Endocrinology. First published ahead of print April 19, 2007 as doi:10.1210/en.2007-0169
STEROID-PRODUCING CELLS REGULATE ARTERIAL TONE OF ADRENAL CORTICAL
ARTERIES
Short Title: ACTH and adrenal cortical arterial tone
1
1
David X. Zhang, 1Kathryn M. Gauthier, 2John R. Falck, 2Anjaiah Siddam, and
William B. Campbell*
1
Department of Pharmacology and Toxicology
Medical College of Wisconsin
Milwaukee, WI 53226
2
Department of Biochemistry
University of Texas Southwestern Medical School
Dallas, Texas 75235
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Society. From the time of acceptance following peer review, the full text of this manuscript is made freely
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author(s), article title, journal title, year of publication and DOI.
This research was supported by grants from the National Institutes of Health (HL-83297, GM-31278,
DK-58145)) and the Robert A. Welch Foundation. Dr. Zhang was a postdoctoral fellow of the American
Heart Association, Greater Midwest Affiliate and a recipient of the Jenkins Cardiovascular Research
Fellowship.
Send Correspondence and Reprint Requests to:
William B. Campbell, PhD
Department of Pharmacology and Toxicology
Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee, WI 53226
Phone: (414) 456-8267
Fax: (414) 456-6545
Email: [email protected]
Key words: adrenocorticotropic hormone, arachidonic acid, vasodilation, adrenal gland,
epoxyeicosatrienoic acid
Disclosure summary: all authors have nothing to disclose.
Copyright (C) 2007 by The Endocrine Society
Abstract
Adrenal blood flow is coupled to adrenal hormone secretion. Adrenocorticotropic hormone (ACTH)
increases adrenal blood flow and stimulates the secretion of aldosterone and cortisol in vivo. However,
ACTH does not alter vascular tone of isolated adrenal cortical arteries. Mechanisms underlying this
discrepancy remain unsolved. The present study examined the effect of zona glomerulosa (ZG) cells on
cortical arterial tone. ZG cells (105-107 cells) and ZG cell conditioned medium relaxed preconstricted
adrenal arteries (maximal relaxations = 79±4% and 66±4%, respectively). In adrenal arteries coincubated
with a small number of ZG cells (0.5-1x106), ACTH (10-12-10-8 M) induced concentration-dependent
relaxations (maximal relaxation = 67±4%). Similarly, ACTH (10-8 M) dilated (55±10%) perfused arteries
embedded in adrenal cortical slices. ZG cell-dependent relaxations to ACTH were endotheliumindependent and inhibited by high extracellular K+ (60 mM), the K+ channel blocker, iberiotoxin (100
nM), the cytochrome P450 inhibitors SKF 525A (10 µM) and miconazole (10 µM) and the
epoxyeicosatrienoic acid (EET) antagonist 14,15-EEZE (2 µM). Four EET regioisomers were identified
in ZG cell conditioned media. EET production was stimulated by ACTH. We conclude that ZG cells
release EETs and this release is stimulated by ACTH. Interaction of endocrine and vascular cells
represents a mechanism for regulating adrenal blood flow and couples steroidogenesis to increased blood
flow.
2
Introduction
The secretion of steroid hormones in the adrenal
gland is modulated by adrenal blood flow (1-4).
Blood flow to the adrenal cortex delivers
stimulants, nutrients and oxygen to
steroidogenic cells and exports secretory
products into the systemic circulation. In
addition, increases in adrenal blood flow per se
may stimulate steroidogenesis. Despite the
importance of blood flow in adrenal function,
mechanisms responsible for blood flow
regulation are not well understood. Current
evidence indicates that the regulation of adrenal
blood flow is complex and involves neural,
humoral and local mediators. For example,
adrenal blood flow responds to changes in O2
tension and endogenous vasoactive substances
such as endothelin-1, adrenomedullin, nitric
oxide (NO), prostacyclin, epoxyeicosatrienoic
acids (EETs), and acetylcholine (5-10).
As a primary humoral regulator of the
adrenal gland function, adrenocorticotropic
hormone (ACTH) dilates the adrenal vasculature
and increases adrenal flow in vivo and in
perfused adrenals (6,11-14). Intriguingly, ACTH
does not alter vascular tone of isolated adrenal
cortical arteries in vitro (15). Hinson et al
suggested that ACTH-induced relaxation is due
to the release of histamine and serotonin from
perivascular mast cells (11,12). Stimulation of
histamine and serotonin release with compound
48/80, a mast cell degranulator, mimics ACTHinduced increase in adrenal blood flow and
steroidogenesis in the rat adrenal gland. Sodium
cromoglycate, a mast cell stabilizer, blocks the
effects of ACTH. Histamine and serotonin
antagonists were not tested in these studies.
