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Clayton Foundation Laboratories for Peptide Biology, The Salk Institute (S.L., C.R.), La Jolla, California 92037
Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Salk Institute, The Clayton Foundation Laboratories for Peptide Biology, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu
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-nitro-L-arginine-methylester
(L-NAME; 50 mg/kg, sc), which attenuated the ACTH response to LPS,
virtually abolished basal NOS activity in the PVN, as well as anterior
and neurointermediate lobes of the pituitary, and prevented the
appearance of citrulline in the PVN of rats injected with LPS. Collectively, these results show that LPS-induced activation of the HPA axis correlates with the activation of the PVN NOergic system, and supports a stimulatory role for NO in the modulation of the HPA axis in response to immune challenges.
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Interest in NO as a putative modulator of the hypothalamic-pituitary-adrenal (HPA) axis first stemmed from the discovery that NOS I was present in hypothalamic neurons known to produce two peptides important for ACTH release, namely corticotropin-releasing factor (CRF) and vasopressin (VP) (15, 16, 17, 18, 19, 20, 21). Subsequently, the finding that blockade of NO formation with arginine derivatives significantly altered the ACTH response to homeostatic threats such as inflammation, mild electrofootshocks, water avoidance, and immobilization (see references in Ref. 22) strengthened the concept that NO played an important role in regulating the response of the HPA axis to various stresses. The next step was to show that stresses that stimulated ACTH release through an NO-dependent mechanism augmented NO production in brain areas that were important in controlling the HPA axis. In situ hybridization histochemistry for NOS transcripts was initially used because of the difficulties encountered in measuring the highly reactive NO. Using probes specific for NOS I messenger RNA (mRNA) levels, we and others reported that both immune and nonimmune stimuli were able to up-regulate the gene expression of this enzyme in the paraventricular nucleus (PVN) of the hypothalamus (23, 24, 25, 26, 27, 28, 29). However, these studies indicated that changes in NOS I transcripts were modest relative to the high levels of constitutively expressed NOS I, as well as significantly delayed vis-à-vis changes in plasma ACTH levels. The extent to which stress indeed up-regulates NO formation and possible relationships in the time domain of ACTH and NO release, therefore, remained unresolved issues. What was required were sensitive and quantitative methods to measure acute and discrete changes in NO release as well as concomitant increases in NOS activity.
In the present work, we tested the hypothesis that mild endotoxemia
increased NO levels and NOS activity in the PVN and functionally
related regions. We chose three approaches to quantify NO production.
First, we used the microdialysis hemoglobin-trapping technique, a
methodology that provides real-time changes in PVN NO levels in
response to lipopolysaccharide (LPS) and is sensitive enough to study
NO release in vivo (30, 31). With this approach, we observed
a significant, but short-lived, increase in NO production in the PVN in
response to the iv injection of 100 µg/kg LPS. We then corroborated
changes in NO concentrations with increases in NOS activity by
determining the conversion of [14C]arginine to
[14C]citrulline in specific brain tissues. We showed that
LPS induced a transient, but significant, increase in NO levels and NOS
activity in the PVN, but not in the medial basal hypothalamus/median
eminence or the pituitary. These changes showed a good correlation with
elevated plasma ACTH levels. Thirdly, immunohistochemical detection of
citrulline, which is formed stoichiometrically with NO, indicated that
LPS up-regulated concentrations of this compound in the PVN.
Furthermore, citrulline-producing cells in the PVN were colocalized
with NOS I mRNA, providing evidence for the concept that LPS induced NO
formation through the isomer Ca2+-dependent NOS I. Finally,
we showed that the arginine derivative
N-nitro-L-arginine-methylester
(L-NAME), a known inhibitor of NOS activity (see references in Refs. 32, 33), decreased basal NOS levels in all brain areas studied,
abolished LPS-induced citrulline production, and blunted the ACTH
response to LPS.
