Nociceptin and nociceptin receptor, which show
structural similarities to opioid peptides and opioid receptors,
respectively, have been recently found to constitute a novel
neuromodulatory system. In the brain, however, the physiological role
of the modulation via the nociceptin receptor is still unclear.
Administered nociceptin produces hyperalgesia and hypolocomotion,
whereas the nociceptin receptor-knockout mice show no significant
abnormalities in nociceptive thresholds and locomotion. To clarify
possible involvement of the nociceptin receptor in the regulation of
nociception and locomotion, we made use of the knockout mice and
naloxone benzoylhydrazone (NalBzoH) identified originally as a ligand
for opioid receptors. Experiments on the cultured cells transfected
with the nociceptin receptor cDNA showed that NalBzoH competed with
[3H]nociceptin binding and attenuated the
nociceptin-induced inhibition of cAMP accumulation. Furthermore,
behavioral studies demonstrated that NalBzoH completely inhibited
nociceptin-induced hyperalgesia and hypolocomotion. It is therefore
likely that NalBzoH can act as a potent antagonist for the nociceptin
receptor in vivo. In wild-type mice, NalBzoH induced
antinociception but did not affect locomotor activity. In contrast, in
the knockout mice, no significant changes in nociception and locomotion
were induced by NalBzoH. These results clearly suggest that the
nociceptin system takes part in the physiological regulation of
nociceptive thresholds but not in the basal modulation of
locomotion.
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INTRODUCTION |
Modulation by biologically active peptides is an essential feature
in neurons. The opioid receptors exhibit a widespread distribution through the central and peripheral nervous systems and mediate physiological effects of endogenous opioid peptides and pharmacological actions of opioid analgesics. Recent DNA cloning studies have shown
that the G protein-coupled opioid receptor family is comprised of three
distinct opioid receptors (
-, µ-, and
-opioid receptors) and
the nociceptin/orphanin FQ receptor (1-3). On the other hand, in
previous pharmacological experiments, further refined classification of
the opioid receptor subtypes has been suggested, for example the
-receptor was classified into
1-,
2-, and
3-subtypes
(4-6). The subclassified opioid receptor subtypes are still unknown at the molecular level, and the possibility that these are based on
alternative splicing or derived from differences in post-translational modifications was proposed (1).
In contrast to the opioid peptides, nociceptin induces hyperalgesia and
hypolocomotion by interacting specifically with the nociceptin
receptors in the central nervous system (2, 3). To study the
physiological roles of the nociceptin system, we have generated mutant
mice lacking the nociceptin receptor. The knockout mice, lacking the
responses of nociceptin-induced hyperalgesia and hypolocomotion, show
no significant differences in nociceptive thresholds and locomotor
activity to control mice (7). This suggests that the nociceptin
receptor plays no essential role in regulating either nociceptive
sensitivity or locomotor activity. However, these experimental results
cannot eliminate the possibility that the nociceptin receptor is
involved in the modulation of both the mechanisms on the basal level
because the redundancy of other regulatory systems may compensate for
the abnormalities caused by the deficiency of the nociceptin receptor.
On the other hand, the loss of the nociceptin receptor results in
abnormal hearing ability, demonstrating that the nociceptin system is
essential for the regulation of the auditory system physiologically
(7). Although many pharmacological actions have been suggested so far based on effects of nociceptin administration, the physiological roles
of the nociceptin receptor have not yet been fully elucidated (8). One
major reason for this is that specific non-peptide drugs modulating the
nociceptin receptor activity in vivo are not available.
Naloxone benzoylhydrazone
(NalBzoH)1 is a derivative
compound of the µ-opioid receptor antagonist, naloxone, and produces
antinociceptive effects in vivo (9). Previous ligand-binding
studies suggested that NalBzoH interacts with the µ-,
1-, and
3-opioid receptor subtypes (4, 5). Because the
-opioid receptor
defined by cloning studies shows the pharmacological characteristics of
the
1-subtype when the cDNA is functionally expressed in
cultured cells, the molecular profile of the
3-subtype has yet to be
elucidated. Recent studies have suggested a close relationship between
the pharmacological
3-subtype and the molecular biological
nociceptin receptor, but some data have negated the relationship (10,
11). Thus, the pharmacological characteristics of NalBzoH remained to
be investigated.
