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The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu
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Abstract |
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-nitro-L-arginine-methylester
(L-NAME; 50 µg) or the HO inhibitor tin protoporphyrin (SnPP; 20–25
µg) significantly blunted the plasma ACTH response to a 45-min
session of intermittent electroshocks. Importantly, in these same
animals there were concurrent decreases in hypothalamic NOS or HO
activities, respectively. There were little or no effects of these
inhibitors on anterior pituitary NOS or HO activities, indicating that
there was only minimal leakage of the drug from the brain after icv
administration. The specificity of action of these inhibitors was
confirmed by the fact that SnPP did not affect NOS activity, and L-NAME
did not affect HO activity. Finally, L-NAME produced no effect, whereas
SnPP produced only transient increases in blood pressure, suggesting
that these inhibitors do not affect activity indirectly through
alterations in blood pressure. These data support the hypothesis that
in the whole animal, both NO and CO exert a stimulatory influence on
the acute ACTH response to physico-emotional stressors, and that the
hypothalamus is the critical site of their actions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HPA axis is regulated by multiple stimulatory and inhibitory inputs (1, 2, 3). CRF, ACTH, and especially glucocorticoids themselves have regulatory roles through various negative feedback loops at different levels of this axis. At the PVN level, many of the classic neurotransmitters and other substances, such as PGs and opiates, alter CRF/VP synthesis and/or release. Recently, there has been recognition that the newest class of brain neuromodulators, the short-lived and highly diffusable gases nitric oxide (NO) and carbon monoxide (CO), may also play a role in HPA regulation (4, 5, 6). NO is formed during the conversion of arginine to citrulline by NO synthase (NOS) (7, 8, 9). There are three forms of this enzyme: two constitutive forms whose activities are dependent on the presence of Ca2+ [neuronal (n) NOS or NOS I, and endothelial (e) NOS or NOS III] and an inducible form that is Ca2+-independent (iNOS or NOS II). CO is produced by heme oxygenase (HO) during the conversion of iron-protoporphyrin to biliverdin and free iron (7, 8, 10). There are three forms of this enzyme: constitutive HO-2, inducible HO-1, and the recently discovered HO-3 (11). Of particular relevance, NOS (12, 13, 14, 15) and HO (16, 17) are found in hypothalamic nuclei known to control the release of CRF/VP. Specifically, NOS has been shown to be colocalized with CRF- and VP-producing neurons in the PVN (18, 19).
Studies from other laboratories that have examined the roles of NO and
CO in the HPA stress response have yielded contradictory results.
Experiments using tissue extracts have reported stimulatory and
inhibitory roles for both NO (20, 21, 22, 23, 24, 25, 26, 27, 28, 29) and CO (30, 31, 32) in CRF and/or VP
release. Experiments using the whole animal have also reported
stimulatory and inhibitory roles for NO (27, 33, 34, 35, 36, 37, 38, 39) in CRF, VP, ACTH,
and/or corticosterone release. Due to these discrepancies, our
laboratory has conducted a comprehensive series of studies over the
past several years using the rat model to determine the role of NO and
CO on the HPA stress response. Our initial studies demonstrated that
the systemic (sc or iv) administration of NOS inhibitors such as
N-nitro-L-arginine-methylester
(L-NAME),
N-nitro-L-arginine,
or
NG-methyl-L-arginine
augmented the ACTH response to systemic injections of cytokines such as
interleukin-1ß, suggesting an inhibitory role for NO (40, 41, 42, 43). As the
acute ACTH response to blood-borne cytokines is thought to primarily
represent increased CRF and VP release from nerve terminals in the
median eminence as well as the stimulatory effect of these peptides on
the corticotrophs, our results suggested that NO might interfere with
both events. In contrast, we found that NOS (L-NAME,
N-nitro-L-arginine,
or
NG-methyl-L-arginine)
or HO [tin protoporphyrin (SnPP) or tin mesoporphyrin (SnMP)]
inhibitors attenuated the ACTH response to physico-emotional
stressors such as electroshock or water avoidance (44, 45). As these
stressors are thought to release ACTH by initially stimulating CRF and
VP synthesis and release from the PVN, these results suggested a
stimulatory role for NO and CO in the PVN. This idea was subsequently
substantiated by our finding that intracerebroventricular (icv)
injection of the NO donor 3-morpholino-sydnonimine (SIN-1) up-regulated
PVN CRF and VP gene expression and increased plasma levels of ACTH
(46).
