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Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Address all correspondence and requests for reprints to: Dr. William B. Campbell, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail: wbcamp{at}mcw.edu
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Introduction |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bovine adrenal capillary ECs were isolated as described by
Gospodarowicz et al. (26). Tissue fragments from the
cortical zone were incubated at 37 C in 0.5% collagenase in PBS. The
supernatant containing the dispersed cells was collected, and the cells
were washed three times with DMEM containing glucose (4.5 g/liter),
calf serum (10%), and gentamicin (50 µg/ml; plating medium). The
cells were plated on 60-mm gelatin-coated petri dishes in PBS and
incubated at 37 C in an atmosphere of 5% CO2 in air. After
30 min and again at 2 h, cells that did not adhere were removed,
and plating medium was added. After 1 day, colonies of ECs appeared as
groups of loosely connected cells growing in swirls. The cells were
subsequently grown in DMEM containing glucose (4.5 g/liter), calf serum
(10%), endothelial mitogen (25 ng/ml), and basic fibroblast growth
factor (5 ng/ml; endothelial cell growth medium). The medium was
changed every 2–3 days. The purity of endothelial cell cultures was
assessed as greater than 95% by their uniform cobblestone morphology
as well as positive staining for
(1,1’-dioctadecyl-3,3,3’,3’-tetramethyl indocarbocyanine)
diI-acetylated low density lipoprotein (diI-Ac LDL) and factor
VIII antigen and negative staining for smooth muscle 
-actin by
immunofluorescence. Furthermore, EC cultures did not produce detectable
amounts of aldosterone or cortisol. Cells were transferred
nonenzymatically by incubation in Puck’s/EDTA medium or by allowing
them to migrate to microcarrier beads (see below). Cells from passages
2–6 were used.
Pericytes were present in some early passage cultures of adrenal ECs. These cells were identified by a unique morphology with longitudinal and circumferential processes (27). Mixed cultures were separated and enriched by exposing them to a Puck’s/EDTA solution. ECs detached in this medium and were replated in bovine adrenal endothelial cell growth medium. The adherent pericytes were then incubated in Puck’s/EDTA with trypsin (0.08%) and plated in DMEM containing bovine calf serum (10%) and antibiotic-antimycotic mixture (1%), but without endothelial mitogen or basic fibroblast growth factor (pericyte growth medium). Enriched cultures of pericytes were characterized by their morphology, slow (10- to 14-day) doubling time, and lack of staining with diI-Ac-LDL. Cells from passages 3–6 were used.
Adrenal fibroblasts sometimes appeared in overgrown cultures of adrenal capillary ECs. These cells exhibited typical fibroblast morphology and a lack of contact inhibition at confluence. The same method used to enrich pericyte populations was used in isolating fibroblasts. Fibroblasts were also grown in DMEM containing bovine calf serum (10%) and antibiotic-antimycotic mixture (1%; fibroblast growth medium). Subsequent passages of these cells were characterized by morphology and lack of staining with diI-Ac-LDL. Cells from passages 3–5 were used for these studies.
EC-ZG cell interaction
For cell-cell interaction experiments, sterilized Cytodex 3
microcarrier beads were suspended in the appropriate cell growth medium
and added to confluent cell monolayers of ECs, fibroblasts, or
pericytes at a concentration of 1–2 mg beads/ml culture medium (28).