From the results of ligand-binding experiments using the nociceptin
receptor expressed from the cDNA and behavioral studies contrasting
drug-induced responses between the nociceptin-deficient and wild-type
mice, we report here the identification of NalBzoH as an antagonist for
the nociceptin receptor and the contribution of the nociceptin system
to the regulation of the physiological nociceptive sensitivity.
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EXPERIMENTAL PROCEDURES |
Ligand-binding Assay--
Chinese hamster ovary (CHO) cells
expressing the rat nociceptin receptor (ROR-C) were established as
described previously (12, 13). Cells were washed with
phosphate-buffered saline and homogenized in 50 mM
Tris-HCl, pH 7.5, and 5 mM MgCl2. The homogenate was centrifuged at 1,000 × g for 5 min, the
supernatant was centrifuged at 15,000 × g for 30 min,
and the resulting pellet was suspended in the same buffer and used for
the binding assay. The binding reaction was performed at room
temperature for 60 min with the membrane preparations (35-64 µg) in
a solution (0.2 ml) containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and various concentrations of
[3H]nociceptin (45 Ci/mmol; NEN Life Science Products).
After incubation, samples were collected on a GF/C filter (Whatman),
washed with 15 ml of the buffer solution, and then counted for
radioactivity. Nonspecific binding was measured in the presence of an
excess amount of unlabeled nociceptin. The dissociation constant
(Kd) for [3H]nociceptin was determined
by Scatchard analysis, and apparent Kd values for
unlabeled ligands were obtained by measuring displacement of
[3H]nociceptin binding (3 nM) according the
equation Kd = IC50/(1 + S)
where S = (concentration of
[3H]nociceptin)/(Kd of
[3H]nociceptin).
Cyclic AMP Assay--
The cyclic AMP assay was carried out
essentially as described previously (13). CHO cells expressing the
nociceptin receptor were cultured in 24-well plates (2.5 × 105 cells/assay) and preincubated for 5 min with 0.72 ml of
Krebs-Henseleit buffer (111 mM NaCl, 5.9 mM
KCl, 2.5 mM CaCl2, 1.2 mM
MgCl2, 1.2 mM NaH2PO4,
25 mM NaHCO3, 11 mM glucose, pH
7.4) supplemented with 0.5 mM 3-isobutyl-1-methylxanthine.
Reactions were started by addition of 0.08 ml of 100 µM
forskolin-containing test agents. After 10 min, the incubation
reactions were terminated by addition of ice-cold 1 M
perchloric acid solution (0.2 ml), and cell debris was removed by
centrifugation at 15,000 × g for 10 min. The
supernatants were neutralized with 2 M KHCO3
(0.08 ml) and centrifuged at 15,000 × g for 10 min.
The resulting supernatants were subjected to enzyme immunoassays (cAMP
enzyme immunoassay system; Pharmacia Amersham Corp.).
Antinociceptive Tests and Measurement of Locomotor
Activity--
We used the male mice lacking the nociceptin receptor
and wild-type mice (9-12 weeks old) as reported by Nishi et
al. (7).
In the tail-flick test, the latency to withdraw the tail from a focused
light stimulus was measured electronically, using a photocell. The
intensity of the heat stimuli was set as inducing approximately 10-s
base-line latencies in wild-type mice. A maximal latency of 20 s
was assigned to minimize tissue damage. The base-line latencies were
determined at least 1 h before the drug treatment for all animals
as the mean of two trials. Tail-flick latencies were measured at 10, 15, 60, 5, and 10 min after treatment with nociceptin (1 or 10 nmol
intracerebroventricularly), NalBzoH (10, 25, 50 and 75 mg/kg
subcutaneously), morphine (1 or 5 mg/kg subcutaneously), naloxone
(5 mg/kg intraperitoneally), and U-50,488H (1 or 5 mg/kg subcutaneously), respectively, in the wild-type and knockout mice. The
results were expressed as a percentage of the maximal possible effect.
