brain nitric oxide synthases in the interleukin-1b-induced...
TRANSCRIPT
Brain nitric oxide synthases in the
interleukin-1b-induced activation of
hypothalamic-pituitary-adrenal axis
Anna G¹dek-Michalska, Joanna Tadeusz, Paulina Rachwalska,
Jadwiga Spyrka, Jan Bugajski
Department of Physiology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków,Poland
Correspondence: Anna G¹dek-Michalska, e-mail: [email protected]
Abstract:
Background: Interleukin-1b (IL-1b), the major cytokine involved in activation of hypothalamic-pituitary-adrenal (HPA) axis
modulates both central and peripheral components regulating HPA activity. The role of nitric oxide (NO) generated by neuronal ni-
tric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structures involved in HPA axis regulation has not
been elucidated. The aim of the study was to assess the receptor selectivity of IL-1b stimulatory action on HPA axis and to determine
the involvement of nNOS and iNOS in this stimulation.
Methods: Experiments were performed on male Wistar rats which were injected intraperitoneally (ip) with IL-1b (5 µg/kg) or IL-1
receptor antagonist (IL-1ra) (50 µg/kg or 100 µg/kg) 15 min before IL-1b. Rats were sacrificed by rapid decapitation 1, 2 or 3 h after
IL-1b administration. Trunk blood for ACTH, corticosterone and IL-1b determinations was collected and prefrontal cortex, hippo-
campus and hypothalamus were excised and snap frozen. Western blot analyses were performed and IL-1b, nNOS and iNOS protein
were determined in brain structures samples.
Results: IL-1b significantly increased plasma ACTH, corticosterone and IL-1b levels during 2 h after ip administration. IL-1 recep-
tor antagonist was able to abolish the stimulatory effect of IL-1b on plasma ACTH and corticosterone levels and significantly, but
not totally, reduced plasma IL-1b level. The role of NO in prefrontal cortex, hippocampus and hypothalamus in the IL-1b-induced
HPA axis activity alterations was determined by measuring the changes in nNOS and iNOS levels. The highest level of both izoen-
zymes 1 h following IL-1b administration decreased in a regular, parallel manner 2 and 3 h later, approaching control values. These
changes were almost totally prevented by pretreatment with IL-1 receptor antagonist. In the hypothalamus the IL-1b-induced initial
significant increase of nNOS regularly decreased in a modest rate and remained at significant higher level compared to control val-
ues. By contrast, iNOS level gradually increased 2 and 3 h after IL-1b administration in a significant time-dependent manner. The
changes in both NOS izoenzyme levels in hypothalamus were suppressed by pretreatment with IL-1 receptor antagonist. Results also
show that a regular and parallel decrease of nNOS in the hypothalamus and prefrontal cortex are parallel in time and magnitude to re-
spective fall in plasma IL-1b and ACTH levels.
Conclusion: The present study suggests that the IL-1b-induced transient stimulation of HPAaxis activity is parallel in time and mag-
nitude to the respective changes of nNOS in hypothalamus and prefrontal cortex, the brain structures involved in regulation of HPA
axis activity.
Key words:
interleukin-1b, nitric oxide synthases, interleukin-1 receptor antagonist, immuno-endocrine responses, limbic-hypothalamic-adrenal axis
Pharmacological Reports, 2012, 64, 1455�1465 1455
Pharmacological Reports2012, 64, 1455�1465ISSN 1734-1140
Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences
Introduction
Cytokines secreted by cells of the immune system
may also be synthesized and secreted by non immune
cells and act as signal transmitters on neuroimmune
cells [2, 5, 6]. Interleukin-1b (IL-1b) is one of the ma-
jor cytokine involved in a stereotyped set of re-
sponses, including the regulation of HPA axis that is
mediated by the brain [16, 25]. Peripheral administra-
tion of IL-1b in rats activates the HPA axis resulting
in increased plasma levels of ACTH and glucocorti-
coids. IL-1b is expressed in all components of the
HPA axis and affects all its levels stimulating the se-
cretion of corticotrophin releasing hormone (CRH)
from the hypothalamus and ACTH stimulates the se-
cretion of glucocorticoids from adrenal cortex [10, 13,
21]. Furthermore, IL-1b also directly induces ACTH
secretion from the pituitary via IL-1 type I receptors
and also stimulates the secretion of corticosterone
from the adrenal, although IL-1 receptors were not
found in this gland [1, 7, 36].