Recently, we found that serotonin constricts
isolated bovine adrenal cortical arteries (15), and
histamine relaxes the adrenal arteries through
endothelium-derived nitric oxide and
prostaglandins (16). In intact rat or canine
adrenals, ACTH-induced dilation does not
involve NO or prostaglandins (6,13). Although
these discrepancies could be explained by
species variability, it is also possible that other
mechanisms are involved in ACTH-induced
vasodilation.
Previous studies have demonstrated a
functional interaction between vascular and
steroidogenic cells in the adrenal gland.
Endothelial cells, in response to various
vasoactive agents and sheer stress, release a
variety of soluble factors that regulate adrenal
steroidogenesis. For example, prostacyclin and
endothelin stimulate aldosterone release,
whereas NO inhibits aldosterone production (1720). In addition, endothelial cells release a
transferable steroidogenic peptide other than
endothelin that functions as a paracrine regulator
of aldosterone secretion (21). Alternatively, it
remains unknown whether adrenal steroidogenic
cells can modulate vascular tone and adrenal
blood flow. In the present study, we
hypothesize that ACTH stimulates adrenal
steroidogenic cells to release vasoactive factors,
which diffuse to vascular cells to mediate
relaxation. To test this hypothesis, we examined
the effect of zona glomerulosa (ZG) cells and
ZG-conditioned medium on adrenal arterial
tone, and the vascular activity of ACTH with
and without coincubation of ZG cells with
isolated small adrenal cortical arteries.
Furthermore, we isolated and identified
vasoactive factors released by ZG cells.
Materials and Methods
Isometric tension recording. Fresh bovine
adrenal glands were obtained from a local
abattoir. Small cortical arteries closely attached
to the adrenal surface (200-300 µm) were
dissected and mounted on a 4-chamber wire
myograph as described (15). Arteries were
maintained at 37°C in physiological saline (PSS)
containing (in mM): NaCl 119, KCl 4.7, CaCl2
2.5, MgSO4 1.17, NaHCO3 24, KH2PO4 1.18,
EDTA 0.026, and glucose 5.5, gassed with 95%
O2/5% CO2. Arteries were contracted with
submaximal concentrations of the thromboxane
mimetic U46619 (100-300 nM). Where
indicated, the endothelium was removed by
gently rubbing the intimal surface of the artery
with a human hair. The endothelium was
considered intact if acetylcholine (1 µM) caused
>80% relaxation and effectively removed if
acetylcholine induced <10% relaxation.
Arterial diameter measurement. Slices of
adrenal glands (5-8 mm thickness) were pinned
to a Silastic layer of a perfusion dish. A single
cortical artery (250-350 µm o.d.) was cannulated
with heat pulled PE-10 tubing and secured to the
3
cortical surface with 6-0 suture. The adrenal
slice was perfused and superfused with PSS
equilibrated with 21% O2/5% CO2/balanced N2,
and pressurized to 60 mmHg at 37°C. In
separate experiments, arteries were dissected,
freed of adherent tissue, cannulated in a
perfusion/superfusion chamber and pressurized
to 60 mmHg at 37°C (22). The arteries were
equilibrated for 30-45 min and contracted with
serotonin (50-200 nM) prior to the addition of
ACTH. Digital images were captured and
arterial diameters were measured using MetaVue
software. At the end of the experiments, arteries
were perfused and superfused with calcium-free
PSS to determine maximum passive diameter.
For adrenal slices, India ink (3% in calcium-free
buffer) was added to the perfusate to determine
perfused cortical area.
Preparation of ZG, zona fasciculata (ZF) and
fibroblast cells. ZG and ZF cells were prepared
by enzymatic dissociation of adrenal cortical
slices as previously described (23). Briefly,
adrenal glands (8-12) were bisected and a 500µm slices were obtained from the outer surface
of the gland (first slice for ZG cell isolation and
second slice for ZF cell isolation). The tissue
was finely minced and digested for 30-45 min at
37ºC in Earle's balanced salt solution buffer
containing dissociating enzymes. Cells from
four or five digestion cycles were pooled, rinsed
in HEPES buffer and stored on ice until use. ZG
cells were 95-98% ZG cells and 2-5% ZF cells.
ZF cells were 98% pure. Adrenal fibroblasts
were selectively isolated from mixed cultures of
adrenal capillary endothelial cells and fibroblasts
by replating the cells under conditions that
favored fibroblast but not endothelial cell
growth (21). Adrenal fibroblasts were
maintained in DMEM medium and passages of
3-5 were used.