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Experimental procedures
The rats underwent aseptic insertion of a right jugular venous
catheter 48–72 h before the experiment (34). The cannulas served both
to deliver LPS and to remove blood samples. LPS was administered iv at
100 µg/kg; control rats were injected with the vehicle. Blood samples
(0.3 ml) were taken 0.5, 1, and 6 h after treatment, after which
time the rats were killed under nonstress conditions. The medial basal
hypothalamus (MBH), containing the median eminence and ventral part of
the arcuate nucleus, as well as the anterior lobe (AL) and
neurointermediate lobe (NIL) of the pituitary were excised and kept at
-80 C; the rest of the brain was immediately frozen in powder dry ice
and kept at -80 C. For PVN dissections, a 1.5-mm coronal slice
containing the PVN area was cut with razor blades from the frozen
brain, and the PVN area was removed from that section using 1-mm
diameter neuropunches (Fine Science Tools, Inc., Foster City, CA) as
reported by Palkoviths and Brownstein (35). For MBH and PVN, three
regions per point were pooled, placed in a microcentrifuge tube, and
kept at -80 C until assayed for NOS activity.
Chronic salt-loading experiments
In view of the reported increase in HPA axis activity as well as
NOS mRNA levels after salt loading (36, 37), these experiments were
aimed at validating the NOS activity measurement used in the present
study. A control group was given food and water ad libitum,
whereas the experimental group was maintained with rat chow and 2%
(wt/vol) NaCl solution as the only source of water for 4 days. Control
and experimental animals were then killed, and tissue dissection was
performed as described above, except that trunk blood was
collected.
Microdialysis and NO measurement by the hemoglobin-trapping
technique
Rats were anesthetized with a sc injection of ketamine (100
mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) and placed in a
stereotaxic apparatus. With the incisor bar set at -3.3 mm, a guide
cannula was implanted at coordinates 0.8 mm lateral, 1.8 mm
anterior-posterior, and 7.5 mm dorsal-ventral. Four days after surgery,
the rats were equipped with an iv cannula and used 2 and 3 days later
in microdialysis experiments. On the experimental day, concentric
microdialysis probes (no. 12, CMA, Stockholm, Sweden; length, 1
mm; molecular mass cut-off, 20,000 Da) were introduced into the brain.
The probe was perfused for 1–1.5 h with artificial cerebrospinal fluid
(aCSF; 145 mM NaCl, 3.3 mM KCl, 1.3
mM CaCl2, 1 mM MgCl2,
and 2 mM sodium phosphate buffer, pH 7.4) at a constant
flow rate of 5 µl/min using a microsyringe pump (CMA). NO levels were
detected by the hemoglobin-trapping technique. Specifically, after an
equilibrium period, the aCSF was changed for a freshly prepared
1-µM oxyhemoglobin solution (HbO2). Bovine
hemoglobin diluted in aCSF was maintained at 4 C (30, 38). After 2–3 h
of perfusion with HbO2 (basal NO levels), the rats were
injected with LPS (100 µg/kg, iv) or the vehicle, and the perfusion
was continued for 2 h. Microdialysis samples were collected every
20 min in a fraction collector and kept at 4 C until the end of the
experiment. Sample absorbency (401 and 421 nM) was recorded
using a Beckman Coulter, Inc. DU-30 spectrophotometer
(Palo Alto, CA). NO levels were calculated using measured absorbency
difference values at 401 and 421 nM and the
HbO2 molar extinction coefficient. We calculated a value of
76 nM (r2 = 0.999) for the extinction
molar coefficient of our hemoglobin preparations, and this value was
used for the calculation of the NO concentration in the perfusates. The
calibration curve for the determination of this extinction coefficient
was obtained by quantitative oxidation of increasing amounts of
HbO2 (0.25–8 µg) using 10 mM sodium
nitroprusside and plotting the measured absorbency differences
(401–421 nM) vs. the concentration of
HbO2 used.