Maximal possible effect (%) = ((latency after drug administration
base-line latency)/(cut-off latency
base-line latency)) × 100 (n = 6-10 in each group). In the writhing assay,
mice received an injection (10 ml/kg intraperitoneally) of 0.7% acetic
acid. Morphine (0.1 or 1 mg/kg subcutaneously), U-50,488H (1 or 5 mg/kg subcutaneously), and NalBzoH (10, 25, 50, and 75 mg/kg subcutaneously), were administered 60, 15, and 15 min, respectively, before the observation. The number of writhes, characterized by a wave of contraction of the abdominal muscles followed by extension of the hind
limbs, was counted for 10 min, beginning 5 min after acetic acid
injection.
In the locomotion test, each animal was placed in a transparent acrylic
cage immediately after the tail-flick test, and locomotor activity was
measured for 30 min using digital counters with infrared sensors
(Scanet SV-10; Toyo Sangyo, Toyama, Japan).
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RESULTS AND DISCUSSION |
Naloxone Benzoylhydrazone as Nociceptin Receptor
Antagonist--
First, we examined the ligand-binding properties of
the nociceptin receptor using membrane preparations from the CHO clone transfected with the rat nociceptin receptor (ROR-C) cDNA (12, 13).
Saturation analysis of [3H]nociceptin binding revealed
that the ROR-C proteins expressed in the cells are capable of binding
[3H]nociceptin, and Scatchard analysis yielded a
Kd of 0.54 nM (Fig.
1A). Displacement tests were
performed to examine the effects of NalBzoH on the nociceptin receptor.
The results in Fig. 1B show that NalBzoH competed with
nociceptin binding effectively and the apparent Kd
of NalBzoH was calculated to be ~25 nM.

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Fig. 1.
Ligand-binding properties of nociceptin
receptor expressed from ROR-C cDNA. A, saturation
analysis of [3H]nociceptin binding to membrane
preparation from CHO cells expressing the rat nociceptin
receptor/ROR-C. Total binding ( ) and nonspecific binding ( ) are
plotted. The inset shows Scatchard plots of the data.
B, effects of naloxone benzoylhydrazone on
[3H]nociceptin binding to membrane preparation from CHO
cells expressing the rat nociceptin receptor/ROR-C. Membrane
preparations were incubated with 3 nM
[3H]nociceptin and various concentrations of unlabeled
ligands. Only specific binding is presented. Data are means of at least
two experiments. In the present data the Kd of
NalBzoH was calculated to be 24 nM (in the three sets of
experiments the Kd = 25.3 ± 2.4).
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The results in Fig. 1B demonstrate that nociceptin and
NalBzoH interact with the expressed nociceptin receptor at the same site. This competitive binding is also supported by a recent report that NalBzoH inhibited [Tyr14]nociceptin binding to
microsomes prepared from the brain (14). These facts suggest that
NalBzoH should act as an agonist or antagonist at the nociceptin
receptor. As we reported previously, in contrast to nociceptin, NalBzoH
(10 pM to 1 µM) does not inhibit
forskolin-induced cAMP accumulation in the CHO clone (13). Therefore it
seems appropriate to conclude that NalBzoH acts as a high affinity
antagonist for the nociceptin receptor. This conclusion was further
supported by the experimental data (Fig.
2), in which NalBzoH blocked the nociceptin-induced inhibition of cAMP accumulation in CHO cells expressing the nociceptin receptor. The Kd value of
NalBzoH in this functional assay was severalfold higher than that
calculated in the binding assay. Although the reason for this is
unclear, a similar discrepancy of Kd values has been
observed in the case of nociceptin as an agonist ligand (2, 3, 13).