Typically, cells migrated onto and divided on the beads so that the
beads were 80–100% covered with cells after 24 h. Confluent
cultures of beads were used to seed new cultures onto tissue culture
dishes or for ZG cell incubations as described below. Before an
experiment, confluent ZG cells were washed twice with 1 ml modified
F-12 medium containing 1 mg/ml BSA (buffer 1) and allowed to incubate
for 2 h in this medium. Before the start of the experiment, cells
growing on microcarrier beads were dislodged from the underlying cell
monolayer by spraying culture medium over the surface of the dish with
a pipette. The beads were collected in a plastic centrifuge tube and
allowed to settle. The culture medium was then removed and replaced
with buffer 1. After two washes in buffer 1, cells were resuspended in
an appropriate volume of F-12 containing 2 mg/ml BSA and 1.8
mM calcium chloride (buffer 2) to give a final
concentration of 1–10 mg beads/ml. Cell-free beads that had been
preincubated in culture medium were rinsed and resuspended in the same
manner. For the concentration-response experiment, suspensions of
endothelial cell-covered beads (5 mg/ml) or cell-free beads were
diluted with increasing volumes of buffer 2. The ZG cells were
incubated at 37 C for 1 or 2 h with 1 ml/well of the various
dilutions. The medium was then removed and stored at -40 C until
assayed for aldosterone. ZF cells were handled in the same manner, and
the medium was assayed for cortisol.
Studies with EC-conditioned medium (EC-CM)
In experiments using CM, microcarrier beads coated with adrenal
ECs, adrenal fibroblasts, or cell-free beads were collected in a
plastic conical tube and washed, and the tube was incubated
horizontally in buffer 2 at 37 C to maximize contact between beads and
medium. In some experiments, the incubation time of the beads with
buffer 2 was varied to determine the effect of conditioning time on
steroidogenic activity. The conditioning time varied from 0.5–3 h. At
the end of the conditioning period, the tubes were placed vertically to
allow the beads to settle. The CM was removed from the beads, and 1 ml
of the CM was transferred to ZG cells that had been washed and prepared
as described above. In some experiments, the EC- or fibroblast-CM was
diluted with various amounts of CM from cell-free beads. One milliliter
of each of the dilutions was added to ZG cells or ZF cells. The cells
were then incubated for 2 h at 37 C, and the buffer was removed
and stored at -40 C until assayed for aldosterone or cortisol.
In other experiments, cell free or EC-CM was collected, various antagonists were added, and the medium was transferred to ZG cells that had been pretreated with the corresponding antagonists. The ZG cells were then incubated at 37 C for 2 h. The medium was removed and stored at -40 C until assayed for aldosterone. The antagonists included the angiotensin II AT1 receptor antagonist losartan (10-5 M), the bradykinin B2 receptor antagonist D-Arg-[Hyp8,Thy5,8,D-Phe7]bradykinin (10-5 M), and the endothelin ETB receptor antagonist PD-145065 (10-5 M).
The protease sensitivity of cell free and EC-CM was tested by incubating CM with pronase immobilized on agarose (pronase-CB). The pronase gel was washed twice with buffer 1 before use. After a 2-h incubation with CM (0.4 U pronase/ml CM) in a shaking 37 C water bath, samples were centrifuged to pellet the pronase, and the supernatant was transferred to ZG cells. ZG cells were incubated for 2 h at 37 C, and the medium was stored frozen at -40 C until assayed for aldosterone.
The proteins contained in EC-CM were separated by gel filtration chromatography on a Pharmacia Superose 12 HR 10/30 column (Pharmacia Biotech, Piscataway, NJ). EC-CM was lyophilized and resuspended in 0.15 M ammonium bicarbonate buffer, pH 7.8, at 10-fold the original concentration. After the injection of 500 µl of this concentrate, the column was eluted with ammonium bicarbonate buffer at a flow rate of 0.5 ml/min. Fractions were collected in 1-ml volumes, and fractions from 6–35 ml were assayed for steroidogenic activity. The assay was conducted by adding 0.1 ml of each fraction to 1 ml ZG cell incubation medium. ZG cells were incubated for 2 h at 37 C, and the medium was removed and stored frozen at -40 C until assayed for aldosterone.