Although hypothalamic CRH is considered a primary
mechanism by which cytokines stimulate HPA axis, evi-
dence indicates a direct action of cytokines at the level
of pituitary and adrenal glands. In addition to circulating
cytokines which may act upon all three levels of the
HPA axis, cytokines are generated in the brain, the ante-
rior pituitary and the adrenal cortex [2, 16].
Although the blood-brain barrier (BBB) presents
a physical barrier to cytokine-CNS interaction, they
may enter the brain 1) via fenestrated endothelium of
the circumventricular organs (CVOs) [8, 27] 2) via
limited active transport system, 3) induction of gen-
eration of cytokines from cells of the BBB, which
then secrete cytokines into the brain parenchyma [31,
32, 34] and 4) a transport across the BBB by infiltrat-
ing leukocytes. Cytokine may act at a distance by af-
fecting the CNS via noradrenergic, serotonergic, do-
paminergic or GABA-ergic pathways [20, 30, 39] and
stimulate ascending visceral peripheral neural path-
ways [29], mainly vagus nerves which convey signals
to endocrine neurons in the paraventricular nucleus
(PVN) and supraoptic nuclei (SON) of the hypothala-
mus [9]. Vagal stimulation significantly activates
magnocellular and parvocellular endocrine neurons
within the PVN [19] and SON in rats to the CRH neu-
rons within the PVN. Viscerosensory inputs to the
CRH neurons within the PVN are preferentially nora-
drenergic. In the hypothalamus the noradrenergic in-
nervation of the paraventricular nuclei has been
shown to mediate the IL-1 activation of the HPA axis.
Cytokine normally produced by peripheral immune
cells are also generated by CNS glial cells. Cytokine
can markedly affect neurotransmission within emo-
tion regulatory brain circuits. Neuroimmune interac-
tions involving peripheral and glial derived cytokines
play an important role in depression [17].
Nitric oxide (NO) as a signaling molecule among
various cell types and systems serves also as a neuro-
transmitter in the brain [20, 22, 23, 35]. It is synthe-
sized by nitric oxide synthase, neuronal (nNOS) and
inducible (iNOS). Under physiological conditions
nNOS is constitutively expressed, and it is the major
isoenzyme of NOS in the brain. Inductible NOS is un-
detectable under basal conditions and it is upregulated
in response to various stimuli such as inflammatory
cytokines and stress [4, 14, 24, 40]. NO generated by
iNOS in the brain is involved in central CRH-induced
elevation of plasma CRH, but it is not clear whether
nNOS plays a role in these CRH induced elevations.
Neuronal NOS is highly expressed by cells of the
hypothalamic PVN, where the sympatho-adrenal sys-
tem, the HPA axis and the hypothalamo-neurohypo-
physeal system originates. These structures regulate
the neuroendocrine stress responses. NO generated by
nNOS plays a significant role in modulating the activ-
ity of these systems under acute stressor exposure [26,
28]. NOS constitutive forms are found in PVN CRH
and AVP neurons as well as in direct and indirect af-
ferents to the PVN such as hippocampus, the dor-
somedial lateral part of the hypothalamus and amyg-
dale [33]. However, there is not always concordance
between the regions of the hippocampus or amygdale
that projects to the PVN.