Our experiments focusing on the roles of NO and CO in mediating the ACTH response to physico-emotional stressors, were initially conducted with systemic injections of NOS or HO inhibitors (44, 45). As indicated above, in response to physico-emotional stressors, hypothalamic activation is accompanied by the release of CRF and VP from the median eminence. Consequently, the ability of these antagonists to blunt physico-emotional stress-induced ACTH release implies that either the stimulatory influence of NO and CO on CRF/VP neurons of the PVN predominates over their inhibitory role at the median eminence, or that the inhibitory influence of NO is only observed in the presence of circulating cytokines. Thus, we thought it important to study the consequence of injecting L-NAME or SnPP centrally (i.e. icv), which would restrict the inhibitors’ effects to the brain and thus eliminate their action on the peripheral NOS and HO systems. Furthermore, if the hypothesis that NO and CO exert stimulatory influences on CRF/VP neurons of the PVN during physico-emotional stressors is correct, we would expect NOS and HO inhibitors to decrease hypothalamic NOS and HO activities, respectively, parallel to the observed attenuation of stress-induced ACTH release.
The main purpose of the present study was, therefore, 2-fold. First, we determined whether icv administration of L-NAME or SnPP would attenuate plasma ACTH responses to intermittent electroshocks, similar to those observed in our previous studies after systemic administration of the inhibitors. Second, we determined whether there was concordance between this ACTH response and hypothalamic NOS and HO activity levels, a finding that would support a role for PVN NO and CO in the HPA axis response to physico-emotional stressors. We also compared these NOS and HO activity responses with those observed after sc administration of L-NAME or SnPP. This was important because, as we mentioned above, we had administered these inhibitors peripherally in our initial demonstrations of decreased stress-induced ACTH release (44, 45). Furthermore, we wanted to confirm the specificity of action of L-NAME and SnPP at inhibiting NOS and HO activities, respectively, by assessing their effects on both hypothalamic NOS and HO activities. Finally, we determined that these inhibitors did not affect the HPA response indirectly through their well known actions as vasoconstrictors (42, 47, 48) by assessing blood pressure changes after icv administration of L-NAME or SnPP over the time course that we tested the animals for ACTH response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drugs
The general NOS inhibitor L-NAME, purchased from
Sigma (St. Louis, MO), was diluted in sterile water and
injected icv (50 µg, 5 µl volume) or sc (50 mg/kg, 0.2-ml volume).
The general HO inhibitor SnPP, purchased from Porphyrin Products
(Logan, UT), was diluted in dimethylsulfoxide (DMSO)/sterile water,
sonicated, and then injected icv (20–25 µg, 5-µl volume of 20%
DMSO/80% water) or sc (60 mg/kg, 0.2-ml volume of 50% DMSO/water).
L-NAME and SnPP can cross the blood-brain barrier to block the activity
of NOS (49) or HO (50), respectively. The sc doses of drugs used in the
present study were therefore based on our previously published studies
(45). Icv doses were based on preliminary dose-response studies: L-NAME
at 25–250 µl, and SnPP at 10–50 µl. Because both drugs are light
sensitive, they were protected from light during handling.
Intracerebroventricular injection
An indwelling icv cannula was implanted using standard
stereotaxic techniques (51) 7–10 days before testing. Animals were
anesthetized with 0.6 ml (sc) of a mixture of ketamine (Fort Dodge
Animal Health, Fort Dodge, IA), xylazine (Bayer Corp.,
Shawnee Mission, KS), acepromazine (Vedco, Inc., St. Joseph, MO), and
sterile water in a volume ratio of 9:9:4:1.4. A 26-gauge stainless
steel guide cannula (Plastics One, Roanoke, VA) was placed into the
right lateral ventricle [0.3 mm posterior, 1.4 mm right, 3.5 mm
ventral to bregma skull surface, with incisor bar 3.3 mm below the
interaural line; coordinates from Paxinos and Watson (52)]. On the
morning of the experiment, the internal cannula [33-gauge, projecting
1 mm beyond tip of the guide cannula (Plastics One)] was attached to
the implanted guide cannula and extended with a 50-cm long catheter
containing the appropriate drug. Rats were placed individually into
opaque buckets in a quiet room, with the catheter extending outside the
bucket to allow drug delivery without handling the animal. Rats were
left undisturbed for 3 h before treatment, the time necessary for
return of ACTH and corticosterone to baseline levels. Drugs were slowly
infused (1 µl/10 sec) via a microinjection syringe (Hamilton Co.,
Reno, NV) attached to the catheter. Control animals received the
appropriate vehicle.
