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Institute of Pharmacology (C.M., G.T., P.P., P.N.), Catholic University Medical School, Largo Francesco Vito 1, 00168 Rome, Italy; and Department of Endocrinology (A.G.), St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom
Address all correspondence and requests for reprints to: Prof. Pierluigi Navarra, Institute of Pharmacology, Catholic University Medical School, Largo Francesco Vito 1, 00168 Rome, Italy.
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Abstract |
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In this study, we have investigated the effects of NO and CO on the release of immunoreactive (ir)-IL-1ß from the rat hypothalamus in vitro. It was observed that the NO donor, sodium nitroprusside (SNP), stimulates ir-IL-1ß release under basal conditions, whereas the increase in CO levels obtained with hemin, the CO precursor through the heme oxygenase pathway, has no effect on basal ir-IL-1ß release but inhibits release stimulated by high K+ concentrations. The opposite effects of the two gases on cytokine release seemed to be caused by the activation of different signaling pathways, because: 1) SNP, but not CO-saturated solutions, is able to increase cyclic GMP levels in hypothalamic tissue; 2) CO-saturated solutions increase PGE2 production and release from the hypothalamic explants, whereas SNP has no effect; 3) SNP-stimulated ir-IL-1ß release is counteracted by a selective inhibitor of soluble guanylyl cyclase, LY 83583, but not by a cyclooxygenase inhibitor, indomethacin; and 4) conversely, indomethacin, but not LY 83583, reverses the inhibitory effect of hemin on K+-stimulated ir-IL-1ß release.
It is concluded that NO and CO signal in the rat hypothalamus via the activation of soluble guanylyl cyclase and cyclooxygenase, respectively.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More recently, a new class of neuromodulatory agents has been shown to play a role in the control of neuroendocrine function: the gaseous compounds nitric oxide (NO) and carbon monoxide (CO) (6). The mode of action of these gases is exquisitely paracrine, because they act only at short distance from their sites of generation. All of the known isoforms of NO synthase and heme oxygenase (HO), i.e. the enzymes leading to NO and CO biosynthesis, respectively, have been detected within the rat hypothalamus (7, 8, 9, 10), providing the basis for the paracrine control of neuropeptide release.
An interplay seems to exist between IL-1 and the gases NO and CO in the control of the HPA axis. Thus, IL-1- and LPS-induced increases in circulating adrenocorticotropic hormone and corticosterone levels in the rat were significantly potentiated by the inhibition of NO synthase activity by L-nitroarginine methyl ester, indicating that the generation of NO induced by IL-1 or LPS serves as a counterregulatory mechanism impeding the exaggerated activation of the HPA axis (11, 12). To date, there is no in vivo evidence of a similar interaction between the cytokines and CO, although hemin, the CO precursor via the HO pathway, has been shown to inhibit the stimulatory effect of IL-1ß on CRH release from rat hypothalamic explants (13). This finding matched similar observations with IL-1ß and NO-donors on the releases of CRH and vasopressin in vitro (14, 15), suggesting that the hypothalamus is the major locus for interactions among IL-1ß, NO, and CO.
To explore such interactions in more detail, we have used a previously characterized in vitro model to investigate the effects of NO and CO on the release of immunoreactive (ir)-IL-1ß from rat hypothalamic explants. Such release was stimulated by high K+ concentrations, and K+-induced release was dependent on Ca2+, suggesting that the fraction of IL-1 released under the depolarizing stimulus belongs to a preformed pool, which is probably neuronal in origin (16).
While previous studies have suggested that the biological actions of CO in the CNS mimic those of NO (17), we unexpectedly found that the generation of NO and CO in hypothalamic explants was associated with opposite effects on ir-IL-1ß release. This also suggested that the two gases may exert the above actions through the activation of different signaling mechanisms. Further studies provided pharmacological and biochemical evidence that NO and CO signal in the rat hypothalamus via the activation of soluble guanylyl cyclase (sGC) and cyclooxygenase (COX), respectively.
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Materials and Methods |
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Hypothalamic dissection
On the day of the experiment, the animals were decapitated
between 0900 h and 1000 h to avoid circadian variation. The
brains were removed, and the hypothalami were dissected within the
following limits: anterior border of the optic chiasm, anterior border
of the mamillary bodies, and the lateral hypothalamic sulci. The depth
of dissection was 4 mm. The hypothalami were then bisected
longitudinally through the midsagittal plane, and two hypothalamic
halves from the same animal were incubated in the same vial. The total
dissection time was less than 2 min from decapitation.
