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Vol. 298, Issue 2, 812-819, August 2001
F. Hoffmann-La Roche AG, Pharma Division, Preclinical Research, Basel, Switzerland
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The novel nonpeptide orphanin FQ/nociceptin (OFQ/N) ligand {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one} (Ro 64-6198) was characterized in vitro and in vivo for its agonistic potential. Ro 64-6198 was 130- to 3500-fold selective for the OFQ/N receptor (ORL1) compared with opiate receptors. In the cAMP inhibition assay, Ro 64-6198 was a full agonist at the ORL1 and a partial agonist at the mu opiate receptor. When human embryonic kidney 293 cells stably expressing the human ORL1 receptor were pre-exposed (30 min) to either OFQ/N or Ro 64-6198, the ability of both agonists to inhibit forskolin-mediated cAMP accumulation was strongly reduced, indicating a functional desensitization of the second messenger cascade. However, acidic washes of OFQ/N-exposed cells fully restored the sensitivity of the ORL1 receptor for agonists. In contrast, the cAMP response in Ro 64-6198-exposed cells remained impaired after acidic washes, suggesting sustained receptor internalization at 30 min. In agreement with this finding, the number of cell-surface ORL1 receptors was significantly reduced after Ro 64-6198 pre-exposure, and this effect could be blocked with high sucrose concentrations. When Ro 64-6198 was chronically administered to rats, no signs of tolerance to its anxiolytic-like effects were detected following 15 days of daily drug exposure. In agreement with the behavioral results, Ro 64-6198 was able to reduce brain ORL1 binding sites in both acutely and chronically treated rats. Full recovery of ORL1 binding sites was observed 24 h after Ro 64-6198 administration with a t1/2 of ~5.5 h. These data show that nonpeptide agonists at the ORL1 receptor have a good clinical potential as anxiolytics without causing tolerance.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
ORL1 receptor (the nomenclature is based on the recommendation of
Dhawan et al., 1996
) was cloned from human, mouse, and rat because of
its high homology (~50%) to the closely related delta
(OP1), kappa (OP2), and mu
(OP3) opiate receptors (reviewed in Meunier et
al., 2000). However, the classical opiate ligands do not bind and
activate ORL1, and until the discovery of its cognate ligand, the
17-amino acid peptide orphanin FQ/nociceptin (OFQ/N) (Meunier et al.,
1995; Reinscheid et al., 1995), the receptor was considered an orphan
opiate-like receptor. ORL1, like OP1, OP2, and OP3, is mainly
coupled to the inhibitory G protein. Thus, activation of ORL1 by OFQ/N
results in the inhibition of cAMP production. Besides its inhibitory G
protein coupling, ORL1 has also been demonstrated to modulate
hippocampal Ca2+ channels (Knoflach et al., 1996)
and to increase a K+ conductance in rat dorsal
raphe, locus coeruleus, and hypothalamic neurons (Connor et al., 1996;
Vaughan and Christie, 1996; Wagner et al., 1998).
Potent nonpeptide ORL1 agonists that mimic the effects of the
endogenous peptide OFQ/N have recently been reported (Wichmann et al.,
2000). The most potent compound, Ro 64-6198 {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one} (Fig. 1A), when given intraperitoneally,
was shown to be active, like the benzodiazepine alprazolam, in several
models of anxiety (Jenck et al., 2000; Wichmann et al., 2000). However,
unlike benzodiazepine anxiolytics, Ro 64-6198 was devoid of antipanic,
anticonvulsant, sedative, and amnestic activity at anxiolytic doses in
the rat.
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A purpose of this study was to determine whether tolerance develops to
the anxiolytic-like effects of Ro 64-6198 following chronic parenteral
treatment for 2 weeks. Tolerance is a common problem seen with several
alkaloid agonists at the OP3 receptor (Keith et
al., 1996; Blake et al., 1997) and with benzodiazepine receptor ligands
(Rundfeldt et al., 1995). For example, morphine and heroin, both
alkaloid OP3 agonists (Blake et al., 1997), cause tolerance, while methadone, a synthetic OP3
agonist, is devoid of such activity (Blake et al., 1997). Recently it
has been shown that in contrast to methadone and
[D-ala2,
N-methyl-phe4,
glyol5][tyrosyl-3,5-3H]-enkephalin
(DAMGO), morphine administration to cells expressing the recombinant
OP3 receptor does not induce its desensitization and internalization (Whistler and von Zastrow, 1998; Zhang et al.,
1998). It is now generally accepted that the lack of acute OP3 desensitization and internalization accounts
for cellular adaptation, which as a long-term consequence results in a
loss of cell-surface OP3 binding sites (Whistler
et al., 1999). Because Ro 64-6198 is a nonpeptide agonist at the ORL1
and because ORL1 is closely related to the opiate receptors, a detailed
pharmacological characterization of Ro 64-6198's action at ORL1 in
vitro and in vivo was initiated.
