Full text of DOI:10.1038/sj.bjp.0704279

British Journal of Pharmacology (2001) 134, 529 ± 534
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
Orphanin FQ/nociceptin attenuates the development of morphine
tolerance in rats
,
1
1
Kabirullah Lutfy, Syed M. Hossain, Imran Khaliq & Nigel T. Maidment
1
1
*
1
Department of Psychiatry and Biobehavioral Sciences, Neuropsychiatric Institute, University of California, Los Angeles,
60 Westwood Plaza, Los Angeles, California, CA 90024, U.S.A.
7
1
Recent evidence from studies in mice lacking the opioid receptor-like (ORL-1) receptor and from
experiments using antibodies raised against orphanin FQ/nociceptin (OFQ/N) suggest that this
peptide may be involved in morphine tolerance. In the present study we sought to investigate if
administration of exogenous OFQ/N would modulate the development of tolerance to the
antinociceptive e€ect of morphine.
71
2
7
Rats were treated for 3 days with either saline or morphine (10 mg kg , s.c.) followed, 15 and
5 min later, by two intracerebroventricular injections of either arti®cial cerebrospinal ¯uid (aCSF)
or OFQ/N. The dose of OFQ/N was doubled each day (7.5, 15, 30 nmol). On day 4, rats were tested
on a hot plate apparatus before and 30, 60 and 90 min after morphine administration.
3
Repeated OFQ/N treatment did not a€ect basal nociceptive responses or morphine-induced
antinociception. However, the same treatment signi®cantly attenuated the development of morphine
tolerance.
4
Since learning and memory could contribute to the development of morphine tolerance, in
subsequent studies, we examined the e€ect of OFQ/N administered in the CA3 region of the
hippocampus, where OFQ/N has been shown to block LTP and impair spatial memory. A greater
attenuation of morphine tolerance with no alteration of baseline hot plate latency or morphine-
induced antinociception was observed when OFQ/N was administered in this area of the rat brain.
5 Taken together, our results demonstrate that OFQ/N may act in the hippocampus to attenuate
morphine tolerance.
British Journal of Pharmacology (2001) 134, 529 ± 534
Keywords: Morphine; orphanin FQ/nociceptin; antinociception; tolerance; CA3 region; hippocampus; rat hot plate test
Abbreviations: aCSF, arti®cial cerebrospinal ¯uid; cyclic AMP, adenosine-3',5'-monophosphate; i.c.v., intracerebroventricu-
lar(ly); LTP, long-term potentiation; OFQ/N, orphanin FQ/nociceptin; ORL-1, opioid receptor-like
Introduction
Opioid analgesics, such as morphine, are widely used for
treatment of moderate to severe pain. In addition to side
e€ects such as respiratory depression and constipation, the
clinical usefulness of these drugs is often hampered by the
development of tolerance with chronic administration (for
review, see Osenbach & Harvey, 2001). The mechanism of
morphine tolerance, de®ned as a decrease in the potency of
the drug with repeated use, is not fully understood. At the
receptor level, possible mechanisms of tolerance may involve
opioid receptor desensitization (Law et al., 1982) and/or
down-regulation (Law et al., 1982; Puttfarcken et al., 1988).
In addition to receptor regulations, a change in the level of
second messenger systems has been reported in opioid-
tolerant subjects. Chronic opioid treatment, for instance,
leads to an increase in intracellular calcium levels (Welch &
Bass, 1995; Smith et al., 1999). Moreover, elevated activity of
certain enzymes, such as adenylyl cyclase (Sharma et al.,
(Mao et al., 1995; Mayer et al., 1995; Narita et al., 1995)
have been associated with opioid tolerance. However, such
mechanisms alone are not sucient to explain some forms of
tolerance. For instance, morphine produces tolerance but
does not induce down-regulation (Tao et al., 1987; Yoburn et
al., 1993; Keith et al., 1996) or internalization of opioid
receptors (Arden et al., 1995; Keith et al., 1996).
Among other proposed mechanisms is the increased
activity of so-called anti-opiate peptides in the brain (for
reviews, see Harrison et al., 1998; Goodman et al., 1998).
One recently discovered peptide with counter-opiate action is
orphanin FQ (OFQ; also known as nociceptin), an
endogenous ligand of the opioid receptor-like (ORL-1)
receptor (Meunier et al., 1995; Reinscheid et al., 1995).