Stimulation of hippocampus lowers glucorticoid
secretion in rats and humans, lesions of hippocampus
increase corticosterone release. Hippocampus damage
also increases PVN CRH and vasopressin gene ex-
pression and extent of PVN neural activation after
stress, suggesting that hippocampal inhibition of the
HPA is mediated via PVN [18].
Medial prefrontal cortex (MPC) is implicated in
HPA axis inhibition. Lesions of prelimbic and anterior
cingulated subregions of the MPC increases ACTH
and corticosterone secretion and PVN c-Fos mRNA
induction after restraint stress. The role of corticoster-
one in HPA integration may depend on subregion and
hemisphere.
1456 Pharmacological Reports, 2012, 64, 1455�1465
This study was designed to evaluate the HPA re-
sponse to a challenge dose of IL-1b 1, 2 and 3 h after
administration. Also the role of NO in prefrontal cortex,
hippocampus and hypothalamus in the IL-1b-induced
HPA axis activity alterations was assessed by measur-
ing the changes in nNOS and iNOS protein levels.
Materials and Methods
Animals
Adult male Wistar rats (190–220 g; Charles River
Laboratories, Sulzfeld, Germany) were used in all the
experiments. The animals were kept in groups of 5 per
cage (52 × 32 × 20 cm) under controlled conditions
(a 12/12 h light/dark cycle with the light on at 7:00 a.m.,
a room temperature of 22 ± 2°C), with free access to
standard laboratory food and tap water. Rats were
given 1-week acclimation period before experimental
session. All experiments were carried out in accor-
dance with bioethical requirements and protocols were
approved by the Local Bioethics Commission for
Animal Experiments at the Institute of Pharmacology,
Polish Academy of Sciences, Kraków, Poland.
Experimental procedure
Experiments were performed on 3 main groups of
rats: control animals injected intraperitoneally (ip)
with saline, animals injected ip with a single dose of
IL-1b (5 µg/kg, Sigma-Aldrich) and animals injected
ip with a dose of IL-1 receptor antagonist (50 µg/kg
or 100 µg/kg, Sigma-Aldrich) followed by IL-1b ad-
ministration 15 min later. Rats were sacrificed by
rapid decapitation 1 h, 2 h or 3 h after IL-1b admini-
stration. Trunk blood for plasma corticosterone and
IL-1b determinations was collected in the presence of
EDTA (2.5 mg/ml of blood, Sigma-Aldrich) or for
ACTH immunoassay in the presence of EDTA and
aprotinin (0.6 TIU/ml of blood; Sigma-Aldrich) and
centrifuged in 4°C within 30 min after collection.
After decapitation, the brain was rapidly removed
from the skull and prefrontal cortex, hippocampus
and hypothalamus were excised on ice-cold glass
plates. The tissues were immediately frozen on dry
ice and stored at –70°C. Protein extracts were pre-
pared according to G¹dek-Michalska et al. [15]. Total
corticosterone, ACTH and IL-1b levels were measured
in plasma using commercially available Rat/Mouse
Corticosterone EIA kit (Immunodiagnostic Systems),
ACTH Rat/Mouse EIA kit (Phoenix Pharmaceuticals)
and Rat IL-1b Platinum ELISA kit (eBioscience).
