Me ´dicale–Universite ´LouisPasteur,67404IllkirchCedex,France,3DepartmentofBiochemistry,TohokuGraduateSchoolofMedicine,Sendai,Miyagi980-
receptor-like (ORL-1) receptors, also known as OP4 receptors. Here we demonstrate that buprenorphine, but not morphine, activates
mitogen-activated protein kinase and Akt via ORL-1 receptors. Because the ORL-1 receptor agonist orphanin FQ/nociceptin blocks
markedly enhanced in mice lacking ORL-1 receptors using the tail-flick assay. Additional support for a modulatory role for ORL-1
phine administration. Our results indicate that the antinociceptive effect of buprenorphine in mice is ?-opioid receptor-mediated yet
The synthetic oripavine buprenorphine is a mixed agonist–an-
see Cowan, 1995; Rothman et al., 1995). Buprenorphine is an
alternative medication to methadone in the treatment of heroin
addicts (Mello et al., 1993; Ling et al., 1996, 1998; Litten and
Allen, 1999; Johnson and McCagh, 2000) and also used as an
analgesic (for review, see Finco et al., 1995; Picard et al., 1997).
Subutex and suboxone tablets, which contain buprenorphine
alone or a buprenorphine–naloxone mixture, respectively, were
recently approved for opiate abuse treatment in the United
States. Buprenorphine is also effective in treatment of refractory
addiction; however, clinical trials have not been persuasive (for
review, see Compton et al., 1995).
Of the clinically used opioid agonists, buprenorphine is con-
sidered exceptionally safe. The partial agonist activity at opioid
ceiling effect with regard to ?-opioid receptor-mediated respira-
reported only in cases of coabuse with other drugs such as ben-
zodiazepines (Reynaud et al., 1998). Overall, the safety aspects of
buprenorphine provide a compelling rationale for many clinical
drug has a unique and complex pharmacology (for review, see
discontinuation of the drug appears to elicit limited withdrawal
symptoms (Martin et al., 1976; Cowan et al., 1977a,b; Jacob and
This study was supported in part by National Institutes of Health Grants DA00411 (K.L.) and DA05010 and
tical Sciences, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766. E-mail
TheJournalofNeuroscience,November12,2003 • 23(32):10331–10337 • 10331
doses, buprenorphine is an effective analgesic, but, at higher
doses, the antinociceptive effect of the drug is often diminished
(Dum et al., 1981; Lizasoain et al., 1991). This bell-shaped dose–
pharmacology of buprenorphine. Partial agonism at opioid re-
submaximal opiate-like pharmacological effects of buprenor-
phine (for review, see Heel et al., 1979; Lewis, 1985). However,
partial agonist activity does not provide a satisfactory rationale
for a bell-shaped dose–response curve.
Activation of the classical opioid receptors produces antino-
ciception, whereas activation of supraspinal opioid receptor-like
(ORL-1) receptors is pronociceptive or, at least, opposes opioid
receptor-mediated antinociception (for review, see Mogil and
Pasternak, 2001). Recently, buprenorphine has been shown to
activate ORL-1 receptors (Wnendt et al., 1999; Bloms-Funke et
al., 2000; Hawkinson et al., 2000; Huang et al., 2001), leading to
we demonstrate that buprenorphine is a partial agonist at
?-opioid receptors and a full agonist at ORL-1 receptors for ac-
ing ? or ORL-1 receptors, along with pharmacological tools, we
show that buprenorphine-induced antinociception is mediated
by ?-opioid receptors and modified by ORL-1 receptor
Subjects. Male ORL-1 receptor knock-out mice (Nishi et al., 1997) and
their wild-type littermates were assayed at 10–12 weeks after birth.
?-Opioid receptor knock-out and wild-type mice, fully backcrossed to
et al., 1996). Mice were housed two to four per cage with access to food
and water ad libitum in a 12 hr light/dark cycle. All experiments were
the Care and Use of Laboratory Animals and were approved by the Insti-
tutional Animal Care and Use Committee. Mice were housed in the
testing room for at least 4 d before experimentation and remained there
until the end of the experiment. All experiments were conducted during
the light phase by a researcher blind to the genotype of the mice.
Drugs. Morphine and OFQ/N were purchased from Mallinckrodt (St.
Louis, MO) and Phoenix Pharmaceuticals (Belmont, CA), respectively.
