedullary chromaffin cells (for review, see Ref.49) stimulates steroidogenesis in bovine adrenocortical cells in culture (this study and Ref.24). This stimulation is mediated via the local release of PGs, which are produced by fibroblasts, leukocytes, and endothelial cells and can be completely abolished by indomethacin (24). Therefore, the stimulation of adrenocortical steroidogenesis by adrenomedullary secretory products involves a paracrine interaction with a small subpopulation of PG-producing cells within the adrenal gland.
ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), epinephrine, norepinephrine, and IL-1 stimulated steroidogenesis 4.3-fold when added together to cortical cells during the entire period of culture. In addition to these factors, chromaffin cells produce and secrete a wide variety of factors that are able to stimulate cortical steroidogenesis (for review, see Ref.3). This complex "cocktail" probably is responsible for the observed stimulation of adrenocortical steroidogenesis.
In summary, we have shown that basal steroidogenic activity is remarkably increased in adrenocortical cells cultured together with medullary chromaffin cells compared with that in isolated cortical cells. It is concluded that the coculture system is a model in which the in vivo conditions are mimicked in an in vitro system. It clearly demonstrates that the close anatomical colocalization of the two endocrine systems in the adrenal is of physiological importance for the full strength of steroidogenic response of the adrenal gland. Although there has been some speculation on the influence of adrenomedullary secretory products on adrenocortical function, this is the first study to prove a direct stimulatory influence on adrenocortical function. Further studies based on these results will fully characterize this effect and the factors involved.
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Acknowledgments |
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Footnotes |
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Received May 30, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase, and
3ß-hydroxysteroid-dehydrogenase messenger ribonucleic acid and
proteins and on steroidogenic responsiveness to corticotropin and
angiotensin-II. J Clin Endocrinol Metab 78:1212–1219[Abstract]
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 E. Zambrano, P. W. Nathanielsz, and T. J. McDonald Prenatal and Postnatal Ovine Adrenal Cell responses to Prostaglandin E2 Reproductive Sciences, May 1, 2001; 8(3): 149 - 157. [Abstract] [PDF]
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S. R. Bornstein, H. Tian, A. Haidan, A. Bottner, N. Hiroi, G. Eisenhofer, S. M. McCann, G. P. Chrousos, and S. Roffler-Tarlov Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo PNAS, December 19, 2000; 97(26): 14742 - 14747. [Abstract] [Full Text] [PDF]
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 O. Lesouhaitier, M. K. Kodjo, F. Cartier, V. Contesse, L. Yon, C. Delarue, and H. Vaudry The Effect of the Endozepine Triakontatetraneuropeptide on Corticosteroid Secretion by the Frog Adrenal Gland Is Mediated by Activation of Adenylyl Cyclase and Calcium Influx through T-Type Calcium Channels Endocrinology, January 1, 2000; 141(1): 197 - 207. [Abstract] [Full Text] [PDF]
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 S. R. BORNSTEIN, T. TAJIMA, G. EISENHOFER, A. HAIDAN, and G. AGUILERA Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice FASEB J, July 1, 1999; 13(10): 1185 - 1194. [Abstract] [Full Text]
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S. R. Bornstein and G. P. Chrousos Adrenocorticotropin (ACTH)- and Non-ACTH-Mediated Regulation of the Adrenal Cortex: Neural and Immune Inputs J. Clin. Endocrinol. Metab., May 1, 1999; 84(5): 1729 - 1736. [Full Text]
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 U. Hilbers, J. Peters, S. R. Bornstein, F. M. A. Correa, O. Johren, J. M. Saavedra, and M. Ehrhart-Bornstein Local Renin-Angiotensin System Is Involved in K+-Induced Aldosterone Secretion from Human Adrenocortical NCI-H295 Cells Hypertension, April 1, 1999; 33(4): 1025 - 1030. [Abstract] [Full Text] [PDF]
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 A. G. Mensah-Nyagan, J.-L. Do-Rego, D. Beaujean, V. Luu-The, G. Pelletier, and H. Vaudry Neurosteroids: Expression of Steroidogenic Enzymes and Regulation of Steroid Biosynthesis in the Central Nervous System Pharmacol. Rev., March 1, 1999; 51(1): 63 - 82. [Abstract] [Full Text] [PDF]
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G. Mazzocchi, P. G. Andreis, R. De Caro, F. Aragona, L. Gottardo, and G. G. Nussdorfer Cerebellin Enhances in Vitro Secretory Activity of Human Adrenal Gland J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 632 - 635. [Abstract] [Full Text]
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 M. Ehrhart-Bornstein, J. P. Hinson, S. R. Bornstein, W. A. Scherbaum, and G. P. Vinson Intraadrenal Interactions in the Regulation of Adrenocortical Steroidogenesis Endocr. Rev., April 1, 1998; 19(2): 101 - 143. [Abstract] [Full Text]
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). The medium was
changed every 24 h, and the removed supernatant was used for
cortisol measurement. Cortisol secretion from isolated cortical cells
was on a near-constant low level, ranging from 4.0–7.9 pmol/well·24
h. In contrast, cortisol secretion from cortical cells treated with
conditioned medium was on a much higher level and increased during
culture, reaching a value of 139.3 ± 10.3 pmol/well·24 h on
days 4–5.