Similarities in the structures of the ORL-1 receptor and the
heptadecapeptide OFQ/N with those of opioid receptors and
endogenous opioid peptides, respectively, indicate a close
evolutionary relationship between the two systems (Moller-
eau et al., 1994; Meunier et al., 1995; Reinscheid et al.,
1995). Despite the resemblance, OFQ/N does not bind to the
classical opioid receptors (Mollereau et al., 1994). More
importantly, although OFQ/N produces the same cellular
e€ects as classical opioids, such as inhibition of cyclic AMP
(Meunier et al., 1995; Reinscheid et al., 1995), inhibition of
1
975; Klee et al., 1984), nitric oxide synthase (Kolesnikov et
al., 1993), adenosine-3',5'-monophosphate (cyclic AMP)-
dependent protein kinase (Wang et al., 1996; Bernstein &
Welch, 1998; Wang & Sadee, 2000) and protein kinase C
*
Author for correspondence; E-mail: klutfy@ucla.edu

530
K. Lutfy et al
Orphanin FQ/nociceptin and morphine tolerance
calcium channel conductance (Kno¯ach et al., 1996; Connor
et al., 1996b), and an increase in potassium e‚ux (Matthes
et al., 1996; Vaughan & Christie, 1996; Connor et al.,
Drug treatment schedule
Rats were allowed 4 days to recover from the surgical
procedure and then treated once daily for 3 days. On day
1, rats received a subcutaneous (s.c.) injection of either
1
996a), intracerebroventricular injection of OFQ/N produces
hyperalgesia (Meunier et al., 1995; Reinscheid et al., 1995;
Calo et al., 1998; Lutfy & Maidment, 2000) and attenuates
the antinociceptive e€ects of opioid analgesics (Mogil et al.,
71
saline (SAL) or morphine (MOR; 10 mg kg ). After
15 min, rats were given an injection of either aCSF or
OFQ/N either in the right lateral ventricle (7.5 nmol in
5 ml) or directly into the CA3 region of the hippocampus
(7.5 nmol in 0.5 ml per side). Based on previous experience
with this peptide, which indicated a short duration of
action in the brain, we administered an additional dose of
aCSF or OFQ/N 1 h later. On days 2 and 3, the same
respective treatments were given, except that the dose of
OFQ/N was doubled each day (15 nmol on day 2 and
30 nmol on day 3) because tolerance develops to the anti-
opioid action of exogenous OFQ/N (Lutfy et al., 1999). On
day 4, rats were tested for baseline nociceptive responses in
the hot plate test (for details, see below). Approximately
30 min later, all groups received an injection of morphine
1
1
996; Tian et al., 1997; Lutfy et al., 1999; Wang et al.,
999).
Regarding the role of OFQ/N in opioid tolerance, a
recent study showed increased tissue and ventricular
cerebrospinal ¯uid levels of OFQ/N in the brains of
morphine tolerant rats (Yuan et al., 1999). On this basis
it has been proposed that continual administration of
morphine accelerates the biosynthesis and/or release of
OFQ/N to antagonize the e€ect of morphine, thereby
contributing to the phenomenon of tolerance. In support of
this idea, intracerebroventricular (i.c.v.) injection of an
antibody raised against OFQ/N partially reversed the
expression of morphine tolerance (Tian et al., 1998).
Moreover, morphine tolerance is partially inhibited in mice
lacking the nociceptin receptor gene (Ueda et al., 1997),
raising the possibility that the ORL-1 receptor may play a
functional role in the development of morphine tolerance.
The present study was designed to evaluate the e€ect of
exogenously administered OFQ/N on the development of
morphine tolerance. The ®rst experiment employed an i.c.v.
route of administration and provided evidence for an OFQ/
N-induced attenuation of the development of morphine
tolerance. Given that morphine tolerance and learning and
memory may share common mechanisms (Siegel, 1976), and
since OFQ/N attenuates long-term potentiation (LTP) in
the hippocampus (Yu et al., 1997) and also impairs spatial
memory in a water-maze test when injected directly into the
CA3 region of the rat hippocampus (Sandin et al., 1997),
the second experiment employed local injection of OFQ/N
in this brain region and similarly showed blockade of
morphine tolerance.
71
(10 mg kg , s.c.) and were tested for post-drug hot plate
latency at 30-min intervals for 90 min.
Nociceptive assay
A
modi®cation of the hot plate assay of Woolfe &
MacDonald (1944) was used. Rats were placed on a warm
plate (528C) surrounded by a Plexiglas cylinder and the
amount of time taken for the rats to lick/¯utter one of the
hind-paws or jump from the plate was measured. A cut-o€
time of 60 s was employed in order to prevent tissue damage.
Verification of guide cannula placement
At the end of each experiment, rats were anaesthetized with
71
pentobarbital (50 mg kg , i.p.) and perfused transcardially
with phosphate bu€ered saline (PBS) containing NaCl
(130 mM); NaH2PO4 (3 mM) and Na2HPO4 (7 mM) fol-
lowed by 50 ml of phosphate bu€ered formalin. Brains were
removed and sectioned (40 mm) using a cryostat (Leica
Instruments Gmbh, Germany). The slices were stained using
cresyl violet and viewed under the microscope. Based on such
examination, ®ve rats were discarded from the data analysis
due to improper placement of the guide cannula.
Methods
Subjects
Male Sprague Dawley rats (180 ± 200 g) obtained from
Harlan (San Diego, CA, U.S.A.) were maintained two per
cage, with free access to food and water. All experiments
were conducted during the light phase of a 12-h/12-h light/
dark cycle.
Data analysis
Hot plate latencies were analysed using a two-way analysis of
variance (ANOVA) with repeated measures on the hot plate
latency at di€erent time points. The Newman-Keuls post-hoc
test was used to examine the signi®cance of di€erences among
Surgery
various groups.
statistically signi®cant.