Buprenorphine and etorphine were kindly provided by the National In-
stitute on Drug Abuse Drug Supply Program (Research Triangle Insti-
tute, Research Triangle Park, NC). The ORL-1 antagonist (?)-J-113397
ethyl-1, 3-dihydro-2H-benzimidazol-2-one) was synthesized (Research
Triangle Park) by a modification of the procedure reported previously
(Kawamoto et al., 1999).
Cell lines. Chinese hamster ovary (CHO) cells were transfected with
the human ORL-1 receptor (CHO-ORL-1) or the murine ?-opioid re-
ceptor (CHO-MOR), as described previously (Kaufman et al., 1995;
Keith et al., 1996). Transfected and nontransfected cells were grown in
penicillin-G, 100 U/ml streptomycin, and 0.25 ?g/ml amphotericin B
(Omega Scientific, Tarzana, CA).
Assays for phosphorylation of mitogen-activated protein kinase and Akt.
Two days before the assay, MOR- or ORL-1-transfected cells were cul-
tured in DMEM containing 1% fetal calf serum, followed by serum star-
50 mM HEPES. Cells were then treated for 5 min (37°C) with varying
concentrations of ligand, followed by extraction with SDS sample buffer
containing 62.5 mM Tris, 2% SDS, 10% glycerol, and 50 mM dithiotritol,
mitogen-activated protein (MAP) kinase were detected using specific
rabbit phospho-Akt and phospho-MAP kinase antibodies (Cell Signal-
ing Technology, Beverly, MA), followed by peroxidase-conjugated sec-
ondary antisera (Bio-Rad, Hercules, CA) and ECL detection. Antibodies
kinase protein levels. For quantification, films were scanned using a
Prism 3 (GraphPad Software, San Diego, CA).
Nociceptive assay. The tail-flick assay was used to examine the antino-
ciceptive effect of buprenorphine in wild-type and ORL-1 or ?-opioid
receptor knock-out mice. Briefly, a beam of light was focused on the
dorsal surface of the mouse’s tail ?2.5 cm from the tip of the tail. The
intensity of the light was adjusted so that the baseline latencies averaged
between 3 and 4 sec. A cutoff time of 15 sec was used as the maximum
possible effect to minimize tissue damage. Initially, mice were tested for
baseline latencies and then, 30 min later, injected with cumulative doses
30 min later, respectively. Each experiment was conducted at least twice.
The antinociceptive effect of buprenorphine was also examined in the
presence of J-113397, an ORL-1 receptor antagonist (Kawamoto et al.,
1999), in mice lacking the ORL-1 receptor and their wild-type litter-
mates. Mice were initially tested for baseline and, after a 30 min delay,
treated with J-113397 (3 mg/kg, i.p.) or vehicle (20% DMSO in saline).
Fifteen minutes later, mice were injected with buprenorphine (0.3 mg/
alone on nociceptive responses was also determined in wild-type mice
(n ? 5). Mice were tested for baseline and injected, 30 min later, with
J-113397 (3 mg/kg, i.p.) and tested 15, 30, and 60 min later.
ner. The effect of J-113397 on this bell-shaped dose–response curve was
also studied. Mice were tested for baseline latencies and, after a 30 min
delay, injected with vehicle (20% DMSO; n ? 8 mice) or J-113397 (2
mg/kg, i.p.; n ? 6 mice). Fifteen minutes later, mice were injected with
buprenorphine (0.3, 1, or 3 mg/kg, s.c.) and tested 15 min later. As
a 30 min delay, received J-113397 (2 mg/kg, i.p.). Fifteen minutes later,
mice were injected with saline and tested after an additional 15 min.
Data analysis. For dose–response studies, a two-way repeated-
measure ANOVA was used. For single-dose studies, data were expressed
as percentage maximum possible effect (%MPE) according to the for-
mula [%MPE ? ((Test latency ? Baseline)/(Cutoff ? Baseline)) * 100]
and analyzed using a two-factor ANOVA. The Newman–Keuls post hoc
test was then used to reveal significant differences between different
genotypes and doses of the drug. A value of p ? 0.05 was considered
In CHO cells expressing the ?-opioid receptors (CHO-MOR
cells), buprenorphine and etorphine activated both MAP kinase
and Akt at low nanomolar concentrations (Fig. 1). MAP kinase
activation was semiquantified as described in Materials and
Methods. The calculated Emax, for activation of MAP kinase, was
only 42.97 ? 4.65% of etorphine (1 ?M). This datum suggests
that buprenorphine acts as a partial agonist for MAP kinase sig-
naling at ?-opioid receptors compared with etorphine. Further-
more, buprenorphine was less potent than etorphine (Fig. 1B).
