Ethanol and nitric oxide modulate expression
of glucocorticoid receptor in the rat adrenal cortex
Dragoslava Djikiæ1, Mirela Budeè1, Sanja Vranješ-Djuriæ2, Vera Todoroviæ1,
Neda Drndareviæ1, Sanja Vignjeviæ1, Olivera Mitroviæ1
1University of Belgrade, Institute for Medical Research, PO Box 39, 11129 Belgrade 102, Serbia
2University of Belgrade, Institute for Nuclear Sciences “Vinèa”, 11001 Belgrade, Serbia
Correspondence: Dragoslava Djikiæ, e-mail: [email protected]
Abstract:
Background: This study was performed to investigate expression and distribution of glucocorticoid receptor (GR) in the rat adrenal
cortex, acute effect of ethanol on its expression and possible role of endogenous nitric oxide (NO) in this phenomenon.
Methods: Adult female Wistar rats showing diestrus day 1 were treated with: a) ethanol (2 or 4 g/kg body weight (b.w.), ip),
b) Nw-nitro-L-arginine methyl ester (L-NAME), well-known competitive inhibitor of all isoforms of NO synthase (NOS), (30 mg/kg
b.w., sc) followed by ethanol (4 g/kg, ip) 3 h later and c) L-NAME (30 mg/kg b.w., sc) followed by saline (ip) 3 h later. Untreated rats
were used as controls. Adrenocortical expression of GR was estimated by immunohistochemistry.
Results: Strong nuclear GR staining was observed throughout the cortex of control rats. Acute ethanol treatment significantly de-
creased the expression of GR in the zona fasciculata and zona reticularis. Blockade of NO formation had no influence on this effect
of ethanol, whereas L-NAME itself induced significant decline in GR immunoreactivity.
Conclusions: Obtained findings are the first to demonstrate localization and distribution of the GR throughout the rat adrenal cortex
and to suggest that ethanol as well as endogenous NO may modulate adrenocortical expression of this steroid receptor.
Key words:
ethanol, L-NAME, glucocorticoid receptor, adrenal cortex
Introduction
Glucocorticoids (GC), steroid hormones synthesized
and secreted at high levels by the adrenal cortex, in-
fluence the activity of almost every cell in the body
[6]. They exert pleiotropic actions which are essential
for the maintenance of homeostasis and responses to
stressors. Because of their antiinflammatory and im-
munosuppressive effects, synthetic glucocorticoid
agonists are widely used in the treatment of autoim-
mune diseases, inflammatory disorders [29], and ma-
lignancies of the lymphoid system [18].
At the cellular level, the physiological and pharmacol-
ogical actions of GC are predominantly mediated through
intracellular glucocorticoid receptor (GR) that belongs to
the nuclear receptor family of ligand-dependent transcrip-
tion factors. In the absence of the ligand, GR is located in
the cytoplasm within a protein complex that includes vari-
ous heat-shock proteins. After activation, GR is released
from the multiprotein complex, dimerizes, and translo-
cates to the nucleus, where it binds to specific DNA se-
quences called glucocorticoid response elements [33].
The importance of GR is emphasized by the finding that
inactivation of the GR gene is incompatible with life [10].
896 Pharmacological Reports, 2012, 64, 896�901
Pharmacological Reports2012, 64, 896�901ISSN 1734-1140
Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences
In addition to other actions, GC through GR-me-
diated negative feedback loop at the hypothalamic and
pituitary levels terminate the HPA (hypothalamic-
pituitary-adrenal) axis response to stress [14]. Simi-
larly, the expression of GR within the adrenal cortex
in humans [27] and ovine fetuses [31] imply that glu-
cocorticoid feedback may occur within the gland it-
self. However, very little information is available
about the expression and distribution of the GR in the
rat adrenal gland [17].
Although ethanol is known as a stressor [2, 25, 30],
data regarding its acute effect on GR expression are
missing. Also, there is evidence that nitric oxide (NO)
may be involved in some effects of alcohol [1, 35] as
well as that this signal molecule affects the activity of
HPA axis [9, 22, 23]. In a view of these observations,
the present study was designed to investigate (a) the
expression and distribution of GR in the rat adrenal
cortex, (b) whether acute ethanol treatment alters its
adrenocortical expression and (c) possible influence
of endogenous NO on this effect of alcohol.