NOS activity measurement
NOS activity in brain tissues was determined by measuring the
conversion of [14C]arginine to
[14C]citrulline following the methods of Bredt and Snyder
(39) or an NOS detect Assay Kit (Stratagene, La Jolla, CA;
catalogue no. 204500). Briefly, frozen tissues (three PVN areas, three
MBH, one NIL, or one AL) were homogenized (50 mg tissue/ml
homogenization buffer) in 25 mM Tris-HCl (pH 7.4), 1
mM EDTA, and 1 mM EGTA, then centrifuged at
8000 x g for 10 min at 4 C. Ten 50 µl of the
supernatant (depending on the brain region) were incubated in a
100-µl final volume reaction containing 25 mM Tris-HCl
(pH 7.4), 3 µM tetrahydrobiopterin (Alexis Biochemicals,
San Diego, CA), 1 µM flavin adenine dinucleotide, 1
µM flavin mononucleotide, 0.6 mM
CaCl2, 1 mM NADPH reduced form, and 0.3 µCi
L-[14C]arginine monohydrochloride
(Amersham Pharmacia Biotech, Arlington Heights, IL; >300
mCi/mmol). The reaction was carried out at 37 C, and 45-µl aliquots
of the reaction mixture were taken at 4 and 8 min. Four hundred
microliters of stop buffer [50 mM HEPES (pH 5.5) and 5
mM EDTA] was added immediately. Two hundred microliters of
AG50W-X8 resin sodium form (Bio-Rad Laboratories, Inc.,
Richmond, CA), previously equilibrated with stop buffer, were added to
each sample and vortexed for 1 min at room temperature. Finally, the
mixture was transferred to spin columns (Bio 101, Vista, CA). To
recover the eluate, samples were centrifuged at 12,000 x
g for 15 min. [14C]Citrulline in the
eluent was quantified by liquid scintillation (Ecolume, ICN) by a
ß-counter scintillation counter (Beckman Coulter, Inc.,
model LS 8000), and NOS activity was expressed as picomoles of
citrulline formed per mg protein/min. Protein quantification for each
sample was performed by the Bradford method using a Bio-Rad Laboratories, Inc. (Richmond, CA), protein assay kit.
Immunohistochemistry and in situ hybridization
The animals received the vehicle or LPS (100 µg/kg, iv). Blood
samples were taken 0.5, 1, 2, 3, and 6 h after treatment, at which
time the rats were deeply anesthetized with 0.8 ml 35% chloral
hydrate. They were perfused transcardially with saline (0.9% NaCl)
followed by a fix solution (5% glutaraldehyde, 0.5% paraformaldehyde,
and 0.2% Na2S2O5 in 0.1
M sodium phosphate, pH 7.4). Brains were removed and
postfixed for 2 h in fix solution at room temperature, then
cryoprotected for 2 days at 4 C in 50 mM sodium phosphate
(pH 7.4), 0.1 M NaCl, and 20% (vol/vol) glycerol.
Thirty-micron frozen sections, ranging from the anterior commisura to
the mamillaris nuclei, were cut on a HistoSlide microtome (Leica Corp., Rockleigh, NJ). The brain sections were recollected in
four series and stored on cryoprotectant at -20 C until processed for
histochemistry and double labeling immunocytochemistry-in
situ hybridization.
Immunocytochemical detection of citrulline in the brain has been used to monitor NOS activity, because other citrulline-producing enzymes have been considered to be absent from the brain (40, 41). In our experiments, citrulline-positive cells were detected following the protocol reported by Eliasson et al. (42). Briefly, free floating brain sections were reduced for 20 min with 0.5% NaBH4 and 0.2% Na2S2O5 in PBS [10 mM sodium phosphate (pH 7.4) and 0.19 M NaCl], washed for 45 min at room temperature in PBS containing 0.2% Na2S2O5, blocked with 4% normal goat serum for 1 h in the presence of 0.2% Triton X-100, and incubated overnight at 4 C with an anticitrulline antiserum (1:6000 dilution in PBS containing 2% normal goat serum and 0.1% Triton X-100) provided by Dr. S. H. Snyder (Johns Hopkins University, Baltimore, MD) (42). Immunoreactivity was visualized with the Vestatin ABC Elite Kit (Vector Laboratories, Inc., Burlingame, CA). To test immunohistochemical specificity, brain sections from rats pretreated with L-NAME (50 mg/kg, iv) for 4 days were incubated with the citrulline antiserum, and no signal was detected.