In this study, the potency of Ro 64-6198 to desensitize the recombinant ORL1 receptor stably expressed in human embryonic kidney 293 (HEK293) cells was investigated and compared with that of the natural peptide ligand OFQ/N. We also provide evidence that chronic treatment with Ro 64-6198 does not produce tolerance in a rat model of anxiety and that in the brains of rats exposed chronically or acutely to Ro 64-6198, the ORL1 receptor undergoes a similar cycle of receptor desensitization and resensitization.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials, Peptides, and Reagents.
All cell culture reagents
were purchased from Life Technologies (Basel, Switzerland). Bovine
serum albumin (BSA, fraction V) was obtained from Sigma (Munich,
Germany). OFQ/N, DAMGO, and naloxone were obtained from Calbiochem (San
Diego, CA). The purity of these peptides and reagents was greater than
95%. Ro 64-6198 (purity >99%) was synthesized in-house (see Jenck et
al., 2000; Wichmann et al., 2000).
Radioligands. The radioligands [leucyl-3H]OFQ/N (specific activity 150 Ci/mmol), [3H]DAMGO (specific activity 78 Ci/mmol), [N-allyl-2--3-3H]naloxone (specific activity 54.5 Ci/mmol), and [Ile5,6-3H]deltorphin (specific activity 72 Ci/mmol) were from Amersham (Little Chalfont, UK).
Transfection of ORL1 and Opiate Receptors.
The cDNAs
encoding human OP3 and ORL1 were inserted into
the pcDNA3.1 vector (Invitrogen, Carlsbad, CA), and the cDNAs coding for OP1 and OP2 were
inserted into the pSFV2 vector. Recombinant Semliki Forest virus stocks
were generated as described previously (Wichmann et al., 2000). Baby
hamster kidney (BHK) cells were infected and harvested 24 h after
infection. ORL1 was stably transfected into HEK293 cells, while
OP3 was stably transfected into Chinese hamster
ovary (CHO) cells using the Transfectam reagent (BioSepra Inc.,
Villeneuve La Garenne, France) as described previously (Dautzenberg et
al., 2000). Two days after transfection with 10 µg of plasmid DNA,
geneticin selection (1 mg/ml) was initiated and stable clones were selected.
Membrane Isolation and Radioligand Binding Assays.
Membranes
from cells expressing hOP1,
hOP2, hOP3, or hORL1 or
from rat brain were isolated as described previously (Dautzenberg et
al., 1998). In the case of the rat brain membrane homogenates, the
membrane preparations were washed once with acidified
phosphate-buffered solution (PBS, pH 4.0) to ensure that membrane-bound
Ro 64-6198 was washed off. Afterward, the homogenates were washed once
more with a PBS solution of physiological pH (pH 7.4), centrifuged at
39,000g, then processed further.
). Briefly, membranes from the receptor-expressing cells
were combined with wheat germ agglutinin Scintillation Proximity Assay beads (0.5-1 mg, Amersham) and the indicated
radioligands. Reactions were performed in 0.25 ml of binding buffer (50 mM Tris-HCl, pH 7.8, 1 mM EGTA, 5 mM MgCl2, 0.1%
BSA) for 60 min at 22°C with shaking, and radioactivity was measured
in a TopCount scintillation counter (Packard, Meriden, CT).
Nonspecific binding was defined in the presence of 1 µM unlabeled
OFQ/N (hORL1), 1 µM DAMGO (hOP3), 10 µM
naloxone (hOP2), or 10 µM deltorphin
(hOP1).
Saturation binding assays with rat brain membrane homogenates were
performed in 1 ml of binding buffer with 10 µg of homogenate and
increasing concentrations (25-800 pM)
[leucyl-3H]OFQ/N. Incubation was performed for
60 min at 22°C. At the end of the incubation, the reaction mixture
was filtered through GF/C filters (Amersham) that had been precoated
(0.3% polyethyleneimine plus 0.1% BSA). Filters were washed three
times with 0.5 ml of cold wash buffer (50 mM Tris-HCl, pH 7.5).
Finally, 60 µl of scintillation fluid (MicroScint, Canberra Packard,
Mississauga, ON, Canada) was added, and samples were counted in a
TopCount scintillation counter. Nonspecific binding was defined in the
presence of 1 µM OFQ/N.
cAMP Inhibition Assay.
The inhibition of forskolin-mediated
cAMP accumulation by OFQ/N and Ro 64-6198 was determined in 96-well
plates. Briefly, 20,000 cells were incubated in
Krebs-Ringer-HEPES-buffered solution (124 mM NaCl, 5 mM KCl, 1.25 mM
MgSO4, 1.5 mM CaCl2, 1.25 mM KH2PO4, and 25 mM HEPES,
pH 7.4) in the presence of 100 µM Rolipram and 1 µM forskolin (both
from Sigma) with increasing concentrations of agonists (10 pM to 100 nM) for 15 min at 37°C. Reactions were stopped by the addition of
0.12 ml of ice-cold ethanol and stored at 
80°C for at least 4 h. The cAMP content was determined from the supernatant using the
Biotrak nonradioactive cAMP kit (Amersham) according to the
manufacturer's instructions.
Desensitization Protocol.