Hypothalamic incubations
Two hypothalamic halves from the same animal were incubated in
7-ml polyethylene vials containing 500 µl incubation medium for
ir-IL-1ß and PGE2 assays and 1 ml for cyclic GMP (cGMP)
assay.
In experiments for the determination of ir-IL-1ß release, the incubation medium was Earle’s balanced salt solution (EBSS; Sera-Lab Ltd, Crawley Down, UK) supplemented with 0.1% BSA (Sigma Chemical Co., St Louis, MO), 50 µg/ml ascorbic acid (Sigma), and 80 IU/ml aprotinin (Trasylol, Bayer, Germany), pH 7.4, in an atmosphere of 95% O2/5% CO2. In the first 60 min of incubation after hypothalamic explant, the medium was replaced every 20 min; during this time, irIL-1ß release rate tended to stabilize (data not shown). Therefore, in all the subsequent experiments, the first hour was taken as preincubation time. Thereafter, the explants were subjected to a 20-min control incubation in plain medium to assess basal ir-IL-1ß release. This was followed by a second 20-min incubation in medium containing test substances or, for the control group, in medium alone. In experiments with KCl, in the second 20-min period, EBSS was replaced by a medium consisting of 56 mM KCl and 67 mM NaCl, with the same concentrations of the other ions as found in EBSS. Such medium was able per se to significantly increase ir-IL-1ß release, with respect to incubations with plain EBSS (16). Medium samples were stored at -30 C until assayed for ir-IL-1ß.
Experiments for the determination of cGMP and PGE2 were conducted according to the same protocol as above, with the following modifications: in experiments with CO, saturated solutions of the gas were prepared by bubbling pure CO (Caracciolossigeno, Rome, Italy) for 1 h before the experiments (i.e. during the preincubation time) and then incubating the tissue in CO-saturated medium in an atmosphere of O2/CO2/CO. The use of EBSS in these experiments was prevented by the observation that gassing of EBSS for 1 h with pure CO increases pH of the solution to about pH 8.8, which inhibits sGC activity (18). Therefore, an incubation medium consisting of Tris (0.1 M) + NaCl (95 mM), pH 7.4, was employed in experiments for the determination of cGMP, and the same medium, with the addition of 0.05% BSA, pH 7.4, was used when PGE2 release was assessed.
Experiments with sodium nitroprusside (SNP) for the evaluation of PGE2 and cGMP were conducted in EBSS supplemented with 0.05% BSA and in Tris (0.1 M) + NaCl (95 mM), respectively.
Incubation media and tissues were stored at -30 C until assayed for PGE2 and cGMP, respectively.
Analytical methods
IL-1ß was measured by RIA as previously described (16). The
detection limit of the assay was 8 pg/tube, and the estimated
EC50 was 170 pg/tube. The intra- and interassay variability
was 2% and 10%, respectively.
PGE2 was measured by RIA as previously described (19). The detection limit of the assay was 2 pg/tube, with an EC50 of 28 pg/tube.
cGMP was assayed in hypothalamic homogenates. Hypothalamic tissues were homogenated in 1 ml Tris (0.05 M) + 4 mM EDTA at 4 C using a Labsonic 2000 sonicator (B. Braun, Melsungen, Germany). Fifty µl of the homogenate were taken for the subsequent protein assay, and the remainder was heated for 3 min in a boiling water bath to coagulate proteins. After 20-min centrifugation at 4,000 rpm at 4 C, the supernatants were assayed for cGMP using a commercial RIA kit (Amersham, Little Chalfont, UK). Briefly, 100 µl of unknown or standard (the latter in the range 0.125–8 pmol/tube) were incubated for 90 min at 4 C with 50 µl of tracer (about 9,500 cpm/tube) and 50 µl of appropriately diluted antiserum. Separation of bound from free cGMP was achieved by precipitation with 1 ml of 60% cold ammonium sulfate. Tubes were then centrifuged at 4,000 rpm for 6 min, and the supernatants were discarded. Pellets were resuspended in 1 ml of cold double-distilled water, and radioactivity was measured in 10 ml of scintillation fluid. The detection limit of the assay was 0.125 pmol/tube, and the EC50 was 1.64 pmol/tube.
Proteins from hypothalamic homogenates were assayed using the bicinchoninic acid method (BCA Protein Assay Reagent, Pierce, Rockford, IL).
Drugs and chemicals
Hemin HCl, biliverdin free base, indomethacin (INDO) free base,
SNP, and 3-isobutyl-1-methyl-xanthine (IBMX) free base were obtained
from Sigma. Tin-mesoporphyrin-9 (SnMP9) was obtained from Porphyrin
Products Inc. (Logan, UT). LY 83583 (6-anilino-5, 8-quinolinequinone),
a selective inhibitor of soluble guanylyl cyclase (20), was purchased
from Calbiochem (La Jolla, CA).