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Fig. 2.
Effects of naloxone benzoylhydrazone on
nociceptin receptor-mediated inhibition of cAMP accumulation in CHO
cells transfected with nociceptin receptor/ROR-C cDNA.
Forskolin (10 µM) elevated the cAMP level to 8-fold the
basal level. Nociceptin (3 nM) inhibited 80% of the
forskolin-induced cAMP accumulation. NalBzoH attenuated the
nociceptin-induced inhibition of the cAMP accumulation in a
dose-dependent manner, but naloxone showed no significant
effects. The results are shown as means ± S.E.
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Previous studies suggested that NalBzoH interacts with the
- and
µ-opioid receptors, although [3H]NalBzoH showed a
single high affinity binding component in the brain (4-6). NalBzoH
could be utilized as a potent inhibitor of the function of nociceptin
in vivo if the nociceptin receptor binds NalBzoH
predominantly in the brain.
Nociception and Nociceptin Receptor--
To compare nociceptive
responses induced by various drugs between the nociceptin
receptor-deficient and wild-type mice, we conducted the heat tail-flick
test. No measurable changes were detected in the basal nociceptive
thresholds between the genotypes in this test as described previously
(7, 15). As expected, morphine (µ-opioid receptor agonist) and
U-50,488H (
1-opioid receptor agonist) induced antinociceptive
effects dose-dependently in wild-type mice (Fig.
3A). These effects were also
seen in the mutant mice, and both drugs showed similar effective doses
in the mutant and wild-type animals. As previously reported (5, 7),
NalBzoH and nociceptin induced antinociceptive and hyperalgesic effects, respectively, in wild-type mice. However, the knockout mice
lacked both the effects induced by NalBzoH and nociceptin. Similar
results were obtained in the acetic acid-induced writhing test, a
method allowing detection of relatively smaller changes in nociceptive
thresholds compared with the tail-flick test (Fig. 4). Furthermore, NalBzoH effectively
inhibited hyperalgesia induced by nociceptin in wild-type mice (Fig.
3B). However, the hyperalgesia was affected by neither
U-50,488H at the dose inducing analgesia in normal conditions nor 5 mg/kg of naloxone which antagonizes the action of morphine.
Furthermore, the antagonistic effect of NalBzoH on the
nociceptin-induced hyperalgesia was not affected by naloxone.

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Fig. 3.
Antinociceptive effects of opioid-related
drugs in the tail-flick test. A, effects of opioid
receptor agonists and nociceptin on nociceptive thresholds in
tail-flick test in the nociceptin receptor-knockout ( / ) and
wild-type (+/+) mice. The test protocol is described in the text. The
mean base-line values were 9.3-12.1 s and 11.1-12.6 s in the
wild-type and knockout mice, respectively. The results were expressed
as the maximal possible effect (%) (n = 5-13 in each
group). Naloxone at a dose (5 mg/kg), which can antagonize morphine (5 mg/kg)-induced antinociception in wild-type mice, was used.
**p < 0.05 versus corresponding base-line
value (Dunnett multiple comparisons test). B, effects of
NalBzoH on nociceptin-induced hyperalgesia in wild-type mice. The test
protocol is described in the text. The mean base-line values were
9.2-12.1 s in wild-type mice. The results were expressed as maximal
possible effect (%) (n = 6-10 in each group).
**p < 0.05 versus corresponding base-line
value,  p < 0.05 versus (nociceptin + vehicle (V))-treated group (Dunnett multiple comparisons
test).
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Fig. 4.
Antinociceptive effects of opioid-related
drugs in the acetic acid induced-writhing assay. The test protocol
is described in the text. The results are expressed as the mean ± S.E. of the writhing counts for each mouse (n = 20-25
in each group). **p < 0.05 versus
corresponding pre-value (Dunnett multiple comparisons test).