Binding of [125I]ET-1 to ZG cells
Binding of [125I]ET-1 to ZG cells was based on the
method of Cozza et al. (29). Cells were cultured for 4 or 5
days in 12-well plates at a density of 200,000 cells/well. The cells
were washed twice in buffer 1 and allowed to incubate for 2 h at
37 C. The buffer was then removed and replaced with modified Ham’s
F-12 containing 0.1% BSA, pepstatin (10 µg/ml), and bacitracin (100
µg/ml). [125I]ET-1 was added to the ZG cells at 5
x 105 cpm/well along with increasing concentrations of
unlabeled ET-1 or EC-CM. The EC-CM was conditioned for 2 h and
subsequently diluted with cell-free CM. ZG cells were incubated for
1 h at 37 C followed by a series of four washes with 1 ml/well
ice-cold PBS. ZG cells were solubilized with 0.5 N NaOH.
The resulting suspension was transferred to 12 x 75-mm culture
tubes and passed through a Brandel cell harvester (Bethesda, MD). The
radioactivity bound to the cell harvester filter membranes was measured
by 
-scintillation spectrometry.
Intracellular calcium measurement
Experiments determining intracellular calcium release in ZG
cells were performed as described by Csukas et al. with
slight modifications (30). Briefly, ZG cells were loaded with fura-2/AM
in 10 mM HEPES buffer (pH 7.4) containing 155
mM sodium chloride, 5 mM potassium chloride,
1.8 mM calcium chloride, 1 mM magnesium
chloride, 5.5 mM glucose, and 1 mg/ml BSA (HEPES buffer).
Fura-2/AM was diluted in HEPES buffer containing 1.25 mg/ml BSA and
0.5% dimethylsulfoxide to a final concentration of 5 µM.
ZG cells were loaded with fura-2/AM for 1 h at room temperature.
After loading, the cells were washed four times with HEPES buffer and
transferred to a Photon Technologies, Inc. (Princeton, NJ), dual
excitation fluorescence microscope. ZG cells were stimulated with
angiotensin II (1 µM) and EC-CM.
RIAs
Aldosterone was measured by RIA as described by Gomez-Sanchez
et al. (31). Cortisol was measured as described by
Rosolowsky and Campbell (24). ET-1 was measured in EC-CM by RIA as
described by Hieda and Gomez-Sanchez (32). The ET-1 standards,
[125I]ET-1, and anti-ET-1 serum were diluted in an assay
buffer consisting of 0.05 M sodium phosphate (pH 7.4)
containing 0.1% BSA, 0.05 M sodium chloride, 0.1% Triton
X-100, and 1 mM EDTA. For the assay, 0.1 ml ET-1 standard
or EC-CM was added to 12 x 75-mm culture tubes containing 0.1 ml
anti-ET-1 serum diluted 1:4000 and 0.1 ml [125I]ET-1
containing 6000 counts/min. This mixture was incubated for 18 h at
4 C. After the addition of 0.1 ml goat antirabbit antibody diluted 1:20
and 0.3 ml 16.6% polyethylene glycol, the samples were incubated for
18 h at 4 C. The antibody-bound counts were removed by
centrifugation, the supernatant was decanted, and radioactivity was
measured using a -scintillation spectrometer. cAMP was measured as
described by Callahan et al. (33) and Csukas et
al. (30).
Statistics
Statistical analysis was performed using a two-way ANOVA for
concentration-response experiments with more than one treatment,
followed by multiple comparisons tests when differences were found to
be significant. A Bonferroni adjustment was used at an overall
significance level of P < 0.05. Otherwise, Student’s
t test was used. Data represent averages of multiple
incubations from at least two cell preparations or are a representative
experiment from multiple preparations.