A value of P50.05 was considered
Rats were anaesthetized with halothane in a 1 : 1 mixture of
oxygen and nitrous oxide, and implanted with a 22-G guide
cannula (3.5-mm long) 0.5 mm above the right lateral
ventricle or the CA3 region of the hippocampus. The
coordinates for intracerebroventricular and intra-hippocam-
pal injection sites were, according to the atlas of Paxinos and
Watson (1986): AP=70.8 mm, ML=+1.4 mm; DV=
Drugs
Morphine sulphate was obtained from Mallinckrodt, Inc.
(St. Louis, MO, U.S.A.) and dissolved in normal saline prior
to s.c. injection. OFQ/N was purchased from Phoenix
Pharmaceuticals, Inc. (Mountain View, CA, U.S.A.) and
dissolved in aCSF prior to i.c.v. application. The composi-
tion of aCSF in mM was: NaCl 125, KCl 2.5, NaH2PO4
74.0 mm; AP=73.3 mm; ML=+2.4 mm, DV=74.0,
respectively. A 30-G needle was used for arti®cial cerebro-
spinal ¯uid (aCSF) or OFQ/N injection.
British Journal of Pharmacology vol 134 (3)

K. Lutfy et al
Orphanin FQ/nociceptin and morphine tolerance
531
0.9, Na2HPO4 5, MgCl2 1, D-glucose 2.5, CaCl2 1.2,
bovine serum albumin 0.025%.
Results
Effects of i.c.v. OFQ/N treatment on the development of
morphine tolerance
Morphine administration on day 4 produced a signi®cant
(F3,105=87.87; P50.05) antinociceptive e€ect (Figure 1), the
magnitude of this response was dependent on prior treatment
on days 1 ± 3 (F3,35=15.94; P50.05). The post-hoc Newman-
Keuls test revealed that treatment with morphine
Figure 2 E€ects of intra-hippocampal OFQ/N treatment on
morphine tolerance in rats using the hot plate test. Rats received
the same treatment and were tested as described in the legend to
Figure 1 except aCSF or OFQ/N was administered into the CA3
region of the hippocampus. Morphine treatment for 3 days induced
tolerance (P50.05, SAL ± aCSF vs MOR ± aCSF) and this phenom-
enon was blocked by administration of OFQ/N (P50.05, MOR ±
OFQ vs MOR ± aCSF) into the CA3 region of the rat hippocampus
71
(
10 mg kg , s.c.) for 3 days induced tolerance to its
antinociceptive e€ect when rats were tested on day 4
P50.05; SAL ± aCSF vs MOR ± aCSF). This tolerance to
(
morphine was signi®cantly attenuated in rats treated for 3
days with morphine followed by OFQ/N (P50.05; MOR ±
OFQ vs MOR ± aCSF). There was no signi®cant di€erence in
baseline values among the four groups (P40.05), indicating
that chronic treatment with morphine or OFQ/N, either
alone or in combination, did not a€ect the basal nociceptive
response in the hot plate test. Moreover, chronic treatment
with OFQ/N alone did not change the antinociceptive e€ect
of morphine in the hot plate test (P40.05; SAL ± aCSF vs
SAL ± OFQ).
(
post hoc Newman-Keuls test). Data were analysed by a two-way
ANOVA with repeated measures on hot plate latencies before and
after morphine administration on day 4.
on prior treatment on days 1 ± 3 (F3,19=16.91; P50.05). Post-
hoc analysis of the data showed that morphine treatment for 3
days induced tolerance to the antinociceptive e€ect of
the drug on day 4 (P50.05; SAL ± aCSF vs MOR ± aCSF)
and the extent of such tolerance was attenuated by concomitant
intra-hippocampal treatment with OFQ/N (P50.05; MOR ±
OFQ vs MOR ± aCSF). Indeed, tolerance was e€ectively
blocked by intra-hippocampal OFQ/N treatment since there
was no signi®cant di€erence between rats treated with morphine
plus OFQ/N and saline-treated control groups (P40.05;
MOR ± OFQ vs SAL ± OFQ or vs SAL ± aCSF).
Effects of intra-hippocampal OFQ/N treatment on the
development of morphine tolerance
A similar pattern of e€ects was observed with intra-hippocam-
pal OFQ/N administration (Figure 2). Again, morphine
produced
(
a signi®cant antinociceptive e€ect on day 4
3,57=79.55; P50.05), the magnitude of which was dependent
F
Discussion
The major ®nding of the present study is that chronic
intracerebroventricular or intra-hippocampal treatment with
OFQ/N attenuated the development of morphine tolerance
without a€ecting the basal nociceptive response or acute
antinociceptive e€ect of the opioid analgesic in the rat hot
plate test. Taken together, our results indicate that repeated
OFQ/N administration selectively interferes with the pro-
cesses involved in the development of morphine tolerance.