A combined immuno- and in situ hybridization histochemical protocol was performed to detect citrulline immunoreactivity in NOS I-expressing neurons. Briefly, free-floating PVN brain sections were washed twice with sterile PBS for 10 min; reduced for 20 min with 0.5% NaBH4 and 0.2% Na2S2O5 in PBS; washed for 45 min at room temperature in PBS; blocked with 1% BSA (fraction V, Sigma Chemical Co., St. Louis, MO), 0.25% heparin sodium salt (Calbiochem, CA), and 0.3% Triton X-100 in PBS for 1 h at room temperature; and incubated for 36–48 h at 4 C with the citrulline antiserum (1:6000 dilution in PBS 1% BSA, 0.25% heparin sodium salt, and 0.1% of Triton X-100). Immunoreactivity was visualized with the Vestatin ABC Elite Kit (Vector Laboratories, Inc.). Thereafter, tissues were rinsed in sterile Krebs PBS, mounted on poly-L-lysine-coated slides, desiccated under vacuum overnight, then fixed in 4% paraformaldehyde for 30 min and digested with proteinase K [10 µg/ml in 50 mM Tris-HCl (pH 7.5) and 5 mM EDTA] at 37 C for 30 min. Sections were acetylated with a solution of 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8, then dehydrated for 1 min through graded concentrations of alcohol (50%, 70%, 95%, and 100%). After the slides were vacuum dried for a minimum of 2 h, they were covered with 90 µl of a hybridization mixture (107 cpm/ml), sealed under a coverslide with a mixture of distyrene, a plasticizer, and xylene (DPX), and incubated at 60 C overnight (15–20 h) on a slide warmer. The coverslips were then removed, and the slides were rinsed in 4 x SSC solution (1 x SSC = 0.15 M NaCl and 15 mM trisodium citrate, pH 7) at room temperature. Sections were digested with ribonuclease A (20 µg/ml, 37 C for 30 min), rinsed in descending concentrations of SSC (2, 1, and 0.5 x SSC), washed in 0.1 x SSC for 30 min at 65 C, and dehydrated through graded concentrations of alcohol. After being dried under vacuum, the sections were exposed overnight at 4 C to x-ray film (Bio-Max, Eastman Kodak Co., Rochester, NY), then defatted with xylene and dipped in NTB2 nuclear emulsion (Eastman Kodak Co.; diluted 1:1 with distilled water). The slides were exposed for 2 weeks, then developed with D19 developer (Eastman Kodak Co.) for 3.5 min at 14–15 C, and fixed for 6 min. Thereafter, tissues were rinsed in running distilled water for 1–2 h, dipped through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX.
Quantification of PVN citrulline-positive cells and percentage of
colocalization of citrulline-positive cells with NOS I mRNA
One series of brain sections containing five sections of PVN was
processed for citrulline immunohistochemistry. The sections were
analyzed under the brightfield illumination of a Leitz
microscope using a x20 objective. The parvocellular and magnocellular
divisions of the PVN were delineated in each section under brightfield
at the rostral/caudal level. The counting was performed by eye on a
single PVN using a grill that covered all of the PVN area.
Citrulline-positive cells were considered to be all of the cells that
were dark brown. The total number of PVN citrulline cells corresponds
to the sum of all of the positive cells in the five sections of PVN per
rat. For colocalization, the sections were analyzed under the
brightfield illumination of a Leitz microscope using a
x40 objective, and double staining neurons were assessed in the PVN by
the presence of both phenotypic markers (brown color for citrulline and
dark silver grains for NOS I mRNA). The counting was performed as
described for citrulline-positive cells.
Reagents
LPS (Escherichia coli serotype O26:B6, code L-3755,
lot 37H4095) and L-NAME were purchased from Sigma Chemical Co.. Both were diluted in apyrogenic saline.
ACTH and corticosterone assays
ACTH concentrations were determined by a two-site
immunoradiometric assay (Allegro, Nichols Institute Diagnostics, San Juan Capistrano, CA) using 50 µl plasma.
This assay has been previously validated for use in the rat (34).
Corticosterone values were measured by RIA with an antiserum provided
by Dr. G. Niswender (Colorado State University, Fort Collins, CO). The
characteristics of both assays have been reported previously (34, 43).
Statistical analysis
All of the results are expressed as the mean ±
SEM. They were analyzed by factorial ANOVA. When the ANOVA
was significant (P < 0.05), post-hoc
comparisons (Duncan’s new multiple range test) were made to determine
the statistical levels of difference between groups.
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). NO levels then returned to
baseline.
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).
Levels had returned to baseline by 1 h and had significantly
(P < 0.05) decreased by 6 h. In contrast, we did
not detect any change in NOS activity in the MBH, NIL, or AL at 0.5 and
1 h after treatment (Table 1).
Plasma ACTH and corticosterone levels showed the expected rises, with
peak responses at 0.5–1.0 h for ACTH and levels still rising at 6
h for corticosterone (Fig. 2).
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Time-dependent changes in the number of citrulline-positive cells
in the PVN and in plasma ACTH and corticosterone levels after LPS
treatment
There was a significant (P < 0.01) increase in
the number (Fig. 3) and color intensity
(Fig. 4) of citrulline-positive cells 1
and 3 h, but not 30 min, after LPS treatment. By 6 h, the
number of citrulline-producing cells had returned to control levels.