Blood samples
An indwelling iv cannula to the right jugular vein was inserted
under isoflurane anesthesia (Abbott Laboratories, Chicago,
IL) 2–3 days before testing (51). On the morning of the experiment,
the iv cannulas were extended with a 50-cm long catheter filled with
heparinized saline. Rats were placed individually into opaque buckets
in a quiet room, with the catheter extending outside the bucket to
allow for blood sample collection without handling the animal. Rats
were left undisturbed for 3 h before treatment. Each blood sample
(400-µl volume) was withdrawn and immediately replaced with an equal
volume of sterile saline. Each blood sample was collected in a 1-cc
syringe, transferred to a plastic tube containing the anticoagulant
EDTA, placed on ice, and centrifuged at 4 C for 10 min at low speed,
and the plasma was stored at -70 C until assayed. This blood-sampling
procedure allows for the collection of up to five samples from the same
animal without affecting parameters of HPA activation (51).
Intermittent electroshock
Electroshocks (1 mA, 1 sec duration, randomized to two shocks
per min for 45 min) were delivered to the animal’s paws using a
Coulbourn E13–08 grid floor shocker controlled by a Macintosh
computer (44). Each shock chamber was a 30 x 26 x 31
cm Plexiglas box housed inside a larger Plexiglas chamber. Because of
the low voltage used, this stressor does not cause injury and produces
only minimal pain. Indeed, the intermittent nature of this stressor
makes it essentially an emotional stressor accompanied by a modest
amount of physical discomfort.
Mean arterial blood pressure (MAP) measurement
At least 7 days after icv cannulation, an indwelling catheter
was inserted into the right femoral artery under isoflurane anesthesia.
On the morning of the next day, the cannula was extended with a 100-cm
long catheter filled with heparinized saline and attached to the blood
pressure apparatus (model RS 3200, Gould, Inc., Valley View, OH). Rats
were placed individually into opaque buckets in a quiet room, with the
catheter extending outside the bucket to allow for MAP monitoring in
the freely moving animals without handling. Rats were left undisturbed
for 3 h before icv drug treatment. MAP was continuously monitored
for 30 min before and up to 1 h and 45 min after icv treatment.
Data were expressed in displacement (mm Hg) from the predrug
baseline.
ACTH RIA
Plasma ACTH levels were determined by a commercially available
two-site RIA (Allegro kit, Nichols Institute Diagnostics,
San Juan Capistrano, CA). The lower detection limit and the intraassay
coefficient of variation were 15 pg/ml and less than 10%,
respectively. Data were expressed in pg/ml plasma. We have validated
this RIA for use in the rat (42).
Tissue preparation for NOS/HO activity assays
Animals were decapitated, and the whole brain and anterior
pituitary were quickly removed, quick-frozen with powdered dry ice, and
stored at -70 C until analyzed for NOS and HO activities. The
hypothalamus was isolated using the landmarks of the optic chiasm on
the ventral surface and the fornices for the dorsal and lateral
boundaries. The anterior pituitary was separated from the posterior
pituitary.
NOS activity assay
This procedure assessed the conversion of
[14C] arginine to
[14C]citrulline using the method of Bredt and
Snyder (53). NO is produced in equimolar concentrations with
citrulline. Tissues were homogenized in buffer (25 mM
Tris-HCl, 1 mM EDTA, and 1 mM EGTA, pH 7.4) and
centrifuged at 8000 x g for 10 min at 4 C. Supernatant
(10–40 µl) was added to incubation buffer [25
mM Tris-HCl (pH 7.4), 3
µM tetrahydrobiopterin (Alexis Biochemicals,
San Diego, CA), 1 µM flavin adenine
dinucleotide (Sigma), 1 µM flavin
mononucleotide (Sigma), 0.6 mM
CaCl2, 1 mM NADPH
(Sigma), and 0.3 µCi
L-[14C]arginine monohydrochloride
(Amersham Pharmacia Biotech, Arlington Heights, IL)] to a
final volume of 100 µl, and incubated at 37 C. Aliquots (45 µl) of
this mixture were removed at 4 and 8 min. To each aliquot, 400 µl
stop buffer [50 mM HEPES (Sigma)
and 5 mM EDTA, pH 5.5] and then 200 µl
AG50W-X8 resin (Bio-Rad Laboratories, Inc., Richmond, CA)
preequilibrated with stop buffer (5 g/10 ml) were added, vortexed for 2
min, and centrifuged at 12,000 x g for 15 min in spin
columns (BIO-101, Vista, CA) to recover the eluant.