Numerous studies have reliably shown that tolerance
develops to the antinociceptive e€ect of morphine after
chronic use of the drug (see, Cox, 1990). The mechanism of
this phenomenon is yet to be fully characterized, but one
proposal, among many, is an increase in the activity of anti-
opioid peptides in the brain (for reviews, see Goodman et al.,
Figure 1 E€ects of intracerebroventricular (i.c.v.) OFQ/N adminis-
tration on morphine tolerance in the rat hot plate test. Rats were
treated for 3 days with saline or morphine (10 mg kg7 , s.c.)
followed, 15 and 75 min later, by two i.c.v. injections of aCSF or
OFQ/N. On day 4, rats were tested 30 min before and 30, 60 and
1
1
998; Harrison et al., 1998). For instance, chronic morphine
treatment has been associated with an increase in immuno-
reactivity for cholecystokinin (Lucas et al., 1999; Becker et
al., 2000) and neuropeptide FF (Malin et al., 1990), both of
which have been shown to oppose actions of opioid
analgesics in the central nervous system. The demonstration
that OFQ/N blocks opioid receptor-mediated stress-induced
antinociception (Mogil et al., 1996) and also decreases the
antinociceptive e€ect of morphine and other opioid receptor
71
9
0 min after morphine (10 mg kg , s.c.) in the hot plate test.
Treatment with morphine for 3 days produced a signi®cant decrease
in the antinociceptive e€ect of the drug (P50.05, SAL ± aCSF vs
MOR ± aCSF) and this tolerance was attenuated by i.c.v. OFQ/N
(
P50.05, MOR ± OFQ vs MOR ± aCSF) administration (post hoc
Newman-Keuls test). Data were analysed by a two-way ANOVA
with repeated measures on hot plate latencies before and after
morphine administration on day 4.
British Journal of Pharmacology vol 134 (3)

532
K. Lutfy et al
Orphanin FQ/nociceptin and morphine tolerance
agonists (Mogil et al., 1996; Morgan et al., 1997; Tian et al.,
997; Lutfy et al., 1999; Wang et al., 1999) has led to the
suggestion that OFQ/N may be acting as an anti-opioid
peptide in the brain (Mogil et al., 1996).
with a subsequent ®rst exposure to morphine. The lack of such
an action renders an e€ect on biosynthesis/release more likely
wherein repeated OFQ/N has little e€ect alone (and therefore
the endogenous OFQ/N response to a subsequent initial
morphine challenge is una€ected) but prevents the upregula-
tion of OFQ/N biosynthesis in response to repeated morphine.
A more simplistic explanation for our ®ndings is that each
OFQ/N administration attenuates the antinociceptive e€ect
of morphine (Mogil et al., 1996; Morgan et al., 1997; Tian et
al., 1997; Lutfy et al., 1999; Wang et al., 1999), thereby
preventing induction of events leading to tolerance, events
that may not involve the endogenous OFQ/N system itself.
However, this is unlikely since there is sucient evidence to
dissociate analgesia itself from analgesic tolerance, i.e.,
morphine tolerance has been reported in the presence of
opioid receptor blockade by naltrexone (Yoburn et al., 1990).
It is plausible, however, that exogenous OFQ/N interferes
with mechanisms of tolerance independent of its anti-opiate
action and independent of an action on endogenous OFQ/N
biosynthesis. For instance, learning and memory may
contribute signi®cantly to the development of morphine
tolerance (Siegel, 1976). Previous studies have shown that
analgesic tolerance readily develops after chronic administra-
tion of morphine when rats are tested in the same
environment in which they received their daily morphine
treatment, whereas tolerance is less evident when rats are
tested in a novel environment (Siegel, 1976). This phenom-
enon is referred to as associative tolerance and a Pavlovian
conditioning model has been suggested to account for it
(Siegel, 1976). Long-term potentiation (LTP) in the hippo-
campus is implicated as a basis of memory formation, and
thus may be important for associating environmental cues
with morphine injection. Mediators of LTP include the N-
methyl-D-aspartate (NMDA) receptor and calcium/calmodu-
lin-dependent protein kinaseII (CaMKII), both of which are
present in high concentrations in the hippocampus (Fan et
al., 1999). Evidence for their necessity in the development of
tolerance is drawn from observations that NMDA receptor
antagonists (Marek et al., 1991; Trujillo & Akil, 1991; Lutfy
et al., 1993) and CaMKII inhibitors (Fan et al., 1999)
attenuate morphine tolerance. Interestingly, OFQ/N has been
shown to block LTP in the hippocampus (Yu et al., 1997)
and intra-hippocampal injection of OFQ/N impairs spatial
learning in rats (Sandin et al., 1997). Moreover, OFQ/N has
been shown to block the e€ects of morphine in the classical
conditioned place preference paradigm (Murphy et al., 1999;
Ciccocioppo et al., 2000) without producing any rewarding or
aversive e€ect of its own (Devine et al., 1996). We observed
an almost complete blockade of morphine tolerance after
repeated OFQ/N administration into the CA3 region of the
hippocampus using the same dose that only partially
attenuated tolerance after i.c.v. administration. Our data is
therefore consistent with the notion that OFQ/N may be
acting in the hippocampus to attenuate morphine tolerance,
perhaps blocking the associative component of this phenom-
enon. The lack of complete blockade of morphine tolerance
by OFQ/N in these studies presumably re¯ects the presence
of additional tolerance mechanisms (perhaps the non-
associative component) that remain una€ected by OFQ/N
treatment. Alternatively, as previously demonstrated (Lutfy et
al., 1999), tolerance develops to the action of OFQ/N and
therefore OFQ fails to completely block morphine tolerance.