Identification of arachidonic acid metabolites.
ZG cells were incubated with [14C] arachidonic
acid (0.05 mCi, 100 nM), and the buffers were
extracted on C-18 solid-phase extraction
columns and extracts dried under a stream of
nitrogen gas. The extracts were redissolved in
acetonitrile and analyzed by reverse-phase
HPLC with a Nucleosil C-18 reverse phase
column (5 mm, 4.5 x 250 mm) as described
(24,25). Solvent A was distilled water and
solvent B contained acetonitrile: glacial acetic
acid (999:1). A linear gradient from 50%
solvent B in A to 100% B over 40 min was used
with a flow rate of 1 ml/min. Column effluent
was collected in 0.2-ml fractions and analyzed
for radioactivity by liquid scintillation
spectrometry. Normal phase-HPLC with a
Nucleosil silica column (5 mm, 4.6 x 250 mm)
was used to separate EET regioisomers.
Solvents were hexane containing 0.4%
isopropanol and 0.1% glacial acetic acid. The
flow rate was 2 ml/min and column eluate was
collected in 0.4 ml fractions. An additional set
of samples was analyzed on by LC/MS (Agilent
1100 LC/MSD, SL model) as described (26).
Arachidonic acid metabolites were resolved
using a C18 (Kromasil, 250 x 2 mm) column
using water/acetonitrile with 0.005% acetic acid
at a flow rate of 0.2 ml/min. The separation
used a 60-80% acetonitrile in water linear
gradient over 30 min followed by an increase to
100% over 5 min and held at 100% for 5 min.
Drying gas flow was 12 liters/min, at 350ºC,
nebulizer pressure was 35 psig, vaporizer
temperature was 325ºC, and capillary voltage
was 3000 V. Detection was made in the
negative ion mode.
Data analysis. Relaxations and dilations are
expressed as a percentage relative to U46619- or
serotonin-preconstriction with 100%
representing basal tension or maximum passive
diameter in calcium-free PSS. Data are
presented as mean±SEM. Significance of
difference between mean values evaluated by
Student t test or ANOVA followed by the
Student-Newman-Keuls multiple comparison
test. A value of P<0.05 was considered
statistically significant.
Drugs and chemicals. ACTH, serotonin, Nnitro-L-arginine (L-NA), indomethacin,
iberiotoxin, SKF 525A, and miconazole were
purchased from Sigma (St. Louis, MO).
U46619 was obtained from Cayman Chemical
Company (Ann Arbor, MI). 14,15-EET and
14,15-EEZE (27) were synthesized as previously
described (28). All solvents were HPLC grade
and purchased from Burdick and Jackson. [14CU] Arachidonic acid (920 mCi/mmol) was
4
obtained from New England Nuclear (Boston,
MA).
Results
ZG cell-induced relaxations. Since the
cortical, subcapsular arterioles are embedded in
ZG cells rather than ZF cells, most studies used
ZG cells. Isolated ZG cells (105-107 cells) added
to preconstricted adrenal arteries with or without
intact endothelium caused relaxation responses
that were related to the number of ZG cells
added (maximal relaxations of 79±4%, with
endothelium, and 70±8%, without endothelium;
Fig. 1a). ZF cells relaxed adrenal arteries (data
not shown). ZG cell-induced relaxations were
not altered by indomethacin (10 µM; maximal
relaxation of 64±4%) or N-nitro-L-arginine, (LNA, 30 µM; maximal relaxation of 64±7%), but
were blocked by the large-conductance, Ca2+activated potassium (BKCa) channel inhibitor,
iberiotoxin (100 nM) and the EET antagonist
14,15-EEZE (2 µM).
Effect of ZG cells on vascular responses to
ACTH. We tested whether ZG cells influenced
the vascular responses to ACTH. ACTH did not
relax adrenal arteries preconstricted with
U46619 (maximal relaxation of 5±2%, Fig. 1b).
In the presence of a small number ZG cells (0.51x106 cells), ACTH (10-12-10-8 M) caused a
concentration-related relaxation (maximal
relaxation of 67±4%). Similar to ZG cells alone,
relaxations to ACTH in the presence of ZG cells
were not affected by endothelium removal
(maximal relaxation of 73±3%) or L-NA plus
indomethacin (maximal relaxation of 72±3%),
but were blocked by iberiotoxin and high
extracellular K+ ([K+]o) (60 mM). As a control,
similar experiments were performed with
isolated adrenal fibroblasts. In the presence of
adrenal fibroblasts (106 cells), ACTH did not
cause significant relaxations (maximal
relaxation of 6±8%, data not shown).