[14C]Citrulline was quantified by liquid
scintillation (ICN Pharmaceuticals, Inc., Costa Mesa, CA)
counting in a ß-counter, and values were expressed as pmol/mg
protein/min. Protein quantification of each sample was performed by the
Bradford method using a commercially available kit (Bio-Rad Laboratories, Inc.). The above-mentioned procedure assessed cNOS
(Ca2+-dependent) activity. iNOS
(Ca2+-independent) activity was assessed after
deletion of the Ca2+ source
(CaCl2) from the incubation buffer.
HO activity assay
This procedure, adopted from that described by Maines et
al. (54), relied on the quantification of bilirubin production. HO
converts iron-protoporphyrin to biliverdin, free iron, and CO;
biliverdin reductase then quickly converts biliverdin to bilirubin. CO
and bilirubin are produced in equimolar concentrations. Tissues were
homogenized in buffer (10 mM Tris-HCl and 250
mM sucrose, pH 7.5) and centrifuged at
10,000 x g for 20 min at 4 C, supernatants were
centrifuged at 100,000 x g for 1 h at 4 C, and
pellets were resuspended in buffer (20 mM
Tris-HCl, 0.1 mM EDTA, 20% glycerol, and 0.4%
Triton X-100). For each sample, a reaction mixture was made: 50 µl
resuspended sample, 400 µl assay buffer (0.1 M
potassium phosphate monobasic and 1 mM EDTA, pH
7.4), 10 µl biliverdin reductase (isolated supernatant after the
centrifuge steps, from kidney tissue), 10 µl 1
mM heme solution [6.52 mg hemin
(Sigma), 250 µl 0.1 N NaOH, 100 mg
Tris base, 132 mg BSA, 9.5 ml water, and 200 µl 0.1
N HCl], and 0.02 U cytochrome P450 reductase
(Calbiochem, La Jolla, CA). The reaction mixture (100
µl) was added to a test tube and a reference tube, and 10 µl of
either 2.75 mM NADPH (Sigma) or
assay buffer were added to the test and reference tubes, respectively.
The tubes were incubated at 37 C for 15 and 30 min, placed into an ice
bath to terminate the reaction, then scanned on a spectrophotometer
(model DU 640B, Beckman Coulter, Inc., Fullerton, CA)
using the reference tube as the blank. The change in OD of the test
tube at the peak (530 nm) compared with the trough (465 nm) was divided
by the extinction coefficient of bilirubin (40/mm cm), and
values were expressed as nmol/mg protein/h. Protein quantification of
each sample was performed by the Bradford method using a commercially
available kit (Bio-Rad Laboratories, Inc.).
Statistical analysis
Statistical analyses were performed with t tests or
appropriate one-, two-, or three-way ANOVA followed by Tukey’s
post-hoc test. The P values are only reported
when statistical significance was achieved (P < 0.05,
two-tailed).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Fig. 1
, all nonshocked
animals, whether they received L-NAME or vehicle, displayed basal ACTH
levels throughout. As expected, electroshocks produced significant
(P < 0.01) elevations of ACTH, and L-NAME
significantly (P < 0.01) blunted this response,
suggesting that NO plays a stimulatory role in ACTH release. NOS
activity in the hypothalamus was significantly (P <
0.01) decreased in all animals that received L-NAME regardless of
whether they were shocked. NOS activity in the anterior pituitary was
marginally decreased by L-NAME. In animals that were shocked, those
that received L-NAME showed a modest decrease (P <
0.07) compared with those that received vehicle. This suggests that
icv L-NAME markedly blocked NO production in the hypothalamus, and
there may have been a small amount of leakage of the drug to the
anterior pituitary to marginally inhibit NO production. However, it is
possible that the small decrease in NOS activity in the anterior
pituitary may have been produced by downstream effects mediated by
inhibition at the hypothalamus. Electroshocks exhibited no effects on
NOS activity in either the hypothalamus or anterior pituitary.