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The results clearly show that the knockout mice lack the signal
transduction system mediating both nociceptin-induced hyperalgesia and
NalBzoH-induced antinociception. Thereby the nociceptin receptor is
most likely to correspond to the primary reacting site of
NalBzoH-induced antinociception in wild-type mice. Because we observed
no significant NalBzoH-induced effect on nociceptive thresholds in the
knockout mice retaining sensitivities to µ- and
-opioid receptor
agonists, it seems that NalBzoH at 75 mg/kg in both the tail-flick and
writhing tests did not affect µ- and
1-opioid receptors. Thus, the
specific inhibitory effect of NalBzoH on nociceptin-induced
hyperalgesia strongly indicates that NalBzoH acts as a potent
antagonist for the nociceptin receptor in vivo. Moreover, it
is reasonable to assume that the NalBzoH-induced antinociception in
wild-type mice is due to disregulation of nociceptive thresholds by the
inhibition of the nociceptin receptor. Our findings suggest that the
nociceptin receptor participates in the physiological regulation of
nociceptive thresholds, and also that in the knockout mice
neuroregulatory systems other than the nociceptin system compensate for
abnormalities in nociceptive thresholds caused by the deficiency of the
nociceptin receptor. This should stimulate further investigations to
locate regions responsible for the nociceptive control exerted by the nociceptin receptor. We now know the distribution patterns of both
nociceptin precursor and nociceptin receptor in the central nervous
system (8). Areas relating to perception and modulation of pain include
the periaquedueductal gray and cerebral cortex, where expression of
both nociceptin precursor and nociceptin receptor is found. These
regions may be the best candidate sites responsible for the modulation
of nociceptive sensitivity.
Locomotion and Nociceptin Receptor--
Next, we examined
whether the nociceptin system is involved in locomotor activity. As
described previously (7), the spontaneous locomotion in the nociceptin
receptor-knockout mice was slightly lower than that in wild-type mice,
and nociceptin induced hypolocomotion in wild-type mice while the
knockout mice lacked the effect (Fig. 5A). Administration of NalBzoH
resulted in no significant changes of locomotion in either mouse
genotype (Fig. 5A). In wild-type mice, however,
nociceptin-induced hypolocomotion was efficiently inhibited by NalBzoH
at the dose inducing the antinociceptive effect. In contrast, U-50,488H
at the effective dose for antinociception barely attenuated the
hypoactivity (Fig. 3B).

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Fig. 5.
Locomotor activity. A,
effects of nociceptin and NalBzoH on the locomotor activity in the
nociceptin receptor-knockout ( / ) and wild-type (+/+) mice. The test
protocol is described in the text. Results were analyzed as total
counts for 30 min and are given as the mean ± S.E.
(n = 6-10 in each group). **p < 0.05 versus control (cont.) group (Dunnett multiple
comparisons test). B, effect of NalBzoH on
nociceptin-induced hypolocomotion in wild-type mice. Results were
analyzed as total counts for 30 min and are given as the mean ± S.E. (n = 6-10 in each group). **p < 0.05 versus control (cont.) group.
 p < 0.05 versus (nociceptin + vehicle
(V))-treated group (Dunnett multiple comparisons
test).
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The inhibitory effect of NalBzoH on nociceptin-induced hypolocomotion
is probably due to an antagonistic action at the nociceptin receptor as
discussed above. If the nociceptin receptor contributes to the
regulation of locomotor activity, NalBzoH should induce hyperactivity
in wild-type mice. However, we could not find any significant changes
in locomotor activity when we administered NalBzoH in wild-type mice.
This result strongly suggests that the nociceptin receptor does not
participate in the control of spontaneous locomotion physiologically.
Perhaps nociceptin-induced hypolocomotion is an adverse effect caused
by nociceptin-induced hyperalgesia. Behavioral studies reported
recently showed disruption of balance following intracerebroventricular
administration of nociceptin in rats (16). Since our results here did
not deal with motor coordination, the possible involvement of the
nociceptin receptor in the regulation remains to be investigated.