Role of arachidonic acid metabolites in ZGinduced relaxations. Relaxations to ACTH in
the presence of ZG cells were inhibited by the
cytochrome P450 inhibitors SKF 525A (10 µM;
maximal relaxation of 42±4%, Fig. 2a) or
miconazole (10 µM; maximal relaxation of
41±4%), and also inhibited by the EET
antagonist, 14,15-EEZE (2 µM; maximal
relaxation of 30±2%). SKF 525A did not inhibit
aldosterone synthesis and 14,15-EEZE did not
alter ACTH- or angiotensin II-induced
aldosterone synthesis by bovine ZG cells (data
not shown). In U46619-preconstricted arteries,
14,15-EET caused concentration-dependent
relaxations with a maximal relaxation of 86±1%
(Fig. 2b). The relaxations were blocked by
iberiotoxin and high [K+]o, and inhibited by
14,15-EEZE (maximal relaxation of 62±4%).
Arachidonic acid also relaxed indomethacin (10
µM) treated adrenal arteries with maximal
relaxations of approximately 60% at 10-5 M.
Similarly, these relaxations were blocked by
iberiotoxin, high [K+]o (data not shown) and
endothelium removal and inhibited by 14,15EEZE (Fig 2c).
ZG cells release a transferable relaxing
factor(s). To determine whether ZG celldependent relaxation involves a transferable
factor, ZG cells (2x106 cells/ml) were incubated
for 5-10 min in PSS at 37ºC. The cells were
removed by centrifugation and cell conditioned
medium tested for activity on U46619preconstricted arteries. ZG conditioned medium
relaxed adrenal arteries in a concentrationdependent manner (maximal relaxation of
66±4% at 100% medium (Fig. 3). Relaxations to
ZG-conditioned medium were blocked by
iberiotoxin and 14,15-EEZE.
The ZG conditioned medium was
extracted with ethyl acetate. This extract also
relaxed the adrenal arteries (data not shown),
indicating that the relaxing factor is extractable
by ethyl acetate. Since ZG cells release steroids
that can be extracted by ethyl acetate, we tested
several adrenal steroids for vascular activity.
Aldosterone and dexamethasone were without
effect on U46619-contracted arteries (Fig. 4).
Progesterone relaxed the arteries at 10-5 and 10-4
M; however, the relaxations were not blocked by
high [K+]o.
Metabolism of arachidonic acid by ZG cells.
To provide further evidence that EETs mediate
ZG cell-dependent relaxation, isolated bovine
ZG cells were incubated with 14C-arachidonic
acid and metabolites were extracted and
5
resolved by reverse phase-HPLC. The major
metabolites (fraction 110-120) comigrated with
the EETs (Fig. 5a). Other metabolites
comigrated with the prostaglandins (fractions
20-50), and dihydroxyeicosatrienoic acids
(DHETs), hydration products of EETs. EET
fractions were collected and further resolved by
normal phase HPLC and 14C-peaks comigrating
with 14,15-, 11,12-, 8,9- and 5,6-EETs were
detected (Fig. 5b). LC/MS analysis indicated a
major M-1 ion of 319 m/z for EET (Fig. 5c) and
337 m/z for DHET (Fig. 5d). These mass
spectra are consistent with EET and DHET
structures. In another set of experiments, ZG
cells (106 cells/ml) were incubated in the
absence and presence of ACTH for 10 min in
PSS at 37ºC, and the cells removed by
centrifugation. The supernatant was extracted
and analyzed by liquid chromatography/mass
spectrometry (LC/MS). Basal production of
14,15-, 11,12- and 8,9-EET by ZG cells was
31±7, 28±8 and 21±7 pg/ml, respectively.
Treatment of the ZG cells with ACTH
significantly (P<0.05) increased the production
of these EETs to 56±6, 60±10 and 39±6 pg/ml,
respectively, n = 9. EETs were not detected in
media without ZG cells.
Effect of ACTH on small adrenal arterial
diameter in situ. As a more physiological
approach, an artery in an adrenal cortical slice
was cannulated and perfused in situ (Fig. 6a).
Diameters of adrenal arteries embedded in the
ZG layer (100-300 µm) were measured by
videomicroscopy. Serotonin (50-200 nM) was
added to the perfusate to preconstrict the
arteries. Further addition of ACTH (10-9 M) to
the perfusate caused a significant dilation
(55±10%, Fig. 6b, supplement). Dilations were
similar across the length of the arterial tree. At
the end of experiment, India ink (3%) was
infused into the cannulated artery to demonstrate
the area of vascular perfusion. Perfused arteries
and the ZG cell layer surrounding the arteries
were stained, indicating the close anatomical
association between the perfused cortical arteries
and ZG cells. In addition, adrenal arteries
dissected and freed of adherent tissue, were
cannulated, perfused, and constricted with
serotonin. ACTH failed to dilate these arteries
(maximal relaxation of 10±8%, Fig. 6b).