Materials and Methods
Animals
Adult female Wistar rats (obtained from the Breeding
Colony of the Medical Military Academy, Belgrade),
10–12 weeks of age and 200–250 g of weight, were
used in this study. They were housed five per cage in
temperature-controlled room with light on at 6:00 a.m.
and off at 18:00 p.m. The animals were provided with
standard laboratory diet (Veterinarski zavod, Subotica)
and water ad libitum. The stages of estrous cycle were
monitored every day by vaginal smears, and only the
rats showing the diestrus day 1 were used in this study.
Chemicals
L-NAME (Sigma Co.) was dissolved in apyrogenic
saline just before use. The choice of dose of
L-NAME, regimen of administration and route of in-
jection were based on previous studies [8, 35]. Etha-
nol (SUPERLAB, Belgrade, Serbia) was diluted with
sterile saline up to 35% (v/v) and the animals were
treated with different volumes to reach concentration
of 2 or 4 g/kg.
Experiment
In order to avoid circadian oscillations, the experi-
ments were carried out between 8:00 and 12:00 a.m.
The animals were weighed and injected with: a) etha-
nol (2 or 4 g/kg b.w., ip), b) Nw-nitro-L-arginine methyl
ester (L-NAME), well-known competitive inhibitor of
all isoforms of NO synthase (NOS), (30 mg/kg b.w.,
sc) followed by ethanol (4 g/kg, ip) 3 h later and
c) L-NAME (30 mg/kg b.w., sc) followed by saline
(ip) 3 h later. Untreated rats were used as controls.
The rats were sacrificed by decapitation 30 min after
last injection. Blood samples were collected and left
adrenal glands were promptly removed, fixed in 10%
buffered formalin for 24 h, and embedded in paraffin.
The protocol was in accordance with local institu-
tional guidelines for the care and use of laboratory
animals. The investigation also conformed to the prin-
ciples and guidelines of Conseil de l’Europe (pub-
lished in the Official Daily N. L358/1-358/6, 18th De-
cember 1986), the U.S. National Institutes of Health
(Guide for the Care and Use of Laboratory Animals,
NIH publication no. 85-23) and the Canadian Council
on Animal Care (CCAC).
Corticosterone determination
Serum levels of corticosterone were measured by
Amersham Biotrak rat corticosterone [125I] assay
system using magnetic separation with CV = 5%, per
run. The groups for hormone determination consisted
of five to seven rats.
Immunohistochemistry
Immunohistochemical staining was performed on
6 µm thick paraffin sections of adrenal gland. After
deparaffinization in xylene followed by dehydration
with descending series of alcohols and rehydration in
distilled water, slides were heated for 2 min under
high pressure in 10 mmol/l citrate buffer, pH 6 for
epitope retrieval. The endogenous peroxidase was in-
activated by incubation with 3% hydrogen peroxide in
distilled water for 10 min. Rabbit polyclonal anti-
mouse GR antibody (M-20): sc-1004, Santa Cruz
Biotechnology Inc. diluted (1:200) in DAKO Anti-
body Diluent (S0809) was applied and the sections
were incubated in a humidity chamber for 3 h at the
room temperature. After washing with phosphate
buffered saline (PBS), a refined avidin-biotin tech-
Pharmacological Reports, 2012, 64, 896�901 897
Ethanol, L-NAME and glucocorticoid receptorDragoslava Djikiæ et al.
nique in which a biotinylated secondary antibody re-
acts with several peroxidase-conjugated streptavidin
molecules was employed for amplification using
a DAKO LSAB+/HRP kit. Diaminobenzidine tetrahy-
drochloride (DAB) was used for the visualization of
GR-immunoreactive (ir) cells. Finally, the nuclei were
counterstained with Mayer’s hematoxylin. For control
staining, primary antibody was omitted.
The analysis was performed by the light micro-
scope Olympus AX70 with an objective magnifica-
tion of 40× and software – analySIS Pro 3.1. The ex-
pression of GR in the cortical zones was evaluated on
the basis of the intensity of immunohistochemical
staining: weak (+), moderate (++) and strong (+++).
The results are expressed as the score of intensity and
the percentage of GR-ir cells (number of cells out of
100-stain positive for hormone receptor).
Statistical analysis
Data are presented as the mean ± standard error of the
mean (SE) of each group. One way analysis of vari-
ance (ANOVA) with Fisher’s post-hoc LSD test was
employed for calculation of statistical significance.