Most of the PVN citrulline-positive cells (99 ± 0.45%) were
colocalized with NOS I-synthesizing neurons (Fig. 5). In general, the response to LPS was
stronger in the anterior and medial parvicellular parts of the PVN than
in the magnocellular part. However, 3 h after the treatment, we
observed an increase in the signal in some of the PVN magnocellular
cells. Interestingly, we also observed an important increase in the
number of dendrites and axons stained 1 and 3 h after the LPS
injection. Although plasma ACTH and corticosterone levels also
significantly (P < 0.01) increased in response to LPS,
the initial change preceded any measurable up-regulation in PVN
citrulline-positive cells.
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, acute injection of L-NAME (50 mg/kg,
sc) significantly (87–93%; P < 0.01) lowered basal
NOS activity in PVN, MBH, NIL, and AL. As expected, LPS (100 µg/kg,
iv) produced a marked (P < 0.01) increase in the
number of citrulline-positive cells in the PVN (Figs. 6 and 7).
L-NAME decreased this number below control levels. L-NAME also
partially inhibited the ACTH response to LPS treatment (Fig. 7).
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Discussion |
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The observation that systemic LPS injection significantly increased NO levels in the PVN requires several comments. First, we consistently observed that basal NO levels showed a time-related increase in the afternoon. This suggests the possibility that, like the activity of the HPA axis, NO production may follow a circadian pattern. Indeed, it would be of interest to determine whether blockade of the formation of this gas might interfere with the known increase in PVN CRF production as well as with the ACTH and corticosterone secretion that takes place shortly before lights off (47, 48, 49). Should this be the case, it would provide additional support for our hypothesis that NO exerts a stimulatory effect on the HPA axis under a variety of circumstances. The second comment regards the magnitude of the increase in NO levels. Using measurement of NO metabolites by HPLC, Ishizuka et al. (45) recently reported that the ip injection of a high dose of IL-1ß increased nitrite and nitrate levels in the PVN (45). The magnitude of the change reported by these investigators is comparable to the one we show here, and in both cases it is modest. However, it is important to remember that there are no valid criteria to determine what this magnitude should be to ensure that NO release is temporarily and/or functionally correlated with activation of the HPA axis. For example, the presence of NOS in CRF cell bodies (17, 18) suggests that this gas may influence CRF synthesis in an autocrine fashion. If this is the case, we cannot determine, in a live system, what NO concentrations are necessary to activate CRF transcription pathways. To provide additional support for our hypothesis that LPS-induced NO production was indeed important for the ACTH response, we decided to corroborate data obtained with the microdialysis technique with citrulline production. In these experiments, we took advantage of the fact that the enzymatic reaction that produces NO also generates citrulline in equimolar concentrations. As, in contrast to NO, antibodies against citrulline are available, and detection of citrulline in the brain by immunohistochemistry provides a convenient as well as reliable method to measure changes in levels of this compound. We report here that LPS induced a significant increase in the number of PVN citrulline-positive cells, with a peak response observed at the 1 h point. The time course of these changes is in partial agreement with changes in NOS activity, with differences in peak levels for the enzyme activity and citrulline detection, possibly due to differences in sensitivity of the two assays. These results support our hypothesis that LPS induced NO production in the PVN. Furthermore, the observation that NOS I mRNA expression was found in citrulline-positive cells in the PVN provides evidence that NO was formed via the constitutive isoform of the enzyme.
The third comment pertains to the duration of LPS-induced NO release. In the work of Ishizuka et al. (45), IL-1ß increased nitrite and nitrate levels in the PVN for several hours. However, a direct comparison of our study and theirs is difficult. Although systemic LPS treatment is expected to increase IL-1ß levels in the brain (50), it probably also up-regulates the levels of many other cytokines and related secretagogues, some of which might exert an inhibitory influence on NO production. Indeed, we observed a significant decrease in NOS activity 6 h after LPS treatment. A number of mechanisms might be responsible for this phenomenon, including NOS protein degradation [which has been reported to occur in cell cultures exposed to cytokines (51)], inhibition of the enzyme by NO itself (52, 53, 54), association with regulatory proteins (55, 56), or an inhibitory influence of increased corticosterone release (57). Although some of these mechanisms might be expected to also have played a role in the experiments conducted by Ishizuka et al. (45), it is of interest to note that NO has been reported to specifically inhibit LPS-induced NOS gene expression (54).