Discussion
This is the first study to explore the role of ZG
cells in the regulation of adrenal vascular tone.
Our results demonstrate that these steroidsecreting cells produce a transferable vasoactive
factor(s) that relaxes small adrenal arteries and
ACTH stimulates the release of this factor(s).
Importantly, we provide evidence that the
vasorelaxing factor is a cytochrome P450
metabolite of arachidonic acid, the EETs and/or
related epoxy-metabolite(s). This interaction of
steroidogenic and vascular cells represents a
previous unknown mechanism for the regulation
of blood flow in the adrenal gland and represents
a mechanism coupling steroidogenesis to
increases in blood flow.
Three major vascular relaxing factors,
namely NO, prostacyclin and endotheliumderived hyperpolarizing factors that include
EETs, act in a paracrine manner. They are
produced by the endothelium and diffuse to the
smooth muscle to cause relaxation (24,29,30).
In the present study, addition of ZG cells to
preconstricted adrenal arteries caused
relaxations that were similar for endotheliumintact and denuded arteries. In addition, ZG
conditioned medium relaxed adrenal arteries.
These results indicate that ZG cell-dependent
responses involve a transferable relaxing
factor(s) that directly acts on vascular smooth
muscle cells to cause relaxation (Fig. 6c). The
relaxing factor(s) is not NO and prostacyclin,
since ZG cell-induced relaxations were not
altered by L-NA and indomethacin. In addition,
the short half-life of NO- and prostacyclin in
solution excludes their role as transferable
factors under our experimental conditions. The
lack of NO involvement is also consistent with a
previous report of our laboratory that bovine ZG
cells do not express NO synthase (NOS)
isoforms by the use of Western blot or
enzymatic activity assay (31). The expression of
NOS has been reported in adrenal cells of other
species. For example, both neuronal and
endothelial NOS mRNA and immunoreactivity
have been detected in rat adrenal zona
fasciculata cells, and endothelial NOS
expression in rat and human ZG cells (19,32,33).
Rat adrenal ZG cells metabolize arachidonic
acid to a number of oxygenated metabolites such
6
as prostaglandins, hydroxyeicosatetraenoic acid
(HETE) and EETs (34). In several blood vessels
including bovine small adrenal arteries, EETs
are vasoactive and mediate agonist-induced
relaxation responses via the activation of smooth
muscle BKCa channels (10,24). A recent study
by our laboratory of bovine coronary arteries
demonstrates that endothelium-derived EETs are
transferred to smooth muscle and cause dilations
(35). Similarly, we found that ZG cell-induced
relaxations were transferable, and blocked by the
BKCa channel blocker and the EET antagonist.
Using HPLC and LC/MS analysis, EETs were
identified in ZG cell conditioned medium.
Therefore, EETs represent the transferable
relaxing factor(s) released by ZG cells.
Extracellular release of arachidonic acid
provides for the exchange of free arachidonic
acid between vascular and other cells, which
could contribute to vascular tone regulation. For
example, platelet microparticles transfer
arachidonic acid to vascular endothelial cells,
resulting in increased production of the
vasodilator prostacyclin (36). Another recent
study showed that arachidonic acid released
from astrocytes, diffuses to adjacent vascular
smooth muscle cells, is converted to 20-HETE
and induces cerebrovascular constrictions (37).
However, the exchange of arachidonic acid does
not mediate ZG cell-induced relaxations of
adrenal arteries since arachidonic acid did not
relax endothelium-denuded adrenal arteries,
smooth muscle cells do not synthesize EETs
(data not shown) and ZG cells relax
endothelium-denuded adrenal arteries. Adrenal
steroids such as aldosterone, cortisol and
progesterone are transferable and vasoactive in
some vascular beds (38-40). In adrenal arteries,
aldosterone and cortisol were not active.
Progesterone relaxed adrenal arteries, but the
relaxation was not inhibited by the BKCa channel
blocker, iberiotoxin or high [K+]o. Therefore,
these steroids could not mediate the ZG cellinduced relaxations.