1
The involvement of OFQ/N in morphine tolerance was
recently supported by the observation that the tissue
concentration of endogenous OFQ/N increased in the rat
periaqueductal gray and amygdala in a time-dependent
manner during the development of morphine tolerance (Yuan
et al., 1999). The OFQ/N concentration was also elevated in
the ventricular cerebrospinal ¯uid of these animals (Yuan et
al., 1999). Such a combination of results indicates a gradual
increase in OFQ/N biosynthesis and/or release during the
development of morphine tolerance. The result of a previous
study (Tian et al., 1998) showing that the expression of
morphine tolerance was attenuated by acute administration
of an antibody raised against OFQ/N is consistent with a
causal relationship between elevated OFQ/N release and
morphine tolerance. Such a conclusion is further strengthened
by the observation that ORL-1 knockout mice display only
partial tolerance to morphine compared to wild-type mice
(Ueda et al., 1997).
Our data show that exogenous OFQ/N, administered
concomitantly with repeated morphine injections, attenuated
the development of morphine tolerance. On the surface, this
may seem to con¯ict with the data of Tian et al. (1998) using
an OFQ/N antibody. However, it should be noted that these
authors observed an attenuation of the expression of
morphine tolerance with the antibody to OFQ/N. The
antibody was administered once only, on the day of
morphine challenge, whereas we administered OFQ/N
repeatedly during the development phase, but not on the
test day. Our data are, in fact, consistent with the idea that
repeated morphine administration repeatedly activates the
endogenous OFQ/N system as a homeostatic mechanism, and
that a resultant sensitized response of this system upon
morphine challenge is responsible for morphine antinocicep-
tive tolerance. Thus, repeated OFQ/N administration during
the development phase may reduce the activation of
endogenous OFQ/N biosynthesis/release, essentially blocking
the progressive sensitization of the OFQ/N system observed
in the previous investigation (Yuan et al., 1999). This would
result in an attenuated endogenous OFQ/N response to a
subsequent morphine challenge. Alternatively, high concen-
trations of exogenously applied OFQ/N may cause long-term
down-regulation/desensitization of the ORL-1 receptor. Both
would potentially be manifested as attenuated antinociceptive
tolerance to morphine.
Interestingly, repeated OFQ/N administration in conjunc-
tion with saline, as opposed to morphine, did not a€ect the
antinociceptive response to a subsequent morphine challenge.
Neither did it a€ect baseline hot plate latencies. Thus, repeated
OFQ/N selectively interfered with the processes involved in the
development of morphine tolerance. Given the known action
of acute OFQ/N administration to attenuate morphine-
induced antinociception (Mogil et al., 1996; Morgan et al.,
1
997; Tian et al., 1997; Lutfy et al., 1999; Wang et al., 1999),
and the recently reported potentiation of morphine analgesia
by an ORL-1 receptor antagonist (Rizzi et al., 2000), one would
predict that if repeated OFQ/N administration causes
desensitization/downregulation of the ORL-1 receptor, such
treatment should potentiate the antinociception associated
British Journal of Pharmacology vol 134 (3)

K. Lutfy et al
Orphanin FQ/nociceptin and morphine tolerance
533
We are grateful to Dr Shridhar Narayanan for helpful discussions
and to Daisuke Arai and Gulpana Mohammad for technical
assistance. K. Lutfy was supported by NIDA grant DA00411 and
a NARSAD Young Investigator Award. Special thanks to Mr and
Mrs Staglin for their support of the NARSAD grant.
In conclusion, our results demonstrate that chronic
administration of OFQ/N attenuated the development of
morphine tolerance in rats after i.c.v. or intra-hippocampal
administration without having any signi®cant action on basal
nociceptive responses or on acute morphine-induced anti-
nociception.
References
ARDEN, J.R., SEGREDO, V., WANG, Z., LAMEH, J. & SADEE, W.
(
LAW, P.Y., HOM, D.S. & LOH, H.H. (1982). Loss of opiate receptor
activity in neuroblastoma X glioma NG108-15 hybrid cells after
chronic opiate treatment. A multiple-step process. Mol. Pharma-
col., 22, 1 ± 4.
1995). Phosphorylation and agonist-speci®c intracellular traf-
cking of an epitope-tagged mu-opioid receptor expressed in
HEK 293 cells. J. Neurochem., 65, 1636 ± 1645.
®
BECKER, C.M., POHL, M., THIEBOT, M.H., COLLIN, E., HAMON, M.,
CESSELIN, F. & BENOLIEL, J.J. (2000). Delta-opioid receptor-
mediated increase in cortical extracellular levels of cholecystoki-
nin-like material by subchronic morphine in rats. Neuropharma-
cology, 39, 161 ± 171.
