Ethanol and nitric oxide modulate expression of glucocorticoid...

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


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-



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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