The differences between group means were consid-
ered significant if p < 0.05.
Results
Immunohistochemical method was used to investigate
the presence of GR in the cortex of the rat adrenal
gland. Analyzing the slides obtained from normal
rats, we detected diffuse staining pattern restricted to
the nuclei of the steroidogenic cells. The intensity of
the staining was moderate to strong with almost the
same immunoreactivity in the zona fasciculata (ZF)
and zona reticularis (ZR).
Figures 1 and 2 illustrate the expression of GR in
the ZF and the ZR of control and treated rats. Both
doses of ethanol (2 and 4 g/kg) induced decline in the
level of immunoreactivity (Fig. 3 A and B). Treatment
898 Pharmacological Reports, 2012, 64, 896�901
Fig. 1. Micrographs display GR nu-clear staining in cells of the zona fas-ciculata. In control rats (a), GR-im-munorectivity is more frequent than inrats treated with ethanol 2 g/kg, ip (b);ethanol 4 g/kg, ip (c); L-NAME, 30 mg/kg, sc, followed by ethanol 4 g/kg, ip(d) and L-NAME 30 mg/kg, sc, fol-lowed by saline, ip (e). The sectionwhere the primary antibody was omit-ted served as control staining (f)
Fig. 2. Micrographs display GR nu-clear staining in cells of the zona re-ticularis in control rats (a), and aftertreatment with ethanol 2 g/kg, ip (b);ethanol 4 g/kg, ip (c); L-NAME, 30 mg/kg, sc, followed by ethanol 4 g/kg, ip(d) and L-NAME 30 mg/kg, sc, fol-lowed by saline, ip (e). The sectionwhere the primary antibody was omit-ted served as control staining (f)
with L-NAME (30 mg/kg) had a similar effect on
the number of immunoreactive cells in both cortical
zones. The largest decrease of immunoreactivity
was observed in the group treated with L-NAME
(30 mg/kg) followed by ethanol (4 g/kg) (Fig. 3 A and
B). All experimental groups showed a significant dif-
ference in the expression of GR compared with con-
trols (ZF: F = 10.439, p = 0.000; ZR: F = 5.937, p =
0.002).
In order to get insight into the function of the adre-
nal cortex, we measured concentrations of the serum
corticosterone in rats. As can be seen in Figure 4, hor-
mone level was elevated in all experimental groups.
Statistical analysis demonstrated significant differences
between control and treated animals (F = 28.855,
p = 0.000). Alcohol increased the level of corticoster-
one in dose-dependent manner and pretreatment with
L-NAME (30 mg/kg) had no influence on this effect of
ethanol. Administration of L-NAME prior to saline
also elevated the hormone concentration.
Discussion
To the best of our knowledge, we demonstrated for
the first time strong nuclear GR staining in the corti-
cal zones of control rats. This finding supported an
earlier study showing dexamethasone-binding sites in
the rat adrenal cortex [21]. Illera et al. [17] did not
find any GR immunoreactivity in the adrenal cortex
of saline-treated Long Evans rats. Discrepancy be-
tween their study and our could be due to methodo-
logical differences such as the rat strain [24] and pri-
mary antibody [34]. Immunohistochemical analysis
revealed GR staining in the ovine fetal zona glomeru-
losa and in the transitional zona between the ZF and
the ZR [31]. In normal human adrenal cortex, the ex-
pression of the GR was also described [27] with
strong immunostaining especially in the ZR. Under
pathological conditions, increased expression of the
GR was detected in both human cortisol-producing
adenoma [5] and malignant adrenocortical tumors
[36] suggesting a possible role of this steroid receptor
in their pathogenesis.
As we showed previously [7, 25] that acute ethanol
treatment stimulated the activity of adrenal cortex, in
the current study, we wanted to determine whether al-
cohol influences the expression of the GR in this
gland. Using the same experimental model, we re-
Pharmacological Reports, 2012, 64, 896�901 899
Ethanol, L-NAME and glucocorticoid receptorDragoslava Djikiæ et al.