LUCAS, G.A., HOFFMANN, O., ALSTER, P. & WIESENFELD-HALLIN,
Z. (1999). Extracellular cholecystokinin levels in the rat spinal
cord following chronic morphine exposure: an in vivo micro-
dialysis study. Brain Res., 821, 79 ± 86.
LUTFY, K., HURLBUT, D.E.
& WEBER, E. (1993). Blockade of
BERNSTEIN, M.A. & WELCH, S.P. (1998). mu-Opioid receptor down-
regulation and cAMP-dependent protein kinase phosphorylation
in a mouse model of chronic morphine tolerance. Mol. Brain
Res., 55, 237 ± 242.
CALO, G., RIZZI, A., MARZOLA, G., GUERRINI, R., SALVADORI, S.,
BEANI, L., REGOLI, D. & BIANCHI, C. (1998). Pharmacological
characterization of the nociceptin receptor mediating hyperalge-
sia in the mouse tail withdrawal assay. Br. J. Pharmacol., 125,
morphine-induced analgesia and tolerance in mice by MK-801.
Brain Res., 616, 83 ± 88.
LUTFY, K. & MAIDMENT, N.T. (2000). Blockade of mu-opioid
receptors reveals the hyperalgesic e€ect of orphanin FQ/
nociceptin in the rat hot plate test. Br. J. Pharmacol., 131,
1684 ± 1688.
LUTFY, K., SHARZA, S.A. & MAIDMENT, N.T. (1999). Tolerance
develops to the inhibitory e€ect of orphanin FQ on morphine-
induced antinociception in the rat. NeuroReport, 10, 103 ± 106.
LUTFY, K. & MAIDMENT, N.T. (2000). Blockade of mu-opioid
receptors reveals the hyperalgesic e€ect of orphanin FQ/
nociceptin in the rat hot plate test. Br. J. Pharmacol., 131,
1684 ± 1688.
MALIN, D.H., LAKE, J.R., HAMMOND, M.V., FOWLER, D.E.,
ROGILLIO, R.B., BROWN, S.L., SIMS, J.L., LEECRAFT, B.M. &
YANG, H.Y. (1990). FMRF-NH2-like mammalian octapeptide:
possible role in opiate dependence and abstinence. Peptides, 11,
969 ± 972.
3
73 ± 378.
CICCOCIOPPO, R., ANGELETTI, S., SANNA, P.P., WEISS, F. & MASSI,
M. (2000). E€ect of nociceptin/orphanin FQ on the rewarding
properties of morphine. Eur. J. Pharmacol., 404, 153 ± 159.
CONNOR, M., VAUGHAN, C.W., CHIENG, B.
1996b). Nociceptin receptor coupling to a potassium conduc-
tance in rat locus coeruleus neurones in vitro. Br. J. Pharmacol.,
19, 1614 ± 1618.
& CHRISTIE, M.J.
(
1
CONNOR, M., YEO, A. & HENDERSON, G. (1996a). The e€ect of
nociceptin on Ca2+ channel current and intracellular Ca2+ in
the SH-SY5Y human neuroblastoma cell line. Br. J. Pharmacol.,
MAO, J., PRICE, D.D., PHILLIPS, L.L., LU, J. & MAYER, D.J. (1995).
Increases in protein kinase C gamma immunoreactivity in the
spinal cord of rats associated with tolerance to the analgesic
e€ects of morphine. Brain Res., 677, 257 ± 267.
1
18, 205 ± 207.
COX, B. (1990). Drug tolerance and physical dependence. In
Principles of drug action: The basis of pharmacology. ed. Pratt,
W.B. & Taylor, P. pp. 639 ± 690: New York: Churchill Living-
stone, Inc.
DEVINE, D.P., REINSCHEID, R.K., MONSMA JR., F.J., CIVELLI, O. &
AKIL, H. (1996). The novel neuropeptide orphanin FQ fails to
produce conditioned place preference or aversion. Brain Res.,
MAREK, P., BEN-ELIYAHU, S., GOLD, M.
& LIEBESKIND, J.C.
(1991). Excitatory amino acid antagonists (kynurenic acid and
MK-801) attenuate the development of morphine tolerance in the
rat. Brain Res., 547, 77 ± 81.
MATTHES, H., SEWARD, E.P., KIEFFER, B. & NORTH, R.A. (1996).
Functional selectivity of orphanin FQ for its receptor coex-
pressed with potassium channel subunits in Xenopus laevis
oocytes. Mol. Pharmacol., 50, 447 ± 450.
MAYER, D.J., MAO, J. & PRICE, D.D. (1995). The development of
morphine tolerance and dependence is associated with transloca-
tion of protein kinase C. Pain, 61, 365 ± 674.
MEUNIER, J.C., MOLLEREAU, C., TOLL, L., SUAUDEAU, C.,
MIOSAND, C., ALVINERIE, P., BUTOUR, J.L., GUILLEMOT, J.C.,
FERREARA, P., MONSARRAT, B., MAZARGUIL, H., VASSART,
G., PARMENTIER, M. & CONSTENTIN, J. (1995). Isolation and
structure of the endogenous agonist of opioid receptor-like
ORL1 receptor. Nature, 377, 532 ± 535.