Materials
Earle’s Balanced Salt Solution, horse serum, and antibiotics
(Life Technologies, Inc., Grand Island, NY); FBS
(HyClone Laboratories, Inc. Logan, UT); collagenase and
dispase (Roche Molecular Biochemicals, Indianapolis, IN);
basic fibroblast growth factor (R\|[amp ]\|D Systems, Minneapolis,
MN); and endothelial mitogen (Biomedical Technologies,
Inc., Stoughton, MA) were obtained from commercial sources. Primaria
culture plates were purchased from Becton Dickinson and Co. (Lincoln Park, NJ), and Cytodex 3 beads were obtained from
Sigma Chemical Co. (St. Louis, MO). Other cell culture
plasticware was purchased from Corning, Inc. (Houston,
TX). ET-1 was obtained from Peptides International (Louisville, KY),
Losartan from DuPont Merck Pharmaceutical Co., Inc.
(Wilmington, DE), PD-145065 from Parke-Davis (Detroit,
MI), and
D-Arg-[Hyp8,Thy5,8,D-Phe7]bradykinin
from Peninsula Laboratories, Inc. (Belmont, CA). Fura-2/AM
dye was purchased from Molecular Probes, Inc. (Eugene,
OR). [125I]ET-1 and [3H]aldosterone were
obtained from NEN Life Science Products (Boston, MA). The
antialdosterone antiserum was a gift from the Pituitary Hormone
Distribution Program of the NIH. The anti-ET-1 and anti-cortisol
antibodies were gifts from Dr. Celso Gomez-Sanchez, University of
Missouri (Columbia, MO).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Aldosterone release in EC/ZG cell
coincubations was significantly greater than the release from ZG cells
coincubated with cell-free beads. The effect was related to the number
of EC-coated beads added. At EC/ZG cell ratios of 0.08 and 0.4,
aldosterone synthesis was increased by 2.9-fold (P <
0.0001) and 17.5-fold (P < 0.0001), respectively, over
that in the corresponding cell-free bead controls. Aldosterone
production was not detectable from cell free- or EC-covered beads
incubated alone without ZG cells (data not shown). ZG cells incubated
with a maximal concentration of ACTH (3.5 x 10-8
M) produced 7.61 ± 0.69 pg aldosterone/µg
protein·2 h. Thus, the stimulation of ZG aldosterone release by the
highest number of ECs tested was approximately 75% of the maximal
aldosterone released by ACTH. ZG cells incubated with adrenal
fibroblasts or pericytes did not increase aldosterone release compared
with those incubated with cell-free beads (P <
0.0001), and there was no relationship between numbers of fibroblasts
or pericytes and the release of aldosterone. Aldosterone production by
adrenal fibroblasts and pericytes incubated without ZG cells was
undetectable.
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). Incubation of ZG cells with
cell-free CM containing 25% EC-CM increased aldosterone production by
1.6-fold compared with that by ZG cells incubated with cell free-CM
(P < 0.02). One hundred percent EC-CM stimulated
aldosterone production by 3.3-fold (P < 0.0001). The
stimulation of aldosterone release by 100% EC-CM was equal to 25% of
the effect of a maximal ACTH stimulus (18.2 ± 2.7 pg
aldosterone/µg protein·h; data not shown). CM from adrenal
fibroblasts did not stimulate aldosterone release at any concentration.
The CM from ECs or fibroblasts did not contain detectable
concentrations of aldosterone.
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, top). Stimulatory activity could be detected at 15 min
(P < 0.003 vs. cell free-CM) and reached a
maximum at 30 min, after which it appeared to plateau. Medium that was
conditioned by adrenal fibroblasts for up to 4 h did not stimulate
aldosterone release (Fig. 3, bottom). These data indicate
that adrenal ECs release a soluble factor that stimulates aldosterone
release, which we termed EDSF.
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).
Treatment with pronase completely eliminated the stimulatory activity
of EC-CM. The aldosterone release with cell-free CM was not altered by
pronase treatment. These data indicate that EDSF is a protein or
peptide and led us to compare the steroidogenic activity of EDSF to
those of the commonly known steroidogenic peptide agonists, ACTH,
angiotensin II, ET-1, and bradykinin.