In CHO cells expressing ORL-1 receptors (CHO-ORL-1), bu-
Akt. However, buprenorphine was found to be a low-potency
10332 • J.Neurosci.,November12,2003 • 23(32):10331–10337Lutfyetal.•ORL-1ReceptorsCompromiseAntinociceptionbyBuprenorphine
agonist compared with OFQ/N (Fig. 2A,B). Morphine, on the
other hand, was unable to activate MAP kinase or Akt in CHO-
ORL-1 cells, even at concentrations up to 100 ?M (Fig. 2B). Bu-
prenorphine and OFQ/N each failed to activate MAP kinase in
CHO-ORL-1 cells in the presence of 10 ?M J-113397 (Fig. 2C).
No activation of kinases by buprenorphine or OFQ/N was ob-
served in nontransfected CHO cells (data not shown).
?-Opioid receptor knock-out mice and wild-type controls (n ?
5–6 mice per genotype) were tested before and 15 min after bu-
prenorphine (0.1–3.0 mg/kg, s.c.) administration using the tail-
flick assay (Fig. 3). A two-way repeated-measure ANOVA re-
effect of dose (F(3,27)? 3.10; p ? 0.05), and an interaction be-
tween genotype and dose (F(3,27)? 7.80, p ? 0.05). Post hoc
analysis of the data revealed a dose-dependent increase in
buprenorphine-induced antinociception in wild-type mice and
no antinociception at any dose in ?-opioid receptor knock-out
for the antinociceptive effect of buprenorphine.
ORL-1 receptor knock-out mice and their wild-type littermates
buprenorphine (0.1–3.0 mg/kg, s.c.) administration (Fig. 4). A
two-way repeated-measure ANOVA revealed a main effect of
46.83; p ? 0.05), and an interaction between genotype and dose
(F(3,39)? 4.77; p ? 0.05). Post hoc analysis of the data revealed a
at 0.3, 1.0, and 3.0 mg/kg but not at 0.1 mg/kg. These data reveal
Unlike buprenorphine, morphine produced a similar dose-
dependent antinociceptive effect in both wild-type and ORL-1
(F(2,36)? 113.91; p ? 0.05) but not genotype (F(1,18)? 1.16; p ?
0.05) or interaction between dose and genotype (F(2,36)? 1.24;
p ? 0.05). Post hoc analysis of the data revealed that morphine
dose dependently produced antinociception, and there was no
significant difference in morphine-induced antinociception
phospho-specific MAP kinase and Akt antisera (for details, see Materials and Methods). An
using phospho-specific antisera (for details, see Materials and Methods). A shows that bu-
prenorphine, similar to OFQ/N, concentration dependently activated MAP kinase and Akt,
and OFQ/N in activating MAP kinase, albeit OFQ/N was substantially more potent than bu-
Lutfyetal.•ORL-1ReceptorsCompromiseAntinociceptionbyBuprenorphine J.Neurosci.,November12,2003 • 23(32):10331–10337 • 10333
The effect of the ORL-1 receptor antagonist J-113397 (3 mg/kg,
i.p.) on buprenorphine-induced antinociception was then as-
sessed in both wild-type and ORL-1 receptor knock-out mice
(Fig. 6). There was a main effect of treatment (F(1,19)? 4.72;
p ? 0.05) and an interaction between treatment and genotype
(F(1,19)? 5.27; p ? 0.05). Post hoc analysis of the data revealed
increased in the presence of J-113397 in wild-type mice ( p ?
0.05) but not ORL-1 receptor knock-out mice ( p ? 0.05). Im-
portantly, our pilot study showed that J-113397 alone had no
significant effect on nociceptive responses in wild-type mice
[3.48 ? 0.12 (baseline) vs 3.14 ? 0.22 (15 min later) vs 3.24 ?
cological data provide additional evidence that ORL-1 receptors
play a modulatory role in buprenorphine-induced antinocicep-
Buprenorphine has often been reported to produce antinocicep-
a bell-shaped dose–response curve for buprenorphine using low
light intensity in the tail-flick assay. Therefore, we examined
tested for baseline tail-flick latency, injected with buprenorphine (0.1–3.0 mg/kg, s.c.), and
The antinociceptive effect of buprenorphine is significantly enhanced in ORL-1
tail-flick latency, injected with morphine (2.5–10.0 mg/kg, s.c.), and tested for postdrug la-
The antinociceptive effect of buprenorphine was enhanced by J-113397. Mice
10334 • J.Neurosci.,November12,2003 • 23(32):10331–10337Lutfyetal.•ORL-1ReceptorsCompromiseAntinociceptionbyBuprenorphine
whether buprenorphine would produce a bell-shaped dose–
response curve using higher light intensities, and if so, whether
this bell-shaped dose–response curve would be attributed to
ANOVA revealed a main effect of treatment [J-113397 (2 mg/kg,
i.p) vs vehicle] (F(1,12)? 24.04; p ? 0.05) and an interaction
between treatment and buprenorphine doses (F(2,24)? 3.21; p ?