A final comment should be made with regard to the potential mechanisms responsible for the effect of LPS on NO. The ability of immune signals to up-regulate NO production through its inducible enzyme is well recognized (58, 59, 60, 61). However, this enzyme is not believed to be important in our system. As levels of constitutive NOS are high in the PVN, it is possible that NO is released from a preformed pool without an acute increase in NOS transcripts. Indeed, our previous work suggested that no changes in nNOS mRNA levels were detected until 3 h after LPS injection (23), which is significantly later than the increase in NO levels and NOS activity that we report here. Several of the mechanisms that might account for the stimulatory effect of LPS on NO have been elegantly discussed previously (62). In general, it is assumed that changes in NOS activity in hypothalamic neurons are linked to changes in the neurochemical and neurosecretory activities of these neurons (63). However, this concept does not address the question of whether increased NOS gene transcription occurs in parallel with, precedes, or results from the activation of other signals in these neurons. Such signals include corticosteroids (57), transcription factors such as c-fos (64), changes in intracellular calcium (65), and the binding of proteins such as PDZ-95 to N-methyl-D-aspartate (NMDA) receptors (56). Potentially more important for our model is the fact that cytokines increase hypothalamic levels of both NO (45) and norepinephrine (66, 67); this coupled with the observation that abdominal vagotomy blocked both cytokine-induced NO (45) and norepinephrine (68) release in the PVN led to the hypothesis (45) that LPS-induced NO formation might be at least in part mediated by this catecholamine. Another mechanism of interest concerns CRF and/or VP. We previously reported that both peptides significantly up-regulated nNOS gene expression in the PVN (69). In view of the stimulatory effect of LPS on PVN CRF and VP (50, 70), it is tempting to propose that the ability of endotoxemia to increase NO levels in the PVN may also be controlled by CRF and/or VP. Studies conducted with antagonists of these peptides should determine whether this hypothesis is correct.
We found that in our model, LPS did not augment NOS activity in the MBH (which contains the median eminence) or the pituitary. NO is abundantly present in the neural lobe of the rat adenohypophysis (71, 72), where its up-regulation by chronic salt loading parallels its increase in PVN NOS mRNA levels (Ref. 36 and the present work). It is also found in the median eminence (73, 74, 75), where it is thought to regulate the release of GnRH (75). On the basis of our finding that L-NAME augmented the ACTH response to blood-borne cytokines (32, 34), we speculated that NO might act at least in part by altering CRF and/or VP secretion from nerve terminals. As NOS involved in these paradigms was of the constitutive type, the influence of L-NAME does not require new production of the enzyme. The present data therefore do not allow us to determine whether basal NO produced outside the blood-brain barrier played a role in regulating the ACTH response to LPS.
One of the problems previously encountered when trying to correlate increases in plasma ACTH levels with changes in PVN NOS mRNA or protein concentrations, is that many of the methods used did not provide the temporal resolution necessary for this comparison. In the present work, the transient increase in NO or citrulline levels corresponds to the initiation of the ACTH response routinely observed in rats injected with LPS. Although this hormone response typically lasts at least 3 h, changes in NO concentrations were very short-lived. This does not diminish, however, the potential importance of NO in mediating the effect of LPS on the corticotrophs. If, as suggested by previously published work, NO stimulates the neuronal activity of PVN CRF perikarya (76, 77), it is possible that once the early events of signal transduction and nuclear factor activation have been initiated, subsequent gene transcription can proceed independently of additional increases in NO levels. The hypothesis that this gas plays a role in the response of the HPA axis to LPS is further supported by the observation of temporally related increases in the number of citrulline-positive cells and plasma ACTH levels after LPS treatment. However, it must also be noted that the onset of ACTH release preceded detectable changes in citrulline levels. This could be due to methodology difficulties in detecting small increases in concentrations of this compound. It could also mean that NO is not involved in the very early events leading to ACTH secretion, but only becomes functionally significant at a somewhat later time. Regardless of the precise role of NO at the onset of ACTH secretion, our recent observation that microinfusion of the NO donor sodium nitroprusside immediately above the PVN significantly enhanced the ACTH response to iv LPS (Uribe, R. M., and C. Rivier, unpublished) further supports the participation of this gas during the HPA axis response to endotoxemia.