Western blot
Denaturated proteins (10 µg per lane) were separated us-
ing a 7.5% (for nNOS, iNOS) or 15% (for IL-1b) SDS-
polyacrylamide gel electophoresis (Mini PROTEAN®
Tetra Cell, Bio-Rad) under constant voltage (90 V in
stacking gel; 150 V in resolving gel), and were trans-
ferred electrophoretically (90 V, 1 h) onto a nitrocellu-
lose membrane (0.45 µm, Bio-Rad). The membranes
were NOS2 polyclonal antibody in dilution 1:400
(Santa Cruz Biotechnology), and to determine IL-1b
(17.5 kDa) level we used rabbit anti-IL-1b polyclonal
antibody in dilution 1:750 (Thermo Scientific). b-
Actin levels were determined with rabbit anti-b-actin
polyclonal antibody (1:400, Santa Cruz Biotechnol-
ogy) and used for normalization of nNOS, iNOS and
IL-1b bands. After incubation, the membranes were
washed four times for 10 min with TBST (TBS-0.1%
Tween 20) and finally incubated with goat anti-rabbit
horseradish peroxidase-conjugated secondary anti-
body (1 : 10,000, Santa Cruz Biotechnology) in 1%
non-fat dry milk in TBS-0.05% Tween 20 for 1 h at
room temperature. Afterwards, the membranes were
washed four times for 15 min with TBST, and the pro-
teins were detected using Immun-Star HRP Chemilumi-
nescence Kit (Bio-Rad) and visualized by a sensitive
CCD camera system (FujiLas 1000 Imager). The optical
density of appropriate bands was quantified by densi-
tometric analysis of blots using Image Gauge V4.0 Soft-
ware (Fujifilm) and normalized to b-actin levels. All the
values are expressed as a percentage of controls.
Statistical analysis
All results are expressed as the means ± standard error
of the mean (SEM) where n = 6–8 rats per group. Sta-
tistical analyses were performed using GraphPad
Prism 5 (GraphPad Software Inc.). Data were ana-
lyzed by one-way analysis of variance (ANOVA) fol-
lowed by Tukey’s multiple range test. Groups that are
significantly different from control are indicated in
the figures as: + p < 0.05, ++ p < 0.01, +++ p < 0.001,
and significant difference from IL-1b injected group
Pharmacological Reports, 2012, 64, 1455�1465 1457
Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.
is shown as: * p < 0.05, ** p < 0.01, *** p < 0.001 for
1 h time point and ^ p < 0.05, ^^ p < 0.01, ^^^ p <
0.001 for 2 h.
Results
Plasma IL-1b, ACTH and corticosterone
response to systemic IL-1b administration
Exogenous IL-1b (5 µg/kg) administered ip induced
significant increase its plasma level much stronger
after 1 h than after 2 h. This increase was IL-1b selec-
tive, since it was reduced by ip injection of IL-1 re-
ceptor antagonist (IL-1ra) 15 min before IL-1b. This
reduction was dose dependent, stronger after pretreat-
ment with 100 µg/kg than 50 µg/kg of IL-1ra (Fig.
1A). Exogenous IL-1b also significantly increased
plasma ACTH levels 1 and 2 h after administration.
This increase was also abolished by pretreatment with
both doses of IL-1ra 1 h and 2 h after cytokine ad-
ministration (Fig. 1B). IL-1b-induced considerable
increase of plasma corticosterone level which was to-
tally prevented by IL-1ra 2 h following IL-1b injec-
tion (Fig. 1C). These results suggest that the above
changes in plasma IL-1b, ACTH and corticosterone
levels are in a major part IL-1b receptor specific.
Effect of IL-1ra on IL-1b-induced nNOS and
iNOS levels in brain structures
IL-1b (5 µg/kg ip)-induced increases of nNOS and
iNOS levels in prefrontal cortex 1 h after IL-1b admini-
stration gradually declined 3 h following ip injection
(Fig. 2A). The IL-1b-induced significant increase of
nNOS level in prefrontal cortex 1 and 2 h after admini-
stration was almost totally abolished by pretreatment
with IL-1ra (Fig. 2B). Likewise, a marked ip IL-1b-
induced increase of iNOS in prefrontal cortex was
totally abolished by pretreatment with IL-1ra (Fig. 2C).
In the hippocampus IL-1b gradually increased
nNOS level significantly 3 h after its administration
and respective alterations of iNOS levels did not at-
tained the level of significance. (Fig. 3A). IL-1ra sub-
stantially impaired the IL-1b-induced nNOS level 1 h
after its administration and slightly diminished iNOS
level after 1 and 2 h (Fig. 3B, 3C).