Collectively, these results confirm the stimulatory effect of NO on
hypothalamic structures involved in the ACTH response to
electroshocks.
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As shown in Fig. 2, all nonshocked
animals, whether they received SnPP or vehicle, displayed basal ACTH
levels throughout. As expected, electroshocks produced significant
(P < 0.01) elevations of ACTH, and SnPP significantly
(P < 0.01) blunted this response, suggesting that CO
plays a stimulatory role in ACTH release. HO activity in the
hypothalamus was significantly (P < 0.01) decreased in
all animals that received SnPP regardless of whether they were shocked.
HO activity in the anterior pituitary was not affected by SnPP,
suggesting that SnPP was confined to the brain. Electroshocks exhibited
no effect on HO activity at either site. Collectively, these results
confirm the stimulatory effect of CO on hypothalamic structures
involved in the ACTH response to electroshocks.
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, L-NAME significantly decreased NOS
activity in the hypothalamus (P < 0.01) and anterior
pituitary (P < 0.01). This indicates that sc L-NAME
not only acted at the anterior pituitary, but reached the hypothalamus.
Data from rats that were not shocked are not reported here; previous
unpublished data have shown that NOS activity values of shock
vs. no shock animals did not differ significantly after
L-NAME or vehicle treatment.
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, SnPP significantly decreased HO
activity in the hypothalamus (P < 0.01) and anterior
pituitary (P < 0.05). This indicates that sc SnPP not
only acted at the anterior pituitary, but reached the hypothalamus.
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, L-NAME, but not SnPP, significantly
(P < 0.01) decreased NOS activity, and SnPP, but not
L-NAME, significantly (P < 0.01) attenuated HO
activity. These data indicate that L-NAME and SnPP are specific
inhibitors of NOS and HO activities, respectively.
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Effects of icv L-NAME or SnPP on MAP
The purpose of this experiment was to demonstrate that L-NAME and
SnPP did not affect the ACTH response indirectly through their well
known actions as vasoconstrictors (42, 47, 48). Rats were injected icv
with L-NAME (50 µg), SnPP (20 µg), or their respective vehicles,
and displacement of MAP from the predrug baseline was determined over
the time course during which we tested the animals for ACTH response.
The 0 min point is the mean of five measurements taken at 1-min
intervals during the 5 min that immediately preceded drug or vehicle
administration. As shown in Fig. 6, SnPP
produced significant (P < 0.05) increases in MAP, but
this effect subsided within 15 min, well before the time when the rats
were exposed to the electroshock treatment. L-NAME produced no
significant effects on MAP. Although MAP measurements were taken up to
1 h and 45 min after drug injection, these data are not presented
because no effects were observed. These data suggest that these
inhibitors do not alter HPA activity indirectly through alterations in
blood pressure.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stimulatory role of NO and CO that we observed here is consistent with our previous demonstrations of attenuated ACTH response to physico-emotional stressors after systemic administration of NOS or HO inhibitors (44, 45) and increased ACTH response to administration of the NO donor SIN-1 (46). Several other investigators using the whole animal model have demonstrated a stimulatory role for NO on CRF and VP release (27, 33, 36, 37). However, Goyer et al. (34) and Kadowaki et al. (35) observed an inhibitory role of NO on VP release after salt loading. It is possible that NO has different roles in neural circuitries underlying physico-emotional stressors and salt loading. Indeed, physico-emotional stressors predominantly affect VP neurons in the parvocellular (p) division of the PVN, whereas salt loading alters VP neurons in the magnocellular division (55). Another contrasting result is the finding by Tsuchiya et al. (38) that L-NAME delayed the return of plasma corticosterone to baseline levels after 2 h of immobilization stress; the same trend was apparent for ACTH release, but it did not reach statistical significance. Our studies (Refs. 44, 45 and the present study) that demonstrated a stimulatory role for NO examined ACTH changes over the initial 30–45 min of the stressor, which corresponds to the rise and plateau phases of the HPA stress response. As Tsuchiya’s and our studies examined different time domains of the stress response, it is difficult to make direct comparisons. One possibility is that NO has different roles in the different time domains of the HPA response, a hypothesis that we are presently pursuing. Finally, a recent study by Weidenfeld et al. (39) showed that NOR-3 (a NO donor) decreased CRF secretion from the median eminence and decreased serum levels of ACTH and corticosterone at 10 min after acute photic stimulation, whereas L-NAME augmented the ACTH and corticosterone responses to this stressor. As our demonstrations of NO’s effects on the HPA axis have been with physico-emotional stressors, such as electroshocks or water avoidance, it is possible that NO differentially affects photic stress.