0.05). Post hoc analysis of the data showed that buprenorphine
alone produced a bell-shaped dose–response curve, i.e., a signif-
icantly greater antinociception was observed at 1.0 mg/kg com-
pared with 0.3 mg/kg and significantly lower antinociception at
in the presence of J-113397, the bell-shaped dose–responsecurve
dose–response curve for buprenorphine-induced antinociception
can be observed with the increased light intensity in the tail-flick
assay. Furthermore, this bell-shaped dose–response curve was
eliminated after blockade of ORL-1 receptors. As shown above,
J-113397 (2 mg/kg, i.p.), injected 15 min before saline, had no
significant effect on nociceptive responses ( p ? 0.05).
Opioid analgesics are routinely used for treatment of moderate-
to-severe pain. However, side effects associated with the acute
and chronic use of opioids, such as respiratory depression, con-
of prescribing these drugs. Among the many opioid drugs avail-
able, buprenorphine has some unique and attractive properties
for clinical applications. Buprenorphine has only mild effects on
the digestive system, does not itself cause lethality via respiratory
depression, and is a long-lasting drug that reduces craving and
elicits few withdrawal symptoms (for review, see Tzschentke,
drug for maintenance therapy of opiate addicts (Lewis and
Allen, 1999; Johnson and McCagh, 2000), yet the drug has a
complexin vivo pharmacology with aspects not well understood.
In common with most clinically used opioid analgesics, bu-
prenorphine does not have absolute selectivity for one type of
agonist at ?-opioid receptors (Cowan et al., 1977a,b; Dum et al.,
1981; Lizasoain et al., 1991). It also acts as an agonist (Tyers,
?-opioid receptors (Sadee et al., 1982). Agonist-induced phos-
?-opioid receptors (CHO-MOR) supports the notion that bu-
prenorphine, compared with etorphine, is a partial agonist at
?-opioid receptors (Fig. 1). Although interacting with multiple
mice lacking the ?-opioid receptor (Fig. 3), suggesting that the
?-opioid receptors. This finding, however, does not exclude the
potential for other opioid receptors to contribute to the antino-
ciceptive effect of buprenorphine. For example, the signaling ac-
tions at multiple opioid receptors may be synergistic. Alterna-
tively, formation of specific opioid receptor complexes, e.g., via
heterodimerization (Jordan et al., 2001), may be essential for
antinociception induced by some opiate drugs.
In both animals and humans, a hallmark of the antinocicep-
tive action of buprenorphine is the production of a ceiling effect
Rothman et al., 1995). In the present study, using different light
or a bell-shaped dose–response relationship for buprenorphine-
induced antinociception. One very plausible explanation for the
observed ceiling effect is that buprenorphine behaves as a partial
agonist at the ?-opioid receptor, a property clearly evident from
the in vitro signaling data (Fig. 1). However, a bell-shaped dose–
response curve does not fit well with a hypothesis of partial ago-
nist activity. An alternative explanation is that buprenorphine
coactivates receptors other than classical opioid receptors and
this compromises its antinociceptive efficacy.
classical opioid receptors, is the ORL-1 receptor, which shares
tors (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994;
Mollereau et al., 1994). Activation of the ORL-1 receptor was
thought to induce hyperalgesia, an effect that led Meunier et al.
(1995) to name the endogenous ligand of the ORL-1 receptor,
peralgesia (Reinscheid et al., 1995) to blockade of stress-induced
antinociception and proposed OFQ/N to be an anti-opioid pep-
tide with regard to modulation of nociception (for review, see
Mogil and Pasternak, 2001). Thus, activation of supraspinal
ORL-1 receptors by intracerebroventricular OFQ/N administra-
tion can effectively block the antinociceptive actions of ?-, ?-,
and ?-opioid receptor agonists (Mogil et al., 1996). In contrast,
activation of spinal ORL-1 receptors after intrathecal OFQ/N
review, see Mogil and Pasternak, 2001).