In the hypothalamus 1 h following IL-1b adminis-
tration, nNOS reached its highest level (94%) which
gradually decreased 2 and 3 h later and remained at
significantly higher level (68%) compared with saline
control values. Conversely, iNOS level markedly in-
creased 1–3 h after IL-1b administration compared
with saline treated group (Fig. 4A).
1458 Pharmacological Reports, 2012, 64, 1455�1465
Fig. 1. Effect of interleukin-1 receptor antagonist (IL-1ra) onincreased plasma levels of interleukin-1b (IL-1b) (A), ACTH (B) andcorticosterone (C) induced by IL-1b administration. In Figures 1�4rats were injected intraperitoneally (ip) with IL-1ra (50 or 100 µg/kg)15 min before IL-1b ip (5 µg/kg) and decapitated 1, 2 and 3 h later. InFigures 1�6, 8�10 rats per group were used. + p < 0.05, ++ p < 0.01and +++ p < 0.001 vs. saline control group; * p < 0.05, ** p < 0.01 and*** p < 0.001 vs. IL-1b ip 1 h; ^p < 0.05 and^^^p < 0.001 vs. IL-1b ip 2 h
The IL-1b-induced significant increase of nNOS
levels in the hypothalamus was totally abolished by ip
pretreatment with IL-1ra 15 min earlier (Fig. 4B).
Likewise, a marked but somewhat weaker rise of
iNOS 1 h after IL-1b administration was abolished by
IL-1ra after 2 h (Fig. 4C). These results suggest that
the ip IL-1b-induced effects on nNOS and iNOS levels
in examined brain structures, potentially involved in
HPA axis regulation are IL-1b receptor selective.
IL-1b levels in brain structures induced
by exogenous IL-1b administration
Interleukin-1b (5 µg/kg) 1 h after administration con-
siderably and to a similar extent increased IL-1b level
in prefrontal cortex and hippocampus (187 and 192%,
respectively), relative to control values in saline
treated rats. Also marked increase of IL-1b content
appeared in hypothalamus. These increased IL-1b
levels in all three structures sharply diminished to the
lowest levels 2 h after ip IL-1b administration. In the
third hour, IL-1b levels in prefrontal cortex and hypo-
thalamus returned to their high values. Despite these
changes, IL-1b levels in examined brain structures re-
mained significantly higher 1–3 h following exoge-
nous IL-1b administration compared to control levels
(Fig. 5A–C).
NOS levels in brain structures following IL-1b
administration
Interleukin-1b (ip, 5 µg/kg) 1 h after administration
induced a significant increase in nNOS levels, most
pronounced in hypothalamus (92%) and in prefrontal
Pharmacological Reports, 2012, 64, 1455�1465 1459
Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.
Fig. 2. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in prefrontal cortex (A).Effect of IL-1ra (50 µg/kg or 100 µg/kg) on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after IL-1b (5 µg/kg) ip administration. Repre-sentative immunoblot showing the expression of nNOS and iNOS in prefrontal cortex (D). For other details see legend to Figure 1
CORTEX
cortex (51%). This increase gradually dropped in sub-
sequent 2 and 3 h with moderately faster rate in pre-
frontal cortex than in hypothalamus. By contrast, in
hippocampus nNOS level only moderately increased
2 and 3 h after IL-1b administration compared with
control level (Fig. 2A, 3A, 4A). IL-1b administered
systemically also increased iNOS levels in prefrontal
cortex and in hypothalamus 1 h after ip injection com-
pared to respective control levels. This increased
iNOS level in prefrontal cortex sharply declined 2 and
3 h after IL-1b treatment to nearly control level, while
the respective increase of iNOS in hypothalamus fur-
ther linearly raised compared with control level. The
ip IL-1b-induced alterations of iNOS level in hippo-
campus, like these of nNOS, were not significant
compared to control levels (Fig. 2A, 3A, 4A).