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Angiotensin II (10 nM) stimulated a 5-fold increase in
aldosterone release compared with the control value (P
< 0.01; Fig. 5, middle). The
angiotensin II stimulation was inhibited by pretreatment with the
angiotensin AT1 receptor antagonist, losartan (10
µM). EC-CM-stimulated aldosterone release was
approximately 6-fold greater than that stimulated by cell-free
CM, but was not inhibited by losartan pretreatment. A similar
study was performed with bradykinin and the bradykinin
B2 receptor antagonist
D-Arg-[Hyp8,Thy5,8,D-Phe7]bradykinin
(35). Bradykinin (100 nM)-stimulated aldosterone release
was 2.3-fold greater than control release (P < 0.01;
Fig. 5, top). Pretreatment with the bradykinin antagonist
inhibited the stimulation by bradykinin. EC-CM-stimulated aldosterone
release was 2.2-fold greater than cell-free CM, but this stimulation
was not inhibited by the bradykinin antagonist. Aldosterone release
with cell-free CM was not altered by the bradykinin antagonist.
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, bottom). ET-1 (100 nM) stimulated a 2-fold
increase in aldosterone release (P < 0.02), which was
blocked by PD-145065 (P < 0.01). EC-CM stimulated a
9.5-fold increase in aldosterone release, which was not significantly
reduced by the presence of the ET antagonist. To further determine the
role of ET-1, ECs were conditioned for 0–24 h to examine the time
course of ET-1 release. A detectable increase in ET-1 release was not
measured until after 3 h of conditioning (37 ± 1 pg/ml at
0 h vs. 173 ± 28 pg/ml at 3 h; data not
shown). ET-1 release was maximal at 12 h (2023 ± 162 pg/ml)
and appeared to plateau. In comparison, the release of EDSF was near
maximal at 30–60 min (Fig. 3, top). EDSF release was
significantly reduced at 12 h and showed no stimulation above
basal values at 24 h (data not shown). In ligand binding studies
with radiolabeled [125I]ET-1, EC-CM was examined for its
ability to displace [125I]ET-1 from the ET receptor on ZG
cells. Unlabeled ET-1 inhibited the binding of [125I]ET-1
to ZG cells with an IC50 of 2.5 nM. ET-1
displaced 90% of the bound [125I]ET-1 at a concentration
of 100 nM (data not shown). There was no significant
displacement of [125I]ET-1 by EC-CM (data not shown).
EC-CM increased intracellular calcium in ZG cells loaded with fura-2
dye. Angiotensin II (1 µM) increased ZG intracellular
calcium with a characteristic spike, followed by an elevated plateau
phase (Fig. 6B). EC-CM stimulation did
not result in a calcium spike, but caused a sustained elevation of
intracellular calcium to concentrations comparable to those found
during the plateau phase of angiotensin II (Fig. 6A). When summarized,
both angiotensin II and EC-CM caused sustained elevation of
intracellular calcium concentrations within ZG cells, but only
angiotensin II resulted in a rapid calcium spike (Fig. 6C). Treatment
of ZG cells with EC-CM did not increase the production of cAMP.
Accumulation of cAMP was unchanged from 5–120 min in ZG cells treated
with cell-free CM or EC-CM (Table 1).
Maximal stimulation with 35 nM ACTH resulted in cAMP
production of 167 ± 6 pg/µg protein at 120 min.