NG-nitro-L-arginine methyl ester (L-NAME) was
obtained from Bachem Feinchemikalien AG (CH 4416, Bubendorf,
Switzerland).
Hemin and SnMP9 were dissolved in 100 mM NaOH; SNP in normal saline; biliverdin in absolute methanol; and INDO, IBMX, and LY 83583 in absolute ethanol. All substances were further diluted in incubation media to obtain working solutions. The latter had a final pH of 7.4.
All drugs tested were found to produce no shift in the standard curves of the assays for ir-IL-1ß, cGMP, and PGE2.
Statistical analysis
Results are given as means ± 1 SEM, unless
otherwise stated. In the experiments conducted to estimate cGMP content
in the hypothalamic tissue, data are expressed as fmol/mg of protein.
In experiments for assay of ir-IL-1ß and PGE2 released into the
incubation medium, data are expressed as pg/hypothalamus and pg/ml,
respectively. Furthermore, ratios were calculated by dividing the
amount of ir-IL-1ß or PGE2 released in the second 20-min incubation
period by those released in the previous period, the latter providing a
paired control for the second 20-min period in each tissue block.
Expression of data as ratios allows for compensation in irIL-1ß and
PGE2 variations among different tissue explants. To clarify the ratio
calculation procedure, in Table 1
, both
the individual values of IL-1 released in two consecutive 20-min
periods and the ratio calculated for each hypothalamus are reported.
The data were then analyzed by ANOVA and subsequent Newman-Keul’s test
for multiple comparisons among group means. Differences were considered
statistically significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
KCl-stimulated ir-IL-1ß release (Fig. 1B) from the hypothalamus.
Statistically significant increases were obtained with 0.1 and 1
mM SNP.
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, A and B). The inhibitory effect of
hemin was attributed to CO, because it was blocked by the selective HO
inhibitor, SnMP9 (21) (Fig. 2C); and the stable HO end-product,
biliverdin, did not antagonize K+-induced ir-IL-1ß
release (not shown). Hemin also tended to reduce SNP-stimulated
ir-IL-1ß release, although inhibition did not reach statistical
significance (Table 1
).
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The effects of specific antagonists of sGC and COX on NO- and
CO-modulated IL-1ß release
The selective inhibitor of sGC, LY83583, at 1 µM,
significantly reduced the increase in ir-IL-1ß release elicited by 1
mM SNP but failed to counteract the inhibitory effect of 10
µM hemin on K+-stimulated cytokine release
(Table 2). LY83583 given alone had no
effect on basal ir-IL-1ß release but reduced release stimulated by
KCl (Table 2).
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). INDO given alone had no
significant effect on K+-stimulated cytokine release [data
expressed as ir-IL-1ß ratio, µ ± SEM of (n) animals
per group: controls 0.981 ± 0.041 (9); 56 mM KCl
1.164 ± 0.036 (9), P < 0.01 vs.
controls; 56 mM KCl + 1 µg/ml INDO 1.125 ± 0.021
(9), P < 0.01 vs. controls, NS difference
vs. KCl alone].
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). Hemin was also used to increase CO
levels in the tissue; however, the former is known to exert a direct
inhibitory effect on sGC (22). In fact, we found that 10
µM hemin significantly decreased cGMP levels within the
hypothalamic tissue [data expressed as fmol of cGMP/mg of protein, µ
± SEM of (n) animals per group: controls 74.24 ± 10
(6); 10 µM hemin 46.03 ± 11.8 (6),
P < 0.05 vs. controls]. Conversely, 1
mM SNP produced a significant increase in cGMP levels in
the presence of IBMX (Fig. 3B).
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): this finding matches our previous
observations that hemin increases PGE2 production and release by the
rat hypothalamus in vitro (23).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increased cGMP production induced by NO in the CNS is known to elicit both stimulatory and inhibitory responses on the release of neurotransmitters or neurohormones. Stimulation is often associated with an action on neurons signaling via excitatory amino acids (24). Among inhibitory effects attributed to the activation of the NO-cGMP pathway are the release of CRH and vasopressin (14, 25). Many effects of cGMP are thought to be mediated by the stimulation of cGMP-dependent protein kinase (24), but additional effects of the cyclic nucleotide are thought to be caused by activation or modulation of specific cGMP-dependent ion channels, leading to enhanced influx of various cations, including Ca2+ (26). However, it is unlikely that changes seen in brain slices are simply caused by a direct action of cGMP on nerve terminals, because complex interaction between neurons and glia may take place (24). Thus, although the activation of sGC by NO may well account for the observed effect of the NO-donor, SNP, on ir-IL-1ß release in vitro, our experimental approach does not allow us to conclude that neuronal IL-1ß is specifically involved.