MOGIL, J.S., GRISEL, J.E., REINSCHEID, R.K., CIVELLI, O., BEL-
KNAP, J.K. & GRANDY, D.K. (1996). Orphanin FQ is a functional
anti-opioid peptide. Neuroscience, 75, 333 ± 337.
7
27, 225 ± 229.
FAN, G.H., WANG, L.Z., QIU, H.C., MA, L. & PEI, G. (1999). Inhibition
of calcium/calmodulin-dependent protein kinase II in rat
hippocampus attenuates morphine tolerance and dependence.
Mol. Pharmacol., 56, 39 ± 45.
GOODMAN, C.B., HEYLIGER, S., EMILIEN, B., PARTILLA, J.S.,
YANG, H.Y., LEE, C.H., CADET, J.L. & ROTHMAN, R.B. (1998).
Regulation of mu binding sites after chronic administration of
antibodies directed against speci®c anti-opiate peptides. Pep-
tides, 19, 1703 ± 1709.
HARRISON, L.M., KASTIN, A.J.
& ZADINA, J.E. (1998). Opiate
tolerance and dependence: receptors, G-proteins, and antiopi-
ates. Peptides, 19, 1603 ± 1630.
KEITH, D.E., MURRAY, S.R., ZAKI, P.A., CHU, P.C., LISSIN, D.V.,
KANG, L., EVANS, C.J. & VON ZASTROW, M. (1996). Morphine
activates opioid receptors without causing their rapid inter-
nalization. J. Biol. Chem., 271, 19021 ± 19024.
MOLLEREAU, C., PARMENTIER, M., MAILLEUX, P., BUTOUR, J.L.,
MOISAND, C., CHALON, P., CAPUT, D., VASSART, G.
&
KLEE, W.A., MILLIGAN, G., SIMONDS, W.F. & TOCQUE, B. (1984).
The role of adenyl cyclase in opiate tolerance and dependence.
NIDA Res. Monogr., 54, 109 ± 118.
MEUNIER, J.C. (1994). ORL1, a novel member of the opioid
receptor family. Cloning, functional expression and localization.
FEBS Lett., 341, 33 ± 38.
KNOFLACH, F., REINSCHEID, R.K., CIVELLI, O.
(
&
1996). Modulation of voltage-gated calcium channels by
KEMP, J.A.
MORGAN, M.M., GRISEL, J.E., ROBBINS, C.S. & GRANDY, D.K.
(1997). Antinociception mediated by the periaqueductal gray is
attenuated by orphanin FQ. NeuroReport, 8, 3431 ± 3434.
MURPHY, N.P., LEE, Y. & MAIDMENT, N.T. (1999). Orphanin FQ/
nociceptin blocks acquisition of morphine place preference.
Brain Res., 832, 168 ± 170.
orphanin FQ in freshly dissociated hippocampal neurons.
J. Neurosci., 16, 6657 ± 6664.
KOLESNIKOV, Y.A., PICK, C.G., CISZEWSKA, G. & PASTERNAK,
G.W. (1993). Blockade of tolerance to morphine but not to kappa
opioids by a nitric oxide synthase inhibitor. Proc. Natl. Acad. Sci.
U.S.A., 90, 5162 ± 5166.
British Journal of Pharmacology vol 134 (3)

534
K. Lutfy et al
Orphanin FQ/nociceptin and morphine tolerance
NARITA, M., MIZOGUCHI, H. & TSENG, L.F. (1995). Inhibition of
protein kinase C, but not of protein kinase A, blocks the
development of acute antinociceptive tolerance to an intrathe-
cally administered mu-opioid receptor agonist in the mouse. Eur.
J. Pharmacol., 280, R1 ± R3.
TIAN, J.H., ZHANG, W., FANG, Y., XU, W., GRANDY, D.K. & HAN,
J.S. (1998). Endogenous orphanin FQ: evidence for a role in the
modulation of electroacupuncture analgesia and the develop-
ment of tolerance to analgesia produced by morphine and
electroacupuncture. Br. J. Pharmacol., 124, 21 ± 26.
OSENBACH, R.K.
&
HARVEY, S. (2001). Neuraxial infusion in
TRUJILLO, K.A. & AKIL, H. (1991). Inhibition of morphine tolerance
and dependence by the NMDA receptor antagonist MK-801.
Science, 251, 85 ± 87.
patients with chronic intractable cancer and noncancer pain.
Curr. Pain Headache Rep., 5, 241 ± 249.
PAXINOS, G. & WATSON, C. (1986). The rat brain in stereotaxic
coordinates. 2nd ed, New York: Academic Press.
UEDA, H., YAMAGUCHI, T., TOKUYAMA, S., INOUE, M., NISHI, M.
& TAKESHIMA, H. (1997). Partial loss of tolerance liability to
morphine analgesia in mice lacking the nociceptin receptor gene.
Neurosci. Lett., 237, 136 ± 138.