NOS and IL-1b in brain structures compared
with plasma IL-1 b levels
The ip IL-1b induced changes in nNOS, iNOS and
IL-1b levels in brain structures were compared with
the parallel plasma IL-1b levels in order to determine
possible central and systemic significance of IL-1b
1460 Pharmacological Reports, 2012, 64, 1455�1465
Fig. 3. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in hippocampus (A). Effectof IL-1ra on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after ip IL-1b. Representative immunoblot showing the expression of nNOSand iNOS in hippocampus (D). For other details see legend to Figure 1
HIPPOCAMPUS
and nitric oxide synthases in the HPA axis activity.
One hour after ip administration, IL-1b considerably
increased its plasma levels which gradually decreased
2 and 3 h after injection but persisted at significant
higher level compared with control plasma levels in
saline-treated rats. These changes in plasma IL-1b
levels were generally parallel in time but at relatively
much higher levels compared with respective signifi-
cantly lower alterations of IL-1b levels in the prefrontal
cortex, hippocampus and hypothalamus (Fig. 5A–C).
These results suggest similar pattern in transient altera-
tions of IL-1b levels in plasma and brain structures .
Changes in nNOS and iNOS in brain structures
in relation to plasma IL-1b, ACTH and
corticosterone levels
Systemic IL-1b considerably increased its plasma
level parallel with ACTH level. These levels were
most increased 1 h after administration and gradually
diminished in a regular manner in the following 2 h
(Fig. 6A). The IL-1b- induced increased nNOS levels,
higher in hypothalamus than in prefrontal cortex and
iNOS in prefrontal cortex also regularly declined in
a parallel manner to the levels of plasma IL-1b and
Pharmacological Reports, 2012, 64, 1455�1465 1461
Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.
Fig. 4. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in hypothalamus (A). Effectof IL-1ra on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after ip IL-1b. Representative immunoblot showing the expression of nNOSand iNOS in hypothalamus (D). For other details see legend to Figure 1
HYPOTHALAMUS
ACTH levels (Fig. 6B). These results suggest that
both nNOS and iNOS in prefrontal cortex and hypo-
thalamus may be involved in the IL-1b-induced acti-
vation of the HPA axis under basal conditions.
Discussion
In the present study, systemic administration of IL-1b
increased considerably plasma levels of IL-1b like
ACTH and corticosterone. IL-1b administered ip can
stimulate the CNS and HPA axis by direct activation
of the parts of HPA axis outside the blood-brain bar-
1462 Pharmacological Reports, 2012, 64, 1455�1465
Fig. 5. Changes in IL-1b, nNOS and iNOS content in prefrontal cortex(A), hippocampus (B) and hypothalamus (C) in relation to plasmaIL-1b level. For other details see legend to Figure 1
Fig. 6. Comparison of IL-1b-induced changes in plasma IL-1b, ACTHand corticosterone levels (A) with nNOS and iNOS content in hypo-thalamus and prefrontal cortex (B). For other details see legend toFigure 1
rier, the median eminence to release CRH from end-
ings of PVN neurons, and stimulation anterior pitui-
tary adrenocorticotroph cells to release ACTH or di-
rect stimulation of adrenal cortex [1]. Systemically
administered IL-1b also induces the IL-1b expression
and may create an autocrine signaling loop that influ-
ences the adrenal response and may explain the disso-
ciation between plasma ACTH and corticosterone
levels [3, 11]. The strong inhibition of these IL-1b-i-
nduced stimulations by ip pretreatment with IL-1 re-
ceptor antagonist in the present experiment indicates
that these effects depend, at least in a major part, on
IL-1 receptor stimulation.