Buprenorphine has been shown previously to act at ORL-1
receptors as an agonist in several signaling pathways (Wnendt et
al., 1999; Bloms-Funke et al., 2000; Hawkinson et al., 2000;
Huang et al., 2001). Here, we demonstrate that buprenorphine
can activate both MAP kinase and Akt via activation of ORL-1
receptors expressed in CHO cells. However, the efficacy of
is eliminated by J-113397. Mice were tested for baseline tail-flick latency using a high light
Lutfyetal.•ORL-1ReceptorsCompromiseAntinociceptionbyBuprenorphineJ.Neurosci.,November12,2003 • 23(32):10331–10337 • 10335
ORL-1 receptors compared with ?-opioid receptors. Although
activation was observed in both CHO-ORL-1 and CHO-MOR
cell lines at lower buprenorphine concentrations (Figs. 1B, 2B).
Thus, we initially tested the hypothesis that activation of ORL-1
receptors may modulate buprenorphine-induced antinocicep-
tion (Wnendt et al., 1999).
ulation of nociception via either ? or ORL-1 receptors based on
its in vitro pharmacological characteristics is not straightforward
cuitry. We initially addressed the possible importance of ORL-1
receptors in modulating buprenorphine-induced antinocicep-
tion using mice lacking ORL-1 receptors. The ORL-1 receptor
knock-out mice showed dramatically increased buprenorphine-
induced antinociception compared with wild types, suggesting
that ORL-1 receptors were coactivated with ?-opioid receptors
by buprenorphine, and this activity could compromise the an-
tinociceptive efficacy of buprenorphine. An alternative explana-
enhanced in ORL-1 knock-out mice. However, this appears un-
likely because the antinociceptive effect of morphine, demon-
strated previously to be ?-opioid receptor mediated (for review,
see Kieffer, 1999), is indistinguishable at multiple doses between
wild-type and ORL-1 receptor knock-out mice.
knock-out mice, we also assessed the role of ORL-1 receptors in
buprenorphine-induced antinociception using J-113397, an
ORL-1 antagonist (Kawamoto et al., 1999). The antinociceptive
effect of buprenorphine was significantly enhanced by coadmin-
istration of J-113397 in wild-type, but not ORL-1 receptor
knock-out, mice. Moreover, wild-type mice treated with
J-113397 showed indistinguishable buprenorphine-induced an-
tinociception when compared with mice lacking ORL-1 recep-
tors (Fig. 6). Importantly, J-113397 also eliminated the bell-
shaped dose–response curve exhibited by buprenorphine.
and low antinociceptive efficacy of buprenorphine to concomi-
tant activation of ORL-1 receptors. Furthermore, our results, in
conjunction with previous findings, suggest that activation of
receptor-mediated actions of buprenorphine but also overrides
the possible spinal ORL-1 receptor-mediated antinociceptive ef-
fect of buprenorphine.
Many previous studies have reported a bell-shaped antinoci-
ceptive dose–response curve for buprenorphine. Using a low
light intensity in the tail-flick assay, only a ceiling effect was ob-
resulted in a bell-shaped dose–response curve after buprenor-
dose–response curve of buprenorphine was eliminated by the
ORL-1 and ? receptors to buprenorphine-induced antinocicep-
tion may be altered by the intensity of the noxious stimulus.
Indeed, previous studies have shown that there are different pat-
terns of CNS activation in response to different intensities of
noxious stimulation (Derbyshire et al., 1997). Thus, at higher
noxious stimuli, activation of ORL-1 receptors may, to a greater
extent, compromise the ? receptor-mediated antinociceptive ef-
fect of buprenorphine.
The results of this study indicate that ORL-1 receptors can
contribute to the actions of buprenorphine in vivo. Given the
extensive and prominent distribution of ORL-1 receptors in the
CNS and cells of the human immune system, this would imply
that the ability of buprenorphine to activate ORL-1 receptors
It is not clear, however, whether ORL-1 receptors contribute to
tor activation has been shown to decrease respiratory frequency
(Takita et al., 2003).
Our results present a potential clinical strategy for increasing
the analgesic efficacy of buprenorphine by coadministration of
an ORL-1 receptor antagonist. However, ORL-1 receptor inacti-
vation may have widespread adverse consequences and perhaps
disrupt beneficial properties of buprenorphine, e.g., its actions
on mood. A more satisfactory approach for more efficacious an-
develop analogs with no activity at ORL-1 receptors. Finally, our
observation that buprenorphine-induced antinociception is
attractive hypothesis to explain the low efficacy and the bell-
shaped dose–response curve for this widely used opiate drug.
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