VAUGHAN, C.W. & CHRISTIE, M.J. (1996). Increase by the ORL1
receptor (opioid receptor-like1) ligand, nociceptin, of inwardly
rectifying K conductance in dorsal raphe nucleus neurones. Br. J.
Pharmacol., 117, 1609 ± 1611.
PUTTFARCKEN, P.S., WERLING, L.L. & COX, B.M. (1988). E€ects of
chronic morphine exposure on opioid inhibition of adenylyl
cyclase in 7315c cell membranes: a useful model for the study of
tolerance at mu opioid receptors. Mol. Pharmacol., 33, 520 ± 527.
REINSCHEID, R.K., NOTHACKER, H.P., BOURSON, A., ARDATI, A.,
HENNINGSEN, R.A., BUNZOW, J.R., GRANDY, D.K., LANGEN,
H., MONSMA JR., F.J. & CIVELLI, O. (1995). Orphanin FQ: a
neuropeptide that activates an opioidlike G protein-coupled
receptor. Science, 270, 792 ± 794.
WANG, Y.Q., ZHU, C.B., WU, G.C., CAO, X.D., WANG, Y. & CUI, D.F.
(1999). E€ects of orphanin FQ on endomorphin-1 induced
analgesia. Brain Res., 835, 241 ± 246.
RIZZI, A., BIGONI, R., MARZOL, G., GUERRINI, R., SALVADORI, S.,
REGOLI, D. & CALO, G. (2000). The nociceptin/orphanin FQ
receptor antagonist, [Nphe1]NC(1-13)NH2, potentiates mor-
phine analgesia. NeuroReport, 11, 2369 ± 2372.
WANG, Z., ARDEN, A. & SADEE, W. (1996). Basal phosphorylation of
mu opioid receptor is agonist modulated and Ca2+-dependent.
FEBS. Lett., 387, 53 ± 57.
WANG, Z. & SADEE, W. (2000). Tolerance to morphine at the mu-
opioid receptor di€erentially induced by cAMP-dependent
protein kinase activation and morphine. Eur. J. Pharmacol.,
389, 165 ± 171.
WELCH, S.P. & BASS, P.P. (1995). Modulation of free intracellular
calcium levels [(Ca++)i] in brain and spinal cord of morphine-
tolerant rats and mice. Pharmacol. Biochem. Behav., 51, 57 ± 63.
SANDIN, J., GEORGIEVA, J., SCHOTT, P.A., OGREN, S.O.
&
TERENIUS, L. (1997). Nociceptin/orphanin FQ microinjected
into hippocampus impairs spatial learning in rats. Eur. J.
Neurosci., 9, 194 ± 197.
SHARMA, S.K., KLEE, W.A.
& NIRENBERG, M. (1975). Dual
regulation of adenylate cyclase accounts for narcotic dependence
and tolerance. Proc. Natl. Acad. Sci. U.S.A., 72, 3092 ± 3096.
SIEGEL, S. (1976). Morphine analgesic tolerance: its situation
speci®city supports a Pavlovian conditioning model. Science,
WOOLFE, G.
& MACDONALD, A.D. (1944). The evaluation of
analgesic action of pethidine hydrochloride (demerol). J.
Pharmacol. Exp. Ther., 80, 300 ± 307.
1
93, 323 ± 325.
YOBURN, B.C., BILLINGS, B. & DUTTAROY, A. (1993). Opioid
receptor regulation in mice. J. Pharmacol. Exp. Ther., 265, 314 ±
SMITH, F.L., DOMBROWSKI, D.S. & DEWEY, W.L. (1999). Involve-
ment of intracellular calcium in morphine tolerance in mice.
Pharmacol. Biochem. Behav., 62, 381 ± 388.
TAO, P.L., LAW, P.Y. & LOH, H.H. (1987). Decrease in delta and mu
opioid receptor binding capacity in rat brain after chronic
etorphine treatment. J. Pharmacol. Exp. Ther., 240, 809 ± 816.
TIAN, J.H., XU, W., FANG, Y., MOGIL, J.S., GRISEL, J.E., GRANDY,
320.
YOBURN, B.C., SIERRA, V.
& LUTFY, K. (1990). Simultaneous
development of opioid tolerance and opioid antagonist-induced
receptor upregulation. Brain Res., 529, 143 ± 148.
YU, T.P., FEIN, J., PHAN, T., EVANS, C.J.
& XIE, C.W. (1997).
Orphanin FQ inhibits synaptic transmission and long-term
potentiation in rat hippocampus. Hippocampus, 7, 88 ± 94.
YUAN, L., HAN, Z., CHANG, J.K. & HAN, J.S. (1999). Accelerated
release and production of orphanin FQ in brain of chronic
morphine tolerant rats. Brain Res., 826, 330 ± 334.
D.K.
& HAN, J.S. (1997). Bidirectional modulatory e€ect of
orphanin FQ on morphine-induced analgesia: antagonism in
brain and potentiation in spinal cord of the rat. Br. J.
Pharmacol., 120, 676 ± 680.
(
Received March 13, 2001
Revised July 5, 2001
Accepted July 5, 2001)
British Journal of Pharmacology vol 134 (3)