Exogenous IL-1b may also reach central cytokine
receptors in brain structures involved in HPA axis
regulation [27]. IL-1b may trigger increased synthesis
and release of IL-1b second messengers, PGs and NO
from endothelial cells vs. perivascular cells of BBB,
which have complex interaction in regulating the tim-
ing and types of brain responses [34]. In the present
experiment, IL-1b induced considerably higher in-
crease in plasma corticosterone levels 2 h after ad-
ministration compared with also significant response
1 h after injection.
IL-1b, 1 h after ip administration, significantly in-
creased the IL-1b content in prefrontal cortex and hip-
pocampus to a similar extent and moderately aug-
mented its level in hypothalamus. These increased
levels markedly declined 2 h after IL-1b administra-
tion and were parallel but at considerably lower level
compared to the changes in plasma IL-1b levels. Under
basal conditions the rate of alterations in IL-1b con-
tent in brain structures involved in HPA axis regula-
tion seem to be structure and time dependent.
The present investigation suggests the involvement of
NO generated by nNOS and iNOS in prefrontal cortex,
hypothalamus and hippocampus in the ip IL-1b-in-
duced stimulation HPA activity during 1–3 h after ad-
ministration. Our results show that the effects of IL-1b
on nNOS and iNOS activity were almost totally pre-
vented by prior administration of IL-1ra, suggesting
that they are IL-1 specific. Our former investigations
indicated that NO plays essential role in the IL-1b-in-
duced HPA axis stimulation. L-NAME a general NOS
inhibitor considerably reduced the ACTH and corti-
costerone response to ip IL-1b in rats [14]. Repeated
stress markedly increased IL-1b generation in brain
structures involved in HPA axis regulation [15].
IL-1b induced the strongest and persistent increase
of nNOS protein level in the hypothalamus 1–3 h after
administration and a modestly weaker stimulatory ef-
fect in the prefrontal cortex. Conversely, in the hippo-
campus nNOS level increased in a linear manner 2–3 h
after IL-1b administration, which may be connected
with a comparably weaker and delayed action of the
hippocampus on HPA axis activity. In agreement with
our data, NO donor 3-morpholino-syndomine (SIN-1)
microinfused into the PVN, which contains majority
of the CRH neurons regulating HPA axis activity, sig-
nificantly increased plasma ACTH levels [33]. NO
generated in parvocellular neurons in the PVN stimu-
lates CRH and ACTH release [12, 33]. Microinfusion
into the hippocampus also increased plasma ACTH
levels to a smaller extent and a delayed onset com-
pared to that after PVN treatment. In our experiment
IL-1b considerably increased nNOS level in prefron-
tal cortex 1–2 h after administration. Increased NO
level in that structure is parallel with and may be in-
volved in enhanced HPA axis activity.
However, microinfusion of SIN-1 into the prefrontal
cortex was without effect. These data may suggest that
the NO may stimulate HPA axis when applied to different
brain regions. NOS izoenzymes also mediate centrally
CRH-induced sympathetic activation under stressful con-
ditions and may affect the HPA axis activity [37, 38].
Our present data suggest that the IL-1b-induced central
influence of NO on the HPA axis is stimulatory. They also
provide information regarding the role of hypothalamus
and prefrontal cortex in modulating the activity of NOS on
the HPA activity during stimulation with systemic IL-1b.
In the present experiment, the IL-1b-induced
changes of IL-1b levels in plasma were parallel in time
but relatively much larger than respective alterations in
prefrontal cortex, hippocampus and hypothalamus.
Also the alterations of IL-1b levels in plasma were
generally parallel with plasma ACTH and corticoster-
one levels. This suggests that IL-1b both in brain
structures and plasma is involved in the regulation of
HPA axis activity in non-stressed rats.
Acknowledgment:
This research was supported by grant: POIG 01.01.02-12-004/09-00financed by European Regional Development Fund.
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Received: May 22, 2012; in the revised form: September 19, 2012;accepted: September 25, 2012.
Pharmacological Reports, 2012, 64, 1455�1465 1465
Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.