Experiments that have used isolated tissue have also shown contradictory roles for NO (20, 21, 22, 23, 24, 25, 26, 27, 28, 29) and CO (30, 31, 32) in CRF and VP release, but such discrepancies may arise from the particular method of tissue extraction and manipulation. First, this approach isolates the tissue from relevant afferent inputs and the normal hormonal environment. Second, in view of our finding that NO (and possibly CO) exerts opposite effects in the brain (PVN) and the periphery (median eminence and pituitary), it is possible that the net effect of these gases may depend on the proportions of cells from the PVN and from the basal hypothalamus/median eminence.
Within the hypothalamus, we believe that the critical sites for the stimulatory actions of NO and CO are the CRF and VP neurons of the pPVN. This is based on our previous studies demonstrating attenuation of shock-induced expression of the immediate early gene NGFI-B messenger RNA (mRNA) and/or CRF heteronuclear RNA in the pPVN after systemic administration of NOS or HO inhibitors (45, 56), and increased NGFI-B mRNA and CRF/VP heteronuclear RNA levels in the pPVN after icv administration of SIN-1 (46). We have also shown that CRF antibodies abolished and VP antibodies partially decreased the ACTH response to icv SIN-1 administration (46). Furthermore, other investigators have demonstrated an increase in expression of another immediate early gene, fos, in NOS-producing neurons of the PVN after restraint or novel environment stressors (12, 57) and a decrease in restraint-induced fos expression in the PVN after L-NAME treatment (58).
Given that all NOS and HO isoforms are found in the hypothalamus, which isoform(s) is responsible for the HPA response to physico-emotional stressors? With regard to NOS, we believe it to be the constitutive forms, probably both nNOS and eNOS. Evidence for this comes from the present demonstration that the NOS activity that we measured was Ca2+ dependent (i.e. cNOS), and from our previous studies that have shown L-nitroarginine and NG-methyl-L-arginine (which preferentially blocks cNOS) and 7-nitroindozole (which preferentially blocks nNOS) to attenuate the ACTH response to electroshocks, similar to the effect of L-NAME (45, 59). At this time, we cannot conclude which HO isoform is responsible for the HPA response to physico-emotional stressors.
Our working hypothesis that both NO and CO stimulate the HPA axis would suggest that when a physico-emotional stressor occurs, there may be an increase in NO and CO formation that serves to activate the PVN neurons. The question that arises is why there were no measurable increases in NOS and HO activity levels in the hypothalamus that paralleled the ACTH response to the electroshocks. At present, we are considering several possible explanations. First, although the initial response to a stressor is that the existing enzymes become more active, the method that we used to assess NO and CO formation may not have detected this change. The NOS and HO activity assays quantify the maximum amount of gas that a given amount of enzyme is capable of producing, not the actual amount of gas produced in vivo at any given time. With prolonged stress, there would be an induction of NOS and HO mRNA expression, followed by an increase in enzyme protein levels and subsequently of enzymatic activity levels and gas production. Thus, our stress procedure may not allow sufficient time for the synthesis of new enzyme and the subsequent increase in enzymatic activity levels. This idea is confirmed by the observation that nNOS mRNA expression in the PVN is significantly elevated at 2 h of restraint (60, 61), whereas increases in nNOS protein expression were evident by 6 h (62). Similarly, increases in nNOS mRNA levels at the anterior pituitary and adrenal cortex were evident after 2 h of restraint, whereas increases in NOS activity were not observed at 2 h, but were seen by 6 h of restraint (62). Alternate methods of measuring NO and CO production would be useful in testing this idea. A second possibility is that PVN NOS and HO activities did increase in response to electroshock, but because the whole hypothalamus was examined, the change in activity at the PVN was diluted below detection levels. Measurement of PVN NOS and HO activity levels would confirm this idea. Finally, the roles of NO and CO in HPA activation may be mainly facilatory; i.e. basal levels of these gases may be sufficient in themselves to allow stress-induced CRF/VP release. This is somewhat similar to our observation of the interaction between CRF and VP on ACTH release (63).