The pituitary hormone ACTH is a primary
regulator of adrenal cortical function. ACTH
stimulates cortisol and aldosterone secretion and
increases adrenal blood flow in vivo and in
perfused adrenal glands (6,11-14). In conscious
calves, ACTH stimulated the secretion of
cortisol and increased adrenal blood flow in the
same range of concentrations (41). However,
the threshold concentration of ACTH that
increased adrenal blood flow was higher than the
threshold concentration that increased cortisol
secretion. ACTH increased adrenal cortical
blood flow without changing adrenal medullary
blood flow (14). In the perfused rat adrenal
gland, ACTH increased perfusate flow and
corticosterone secretion in similar
concentrations (42). ACTH also increased
adrenal blood flow in anesthetized rats (43). In
contrast, the effect of ACTH is variable in some
other species such as the dog. ACTH was either
without effect or required high concentrations to
increase adrenal blood flow (13,14,44,45). The
reason for this variability between species is not
apparent.
In the ovine fetus, endogenous ACTH
regulates adrenal blood flow. Hypoxia in the
fetus increased the plasma concentrations of
ACTH and cortisol and increased adrenal
cortical blood flow (46,47). Cortisol was
infused during hypoxia to inhibit the pituitary
release of ACTH. Cortisol blocked the increase
in plasma ACTH concentration and inhibited the
increase in adrenal cortical blood flow. These
studies suggest that ACTH mediates the increase
in adrenal cortical blood flow caused by fetal
hypoxia.
While ACTH dilates adrenal arteries and
increases adrenal blood flow in vivo, it
had no effect on isolated adrenal arteries under
basal conditions (15). ACTH also did not relax
preconstricted adrenal arteries, indicating that
ACTH has no direct vasomotor effect on these
arteries. The action of ACTH on its target
tissues or cells depends on the activation of
specific receptors. Cloning of the melanocortin
receptor (MC-R) family has identified five
distinct G protein- and adenylate cyclasecoupled receptors that have a wide distribution
and diverse functions (48). The MC2-receptor
(MC2-R) preferentially binds ACTH. It is
mainly expressed in the ZG and zona fasciculata
of the adrenal cortex, and is hence considered to
be the main ACTH receptor (49,50). The
expression of the ACTH receptor is limited in
extra-adrenal tissues, although Northern blot
analysis has demonstrated its presence in rodent
adipocytes, human skin and human aortic and
umbilical vein endothelial cells (51,52). To our
7
knowledge, the ACTH receptor has not been
identified in adrenal vascular cells. The lack of
a direct vasomotor effect of ACTH on adrenal
arteries is consistent with these findings.
ACTH-induced relaxations of adrenal arteries
were revealed only with the presence of ZG
cells, as indicated by our in vitro isolated arteries
and in situ perfused adrenal slices. Similar to
relaxations induced by ZG cells or ZG
conditioned medium, relaxations were not
affected by endothelium removal, L-NA or
indomethacin, but were inhibited by high K+,
iberiotoxin, cytochrome P450 inhibitors and an
EET antagonist. LC/MS analysis showed that
ACTH stimulated the release of EETs from
adrenal ZG cells. Together, these results
provide strong evidence that ACTH stimulates
the release of a relaxing factor(s) (probably
EET) from ZG cells, which causes relaxations of
adrenal arteries. Interestingly, this indirect
effect of ACTH in the adrenal gland is not
limited to the regulation of adrenal vascular
tone. A recent study has suggested that ACTH
regulates vascular endothelial-cadherin
transcription in mouse adrenal endothelium
secondary to its indirect effect on steroidogenic
cells (53). Similarly, ACTH stimulates
expression and secretion of vascular endothelial
growth factor from adrenocortical cells, which is
responsible for the remodeling of adrenal
vasculature and adrenal cortex mass (54).
Hinson et al previously reported in perfused
rat adrenal glands that stimulation of histamine
and serotonin release with compound 48/80, a
mast cell degranulator, mimics the ACTHinduced increase in adrenal perfusate flow and
steroidogenesis. Additionally, sodium
cromoglycate, a mast cell stabilizer, blocked
ACTH-induced increase in perfusate flow
(2,11). Thus, the release of histamine and
serotonin from mast cells may mediate ACTHinduced increase in adrenal blood flow. We
recently reported that compound 48/80 relaxes
isolated bovine small adrenal arteries,
independent of histamine activation (16).
In the adrenal cortex, there are two cellular
sources of the EETs, i.e., endothelial cells and
ZG cells. We recently reported that
acetylcholine and endothelin B (ETB) receptor
agonists cause relaxation of isolated small
adrenal arteries which are partially mediated by
the activation of the endothelial EET pathway
(10,15). In the present study, ACTH activates
the ZG cell EET pathway. These results indicate
that activation of the specific EET pathway may
depend on the specific agonist involved.