sGC has previously been regarded as the primary signaling mechanism for endogenous CO (27). This does not seem to be the case for the effects of the gas on ir-IL-1ß release from the hypothalamus. Instead, the present evidence suggests that CO acts on cytokine release via the stimulation of COX. We have previously shown that the precursor of CO through the HO pathway, hemin, induces concentration-dependent increases in PGE2 release from hypothalamic explants in the same experimental model as that used in this study (23). This effect was specifically attributed to the generation of CO after HO-dependent hemin catabolism, because it was blocked by HO inhibitors such as SnMP9 and Zn-protoporphyrin-9, as well as by ferrous hemoglobin, which inactivates CO by direct binding. Moreover, the stable HO end-product, biliverdin, did not enhance PGE2 release from the explants (23). The direct evidence obtained here with CO-saturated solutions matches previous findings with hemin, thereby confirming the stimulatory role of CO on PG production in this experimental paradigm. Though PGE2 was taken in these studies as a marker of COX activity, it is not known whether PGE2 itself or other end-products of COX are responsible for the inhibition of K+-stimulated ir-IL-1ß release, although we have previously observed that PGE2 inhibits unstimulated IL-1 release from hypothalamic explants (our unpublished data).
We have been unable to show any significant effect of the NO-donor, SNP, on PGE2 production and release from the hypothalamus. This finding is in conflict with evidence showing that NO stimulates COX in various in vitro models, including short-term incubations of rat medio-basal hypothalami (28, 29). More recently, however, it has been demonstrated that NO can also inhibit COX activity (in particular, the inducible isoform COX2) in microglial cells (30). In our experimental paradigm, we cannot discriminate between the COX isoforms implicated in PGE2 production; but COX2 might play a role, insofar as it is the isoform constitutively expressed in neurons (31). Moreover, the possibility that NO activates COX in the same manner as it does sGC (i.e. binding to the heme moiety of the enzyme) has been questioned (32). Though the issue of the actions exerted by NO on COX remains under dispute, in any case, the COX pathway does not seem to be involved in SNP stimulation of IL-1ß release, because its inhibition by INDO had no significant effect on SNP-stimulated cytokine release.
As far as the lack of CO effect on cGMP production is concerned, a fundamental factor may be that the gas is only a weak activator of sGC, producing only 1- or 2-fold increases in enzymatic activity (33, 34). Therefore, effects of CO mediated by its activation of sCG are likely to be detected only in those brain areas, such as the hippocampus, endowed with high levels of sGC (17). On the contrary, two major studies have shown very low sGC levels in the rat hypothalamus (35, 36), which is consistent with our failure to show any relationship between the effects of CO and sGC activity in hypothalamic explants. In contrast, NO was reported to be more active than CO in stimulating sGC, because the former, under directly comparable conditions, increases cGMP production by a factor of 10 or more (33, 34). This may account for the fact that NO is able to induce significant increases in cGMP levels in the hypothalamus, as shown by us and others (37), in spite of low levels of hypothalamic sGC.
In conclusion, it is apparent that the pattern of signaling mechanisms by the gases, NO and CO, is growing increasingly complex. Though it was previously thought that activation of sGC was the dominant, if not the sole, signal transduction mechanism of NO, subsequent studies have revealed that the latter may also exert biological activities via alternative mechanisms such as ADP ribosylation, activation of COX, protein nitrosylation, and the formation of peroxynitrite ions (38). The same profile seems now to emerge with CO, as alternative signaling pathways, such as the enzymes cytochrome P450 (39) or COX, are being proposed. The picture is even more complex if we consider that the two gases may or may not (as shown in the present study) share a common signaling pathway in different brain regions, depending on the amount of target proteins at the sites of gas generation and the relative efficacy of the gases in binding and activating such proteins. Thus, neuroregulation by gaseous transmitters assumes an increasingly important role in neuroendocrine function as their activities become more specifically defined.
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Footnotes |
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2 Contributed equally to this work.
Received June 12, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-adrenergic receptors.
Endocrinology 136:3597–3603[Abstract]
and nitric oxide down-regulate
lipopolysaccharide-induced prostanoid production in cultured rat
microglial cells by inhibiting cyclooxygenase-2 expression. J
Neurochem 66:1963–1970[Medline]
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