Following are some final comments regarding the specific mechanisms by which NO and CO influence the HPA axis. First, given that both L-NAME and SnPP are potent vasoconstrictors (42, 47, 48), it has been suggested that changes in the HPA response after the administration of these inhibitors might be due to alterations in blood pressure. However, the present observation that SnPP produced only a transient increase in blood pressure that subsided by the time the electroshocks were delivered, argues against this mechanism. This is further collaborated by the observation that increases in blood pressure do not up-regulate PVN activity (64). Also, in the present study, the dose of L-NAME that we used did not have a significant effect on blood pressure, ruling out this potential mechanism. Second, it is possible that the blunted HPA response to physico-emotional stressors after L-NAME administration might be due to increased corticosterone negative feedback. As we reported previously (44, 45), there sometimes is a small, albeit statistically significant, increase in plasma ACTH levels after sc L-NAME. This increase in ACTH levels would produce an elevation in circulating corticosterone levels that, in turn, may exert a negative feedback signal. When a stressor is subsequently applied (i.e. electroshocks), the ACTH and corresponding corticosterone responses would be attenuated by the presence of the stronger feedback signal. However, the present study clearly shows that in contrast to sc treatment, the icv injection of L-NAME produced no elevation in basal ACTH levels, arguing against an increased corticosterone feedback mechanism. Furthermore, neither sc (45) nor icv (present study) delivery of SnPP was associated with increased plasma ACTH levels, ruling out a feedback mechanism.
To summarize, central administration of inhibitors of NO or CO formation (L-NAME or SnPP, respectively) attenuated the plasma ACTH response to intermittent electroshocks and concurrently decreased hypothalamic NOS and HO activities. This suggests that both gases exert a stimulatory influence on the HPA response to physico-emotional stressors, and that the hypothalamus is the critical site of their action. The present study also confirmed the specificity of action of L-NAME and SnPP at suppressing NOS and HO activities, respectively. Finally, we determined that these inhibitors did not affect the HPA response indirectly through their well known actions as vasoconstrictors. However, the issue of whether NO and CO act directly at the PVN to stimulate CRF/VP release or whether there is indirect stimulation of the PVN via neural afferents that terminate at the PVN, remains unsettled. Because the icv administration of an inhibitor produces widespread distribution of the drug throughout the brain, the present study cannot resolve this question. A useful approach may be to identify PVN afferents that contain NOS/HO enzymes and determine whether these pathways are affected by drugs that alter NOS/HO activities and by stressful stimuli. For example, one such pathway may be the PVN afferent from the amygdala, which is thought to contain NOS-positive neurons and influence HPA activity (65). The effects of NOS/HO antagonists microinfused directly into the PVN on the HPA response to stressors represents another way of answering this question. These studies are presently underway in our laboratory and should increase our understanding of the precise mechanisms by which NO and CO modulate the activity of the HPA axis.
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Acknowledgments |
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Footnotes |
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2 Investigator with The Clayton Foundation.
Received November 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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 L. Wu and R. Wang Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications Pharmacol. Rev., December 1, 2005; 57(4): 585 - 630. [Abstract] [Full Text] [PDF]
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 L. Chen, D. Duricka, S. Nelson, S. Mukherjee, S. G. Bohnet, P. Taishi, J. A. Majde, and J. M. Krueger Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases J Appl Physiol, July 1, 2004; 97(1): 17 - 28. [Abstract] [Full Text] [PDF]
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C. L. Rivier, D. E. Grigoriadis, and J. E. Rivier Role of Corticotropin-Releasing Factor Receptors Type 1 and 2 in Modulating the Rat Adrenocorticotropin Response to Stressors Endocrinology, June 1, 2003; 144(6): 2396 - 2403. [Abstract] [Full Text] [PDF]
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 L. Dufourny and D. C. Skinner Influence of Estradiol on NADPH Diaphorase/Neuronal Nitric Oxide Synthase Activity and Colocalization with Progesterone or Type II Glucocorticoid Receptors in Ovine Hypothalamus Biol Reprod, September 1, 2002; 67(3): 829 - 836. [Abstract] [Full Text] [PDF]
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 D. Morse and A. M. K. Choi Heme Oxygenase-1 . The "Emerging Molecule" Has Arrived Am. J. Respir. Cell Mol. Biol., July 1, 2002; 27(1): 8 - 16. [Abstract] [Full Text] [PDF]
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