If the hypothesis that NO is important for ACTH release is correct, one
would expect that blockade of NO formation with L-NAME would both
significantly lower citrulline levels and alter LPS-induced ACTH
release. This was indeed the case, and although the present study
relied on a maximally effective dose of L-NAME, we know that the effect
of the arginine derivative is dose related (32, 78). We have previously
reported that L-NAME increased the ACTH response to the systemic
injection of single cytokines such as tumor necrosis factor-
,
IL-1ß, and IL-6 (32, 34), whereas it decreased it in rats exposed to
electroshocks (33, 76). On the basis of these and other results (77),
we had proposed, as mentioned above, that NO exerted an inhibitory
influence on ACTH secretagogues released in the median eminence and a
stimulatory effect within the PVN. Although the precise mechanisms
responsible for LPS-induced ACTH secretion remain unclear, they are
thought to include both sites and should therefore be under this dual
and opposite influence of NO. Work published by others (79, 80) as well
as preliminary results from our laboratory (Lee, S., and C. Rivier, in
preparation) indeed suggest that doses of LPS such as those used in the
present work have an important PVN component that includes activation
of CRF neurons and PG production. This central component is under the
stimulatory influence of NO, which should therefore prevail. The
ability of L-NAME to decrease ACTH release in our paradigm is in
agreement with this concept. Some investigators (81), but not others
(82), have also suggested that brain interleukin-1ß (IL-1ß) might
participate in the effect of systemic LPS on the HPA axis (for a more
complete discussion, see Ref. 50). Interestingly, however, the ACTH
response to the icv injection of this cytokine does not depend on NO
(78). Even if central IL-ß participates in the stimulatory action of
LPS, it is therefore not expected to be altered by L-NAME.
In summary, we have shown that an immune challenge induced by LPS treatment increased PVN NOergic activity, and that this change was temporally related to ACTH release. Pretreatment with L-NAME, which reduced the ACTH response to LPS, abolished NOS activity and citrulline immunoreactivity. Collectively, our results suggest the presence of a temporal and functional relationship between the response of the HPA axis to LPS, and NO formation in the neurocrine hypothalamus.
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Acknowledgments |
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Footnotes |
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2 Financial support was provided by DGAPA-UNAM. Present
address: Instituto De Biotechnologia/Universidad Nacional
Autónoma de México, Cuernavaca, Morelos
62250, Mexico.
Received April 22, 1999.
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References |
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1-syntropin mediated by PDZ
domains. Cell 84:757–767[CrossRef][Medline]
, bacterial lipopolysaccharide and
NG-monomethyl-L-arginine. Biochem J 316:209–215
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 M. Herman and C. Rivier Activation of a Neural Brain-Testicular Pathway Rapidly Lowers Leydig Cell Levels of the Steroidogenic Acute Regulatory Protein and the Peripheral-Type Benzodiazepine Receptor while Increasing Levels of Neuronal Nitric Oxide Synthase Endocrinology, January 1, 2006; 147(1): 624 - 633. [Abstract] [Full Text] [PDF]
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D. J. Selvage, S. Y. Lee, L. H. Parsons, D. O. Seo, and C. L. Rivier A Hypothalamic-Testicular Neural Pathway Is Influenced by Brain Catecholamines, But Not Testicular Blood Flow Endocrinology, April 1, 2004; 145(4): 1750 - 1759. [Abstract] [Full Text] [PDF]
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 A K Singh and Y Jiang Lipopolysaccharide (LPS) induced activation of the immune system in control rats and rats chronically exposed to a low level of the organothiophosphate insecticide, acephate Toxicology and Industrial Health, March 1, 2003; 19(2-6): 93 - 108. [Abstract] [PDF]
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 A. Beishuizen and L. G. Thijs Review: Endotoxin and the hypothalamo-pituitary-adrenal (HPA) axis Innate Immunity, February 1, 2003; 9(1): 3 - 24. [Abstract] [PDF]
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 J. J. Puder, P. U. Freda, R. S. Goland, and S. L. Wardlaw Estrogen Modulates the Hypothalamic-Pituitary-Adrenal and Inflammatory Cytokine Responses to Endotoxin in Women J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2403 - 2408. [Abstract] [Full Text] [PDF]
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 V. D. Dixit and N. Parvizi Pregnancy Stimulates Secretion of Adrenocorticotropin and Nitric Oxide from Peripheral Bovine Lymphocytes Biol Reprod, January 1, 2001; 64(1): 242 - 248. [Abstract] [Full Text]
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