Blood flow to the adrenal cortex delivers
stimulants (i.e., ACTH and angiotensin) and
substrates (i.e., oxygen and cholesterol) to
steroidogenic cells and exports secretory
products (i.e., corticosterone and aldosterone) to
the systemic circulation. Thus, it would be
advantageous to tightly regulate adrenal blood
flow to steroid hormone synthesis (1,2). Our
results on the regulation of vascular tone by
adrenal ZG cells provide a functional link and
mechanism for this close association of adrenal
blood flow and hormone synthesis. The
regulation of adrenal vascular tone by adrenal
ZG cell EET production may have broader
implications. This may represent a general
mechanism for regulating vascular tone in other
endocrine glands, where blood flow is matched
with hormone synthesis and secretion. EET
synthesis has been documented in the pancreas,
hypothalamus, pituitary gland, ovary and
placenta (55-59). Therefore, EET production by
glandular tissue may represent a paracrine
mechanism of blood flow regulation.
In summary, our data reveal a previous
unknown mechanism by which ACTH increases
blood flow in the adrenal gland. Specifically,
ACTH acts on adrenal ZG cells to stimulate the
release of EETs. EETs diffuse to adjacent
adrenal arteries, activate vascular smooth muscle
BKCa channels and cause relaxation (Fig 6c).
ACTH-induced increase in adrenal blood flow is
explained by this indirect effect on adrenal ZG
cells. This functional interaction of adrenal
steroidogenic cells and vascular cells may
contribute to the close coupling of blood flow
and hormone synthesis in the adrenal gland.
Acknowledgements
This research was supported by a grant from the
National Institutes of Health (HL-83297, GM31278, DK-58145)) and the Robert A. Welch
Foundation. Dr. Zhang is a postdoctoral fellow
of the American Heart Association, Greater
Midwest Affiliate and a recipient of the Jenkins
Cardiovascular Research Fellowship. The
authors thank Gretchen Barg for secretarial
8
assistance and Ms. Stacy Hitt and Andrea
Forgianni for technical assistance.
9
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13
Figure Legends
Figure 1. ZG cell-dependent relaxations of adrenal arteries. a, Tracings, ZG cell-induced relaxations in a
control artery (top) and in an artery pretreated with iberiotoxin (bottom). ZG cell-induced relaxations
were not altered by L-NA (30 µM), indomethacin (10 µM), and endothelium removal, but were blocked
by iberiotoxin (IbTX, 100 nM), and the EET antagonist 14,15-EEZE (2 µM). b, Tracings, effect of
ACTH in an artery without ZG cells (top) and in an artery with ZG cells (bottom). ACTH-induced
relaxations in the presence of ZG cells were inhibited by iberiotoxin (100 nM) and high [K+]o (60 mM)
but not affected by L-NA plus indomethacin, or endothelium removal. Arteries were precontracted with
U46619. n=4-18, * P<0.05 vs. control.
Figure 2. Role of arachidonic acid metabolites in ZG cell-dependent relaxations to ACTH. a, ACTHinduced relaxations after the addition of ZG cells were inhibited by 14,15-EEZE (2 µM), and the
cytochrome P450 inhibitors, SKF 525A (10 µM) and miconazole (10 µM). b, 14,15-EET-induced
relaxations were inhibited by 14,15-EEZE (2 µM), and blocked by high [K+]o (60 mM) and iberiotoxin
(100 nM). c, Arachidonic acid-induced relaxations were inhibited by 14,15-EEZE and blocked by
iberiotoxin or endothelium removal. Arteries were precontracted with U46619. n=4-18, * P<0.05 vs.
control.
Figure 3. ZG conditioned medium-induced relaxations. a, Tracings, relaxations of a control artery (top)
and an iberiotoxin-treated artery (bottom) to ZG conditioned medium. b, ZG conditioned mediuminduced relaxations were blocked by iberiotoxin (100 nM) and 14,15-EEZE (2 µM). Experiments were
performed on endothelium-intact arteries in the presence of L-NA (30 µM) and indomethacin (10 µM).
Arteries were precontracted with U46619. n=4-18, * P<0.05 vs. control.
Figure 4. Effect of adrenal steroids on vascular tone of adrenal arteries. a, In U46619-preconstricted
arteries, aldosterone and dexamethasone had no vasomotor activity. b, Progesterone relaxed U46619contracted arteries at high concentrations. These relaxations were not altered by high [K+]o (60 mM).
n=8-10.
Figure 5. Arachidonic acid metabolism in ZG cells. a, ZG cells were incubated with [14C] arachidonic
acid (AA; 0.05 mCi, 100 nM), and metabolites were extracted and resolved by reverse-phase HPLC.