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). This elution volume corresponds to an
approximate molecular mass of 3000 Da. The arrow in Fig. 7
indicates the elution volume of ET-1 standards analyzed under identical
conditions. EDSF and ET-1 eluted in different fractions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current data indicate that bovine adrenal capillary ECs, but not adrenal fibroblasts or pericytes, stimulate aldosterone release from bovine ZG cells. This effect was concentration dependent either by varying the number of ECs during direct coincubations with ZG cells or by the transfer of varying concentrations of EC-CM to ZG cells. As the direct coincubation of ECs with ZG cells was not required to stimulate aldosterone synthesis, ECs did not modify a precursor from ZG cells to produce aldosterone. The ability of EC-CM to stimulate aldosterone release indicates that the effect is mediated by a soluble factor that is released during EC conditioning. The time course of EDSF release is relatively rapid. The stimulatory factor in adrenal EC-CM was detectable within 15 min of conditioning, peaked at 30 min, and appeared to plateau. Extended conditioning periods indicated that EDSF activity was decreased after 12 h, and no activity was evident after 24 h. In comparison, endothelial release of ET-1 reached a maximum at 12 h and remained at maximal concentrations after 24 h. Therefore, the release of ET-1 does not correlate with EDSF release.
We have demonstrated that EDSF and ET-1 do not compete for a common receptor site, as indicated by the inability of EC-CM to displace the binding of [125I]ET-1 to ZG cells. The ET-1 antagonist, PD145065, decreased EC-CM stimulated aldosterone release only slightly, whereas it was capable of completely inhibiting ET-1-induced aldosterone release. Based on these data, EDSF cannot be ET-1.
The effect of EDSF can be distinguished from other known stimulators of aldosterone release. ACTH stimulated cortisol production from ZF cells at a concentration of 3.5 pM. However, the failure of ECs and EC-CM to stimulate cortisol synthesis from ZF cells indicates that ACTH is not released from ECs. Therefore, EDSF cannot be ACTH. EDSF can also be differentiated from angiotensin II and bradykinin through the use of specific receptor antagonists. Previous studies have demonstrated that the stimulation of aldosterone by angiotensin II is mediated by the AT1 receptor subtype (37). In support of this, the specific AT1 receptor antagonist, losartan, completely inhibited the angiotensin II-stimulated release of aldosterone. However, losartan pretreatment did not inhibit EC-CM-stimulated aldosterone release, indicating that EDSF does not function through the angiotensin AT1 receptor. Bradykinin stimulation of aldosterone was completely inhibited by the B2 receptor antagonist D-Arg-[Hyp8,Thy5,8,D-Phe7]bradykinin (35). However, EC-CM-stimulated aldosterone release was not inhibited by pretreatment with the antagonist. Therefore, EDSF stimulation can also be distinguished from bradykinin.
EC-CM signaling pathways appear to be through increased intracellular calcium and are not dependent on the production of cAMP. The calcium concentrations achieved with EC-CM stimulation are comparable to the plateau phase of a high concentration of angiotensin II. However, angiotensin II responses are characterized by an immediate calcium spike, which is not seen with EC-CM stimulations. This difference in the shapes of the calcium response curves may indicate that the early phase of the angiotensin II response is not stimulated by EDSF. Similar to angiotensin II, EC-CM appears to act only through the calcium pathway and does not involve the accumulation of cAMP.
The separation of lyophilized EC-CM by gel filtration chromatography indicated that the aldosterone-stimulating activity eluted with a retention time corresponding to a molecular mass of approximately 3000 Da based on the elution of molecular mass standards. Its elution volume was less than that of ET-1. These data indicate that EDSF is not a known steroidogenic peptide, so it is either a novel peptide or a known endothelial peptide not previously reported to affect aldosterone release.
The mechanisms regulating EDSF production within the adrenal gland are currently unknown. The immediate and relatively short term release of EDSF suggests that the purpose of this peptide may be the intraadrenal modulation of aldosterone release. The role of the endothelium in the regulation of aldosterone production has been attributed to the production of eicosanoids, ET-1, and, more recently, nitric oxide (19, 22, 23, 38). The production of EDSF by the adrenal endothelium indicates a potent new mechanism for the interaction of the adrenal endothelium and steroidogenic cells. The present study indicates that ECs may alter hormone release via a paracrine mechanism, analogous to their effects on vascular tone.
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Acknowledgments |
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Footnotes |
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Received February 2, 1999.
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, P <
0.02 compared with CF-CM; 