Fig. 3. Effects of ethanol (2 and 4 g/kg, ip), L-NAME (30 mg/kg, sc)followed by ethanol (4 g/kg, ip) or L-NAME (30 mg/kg, sc) followed bysaline, (ip) on the expression of glucocorticoid receptor in zona fas-ciculata (A) and zona reticualris (B) of rat adrenal cortex. Data are ex-pressed as the score of GR immunoreactive cells (the means ± SE).(ANOVA, followed by LSD post-hoc test); * p < 0.05, ** p < 0.01 com-pared with control. EtOH2, ethanol 2 g/kg; EtOH4, ethanol 4 g/kg;LN30, L-NAME 30 mg/kg
Fig. 4. Effects of ethanol (2 and 4 g/kg, ip), L-NAME (30 mg/kg, sc)followed by ethanol (4 g/kg, ip) and L-NAME (30 mg/kg, sc) followedby saline (ip) on concentrations of serum corticosterone in rats30 min after last injection. Data are expressed as the means ± SE.(ANOVA, followed by LSD post-hoc test); ** p < 0.01 comparedwith control. EtOH2, ethanol 2 g/kg; EtOH4, ethanol 4 g/kg; LN30,L-NAME 30 mg/kg
ported earlier [8] that blood ethanol levels, 30 min af-
ter intraperitoneal treatment at doses of 2 or 4 g/kg,
fulfill criterion (> 21.7 mmol/l) for intoxication. The
results, obtained by analysis of GR-ir cells, revealed
that both doses of ethanol (2 and 4 g/kg) significantly
decreased the expression of GR in the ZF and ZR.
Observed phenomenon might be a consequence of
downregulation of this receptor by the increased level
of corticosterone measured in ethanol-treated rats.
A possible explanation for these results is consistent
with dexamethasone-induced downregulation of GR
mRNA, independent of protein synthesis, in both he-
patoma tumor cell line and in the rat liver [26]. This
effect of dexamethasone was confirmed in other rat
tissues, including the adrenal gland where GR mRNA
concentration was found to be reduced by 60% [20].
Our findings cannot be directly compared to those
reported by other authors because as far as we know
there are no studies on the rat adrenal GR following
acute ethanol administration. Decreased expression of
GR in the nucleus paraventricularis and various
amygdaloid structures was associated with chronic al-
cohol exposure [32].
Interactions between ethanol and NO have been
well documented [16] and some effects of ethanol are
mediated by this signal molecule [1]. Ethanol affects
NO synthesis through NOS or other mechanisms [13,
37]. On the other hand, iNOS can catalyze ethanol
oxidation to acetaldehyde via the generation of a-
hydroxyethyl radical [28] and NO interacts with etha-
nol to form ethyl nitrite [15]. Among other actions,
NO is involved in ethanol-induced: release of b-endo-
rphin from hypothalamic cells [4], upregulation of
TLR2 [3], modulation of intestinal IgA [8], and
ACTH response [35]. To assess whether the inhibition
of synthesis of NO alters the acute effect of ethanol on
the adrenocortical expression of the GR, L-NAME
was used. Our results showed that pretreatment with
L-NAME had no influence on ethanol-induced decline
in GR immunoreactivity. Therefore, it seems that this
action of ethanol is not mediated by NO under these
experimental conditions. However, L-NAME itself
decreased the expression of GR suggesting modula-
tory role of endogenous NO. Bearing in mind that rat
adrenocortical cells produce NO [11], it is possible
that this signal molecule affects GR expression in
autocrine/paracrine manner. In addition, enhanced
levels of corticosterone, obtained in all experimental
groups showing decreased score of GR-ir cells, might
be responsible for altered adrenocortical expression of
this steroid receptor.
Since there is no data regarding the influence of en-
dogenous NO on the expression of GR within adrenal
cortex, our findings cannot be considered in view of
others. Only, there is evidence that exogenous NO ac-
tivates endothelial GR in cell culture [19] and in-
creases expression of GR in the lung, liver and kidney
in porcine endotoxin sepsis [12].
Obtained results are the first to demonstrate nuclear
localization and distribution of the GR throughout the
rat adrenal cortex and to suggest that ethanol as well
as endogenous NO may modulate its adrenocortical
expression. Further studies are needed to determine
the mechanisms of observed effects.
Acknowledgment:
This work was supported by a grant (175053) from the Ministry
of Education and Science of Republic of Serbia. We are very
grateful to Mrs. Leposava Jovanoviæ and Mrs. Sne�ana Markoviæ
for their excellent technical assistance.
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Received: August 1, 2011; in the revised form: April 5, 2012;
accepted: April 23, 2012.
Pharmacological Reports, 2012, 64, 896�901 901
Ethanol, L-NAME and glucocorticoid receptorDragoslava Djikiæ et al.