[14C]-Metabolites (fractions 110-120) comigrated with EETs. b, EET fractions were collected and further
resolved by normal phase HPLC. [14C]-metabolites comigrated with 14,15-, 11,12-, 8,9- and 5,6-EETs.
Migrations times of known standards are noted on the chromatogram. c,d, LC/MS analysis confirmed the
molecular identity of 14,15-EET and 14,15-DHET. 14,15-EET and 14,15-DHET produced major ions of
319 m/z and 337 m/z (M-1), respectively.
Figure 6. ACTH relaxes in situ adrenal cortical arteries. a,b, Arteries embedded in adrenal cortical
slices were cannulated, perfused and contracted with serotonin. ACTH (10 nM) was added to the
perfusate. At the end of the experiment, India ink was infused into the cannulated arteries to determine
the extent of the perfused vascular bed. Arteries dissected from the adrenal cortical surface and
constricted with serotonin did not dilate to ACTH. n=4-5, * P<0.05 vs. control. c, Proposed mechanism
of ACTH-induced relaxations. ACTH acts on adrenal ZG cells, mobilizes arachidonic acid from
membrane phospholipids (PL) and EETs are produced via cytochrome P450 epoxygenase. EETs diffuse
to vascular smooth muscle cells and activate BKCa channels to cause relaxation.
14
a
U46
Control
0.1
ZG cells (x106)
1
5
10
b
•
•
ACTH (Log M)
-12 -11 -10 -9 -8
• • • • •
U46
Control
•
•
2 mN 2 min
2 mN
•
IbTX
ZG cells (x106)
0.1 1
5
10
•
•
•
•
% Relaxation
80
60
•
ZG cells
•
•
5 min
•
•
Control
-Endothelium
Indomethacin
L-NA
IbTX
14,15-EEZE
80
60
20
0
*
*
4
6
8
ZG cells (x106)
10
*
-20
0
2 mN
•
40
Fig 1
ACTH (Log M)
-12 -11 -10 -9 -8
2 mN
•
100
U46 ZG
wash
% Relaxation
U46
•
5 min
40
2 min
• •
Control
-Endothelium
L-NA+Indomethacin
IbTX
KCl (60 mM)
-ZG cells
20
*
*
-11 -10 -9
ACTH (Log M)
-8
*
0
*
-20
2
ZG
-12
% Relaxation
a
70
Control
14,15-EEZE
SKF 525A
Miconazole
50
*
*
*
30
*
10
-10
ZG
b
% Relaxation
-8
*
60
40
20
-20
*
*
0
% Relaxation
-11 -10 -9
ACTH (Log M)
Control
14,15-EEZE
KCl (60 mM)
IbTX
100
80
c
-12
-9
80
*
-8
-7
-6
14,15-EET (Log M)
-5
Control
14,15-EEZE
IbTX
Denuded
60
40
*
0
*
*
*
20
*
*
-20
-9
Fig 2
-8
-7
-6
-5
Arachidonic acid (Log M)
U46
Control
% ZG medium
2.5 25 50 100 wash
•
• •
IbTX
•
•
2 mN 2 min
•
Fig 3
Control
IbTX
14,15-EEZE
80
60
•
•
2 mN 2 min
•
b
•
•
•
% Relaxation
a
40
20
*
*
0
*
-20
-40
*
*
0
25
50
75
100
% Conditioned Medium
100
50
b
Dexamethasone
Aldosterone
0
-50
-100
Fig 4
100
% Relaxation
% Change in Tension
a
Control
KCl (60 mM)
80
60
40
20
0
-20
-9 -8 -7 -6 -5 -4
Concentration (Log M)
-9
-8 -7 -6 -5 -4
Progesterone (Log M)
50
500
300
Fig 5
50
100
Fraction
319.3
20
315
m/z
14,15-DHET
320
325
100
100
0
40
d
200
0
60
0
150
Relative Abundance
400
CPM
100
80
150
200
80
60
338.3
50
Fraction
5,6 -EET
b
0
14,15-EET
11,12-EET
8,9 -EET
0
14,15-EET
320.3
Relative Abundance
100
100
337.3
AA
EETs
DHETs
150
CPM
c
6-Keto-PGF1α
a 200
40
20
0
330
335
m/z
340
345
a
Serotonin + ACTH
Serotonin
Serotonin
India Ink
µm
500 µm
% Relaxation
b 80
*
c
ZG Cell
60
40
Adrenal Artery
Smooth Muscle Cell
20
0
Isolated
Arteries
Fig 6
ACTH
Slice
Arteries
PL Arachidonic
Acid
SKF525A
P450 X Miconazole
EETs
14,15-EEZE
X
IbTX
X
K+
Vascular Relaxation