HEK293 cells stably expressing the
hORL1 receptor were incubated with 100 nM OFQ/N or Ro 64-6198 for 30 min at 37°C. At the end of the incubation period, cells were washed
three times with 40 volumes of ice-cold PBS (137 mM NaCl, 2.7 mM KCl,
4.3 mM Na2HPO4, and 1.4 mM
KH2PO4; pH 7.4) as
described previously (Dautzenberg and Hauger, 2000) or once with
acidified PBS (PBS, pH 4.0) followed by two final washes with PBS, pH
7.4. Next, cells were resuspended in Krebs-Ringer-HEPES-buffered
solution and stimulated with 1 µM forskolin and increasing
concentrations of either OFQ/N or Ro 64-6198, as described above.
In Vivo Investigations and Chronic Administration.
Ro
64-6198 or vehicle (0.3% Tween 80 in physiological saline) was
administered chronically to male Sprague-Dawley rats (150-200 g) for
21 days. A dose of 3.2 mg/kg Ro 64-6198 was chosen to induce a robust
anxiolytic-like effect in the elevated plus-maze test, without
significantly impairing sensorimotor functions in the rat (Jenck et
al., 2000). Daily intraperitoneal administration was performed at
variable abdominal injection sites (Ro 64-6198 has low oral
bioavailability) between 8:00 and 9:00 AM. A preliminary subchronic
observation study (5 days, 3 mg/kg i.p., b.i.d.) confirmed that the
drug was well tolerated by rats when given repeatedly and did not
accumulate to toxic levels as predicted by its acute pharmacokinetic
profile. The experimental procedures used in this study received
approval from a local governmental committee based on adherence to
international guidelines and Swiss federal regulations on animal experimentation.
, 2000). Briefly, forelimb grip
strength was quantitatively assessed using a digital strain gauge,
motor performance was measured in a body traction test situation, and
thermal nociception was assessed using a standard tail-flick assay.
Since rats needed to be naive to the elevated plus-maze procedure, no
interim analysis was performed at 7 days, but all animals were exposed
to the plus-maze on day 15, 30 min following drug or vehicle
administration. This test is based on the natural aversion of rodents
for open spaces and heights and uses an elevated maze with two open and
two closed arms. The time spent and the number of entries into open
arms are indices of neophobic anxiety in animals (details in Jenck et
al., 2000). Directly following plus-maze exposure, animals were tested
again for muscular strength, motor performance, and nociceptive
reactivity. Chronic drug or vehicle administration continued for an
additional week, and all animals were sacrificed on day 21 for brain
and liver dissection. Brain samples were used for receptor assays (see
above), and liver samples were analyzed for hepatic enzyme induction
using a standard cytochrome P450 activity assay.
Data Reduction and Statistical Analyses.
OFQ/N- or Ro
64-6198-stimulated cAMP inhibition data were calculated as percentage
of control or percentage of cAMP values, as previously described
(Hauger et al., 1997; Dautzenberg and Hauger, 2000). One-way analysis
of variance (ANOVA) across experimental groups was performed using
StatView Student (Abacus Concepts, Inc., Berkeley, CA) and Xlfit
software (F. Hoffmann-La Roche AG, Basel, Switzerland). If the one-way
ANOVA was statistically significant, planned post hoc analyses were
performed using Bonferroni's multiple comparison tests to determine
individual group differences.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Pharmacological Characterization of Ro 64-6198 at Human OP1, OP2, OP3, and ORL1 Receptors. The binding characteristics of cells transiently expressing hOP1 and hOP2 receptors or stably producing hOP3 and hORL1 receptors were assessed. Saturation binding assays were performed with the four receptor preparations and radiolabeled deltorphin (OP1), naloxone (OP2), DAMGO (OP3), and OFQ/N (ORL1) as ligands. High-affinity binding of the radioligands to OP1 (Kd = 0.09 ± 0.02 nM; Bmax = 1.9 ± 0.3 pmol/mg), OP2 (Kd = 0.6 ± 0.1 nM; Bmax = 1.7 ± 0.4 pmol/mg), OP3 (Kd = 0.2 ± 0.04 nM; Bmax = 0.2 ± 0.02 pmol/mg), and ORL1 (Kd = 0.12 ± 0.03 nM; Bmax = 1.3 ± 0.2 pmol/mg) receptors was observed (not shown).
In competition binding assays, Ro 64-6198 displayed high affinity to the ORL1 receptor only. The compound competed for binding to the ORL1 receptor with subnanomolar affinity (Ki = 0.39 ± 0.05 nM, n = 10), while 130- to 250-fold higher ligand concentrations were needed to compete for binding to the OP3 (Ki = 52.4 ± 5.5 nM, n = 9) and OP2 (Ki = 106 ± 9 nM, n = 7) receptors (Fig. 1B). Finally, Ro 64-6198 displayed very low affinity to the OP1 receptor (Ki = 1.4 ± 0.1 µM, n = 7) and thus differed in its affinity compared with the ORL1 receptor by a factor of 3500 (Fig. 1B). In another set of experiments, the ability of Ro 64-6198 to inhibit forskolin-stimulated cAMP accumulation in cells stably expressing the ORL1 or the OP3 receptor was studied. Like the natural agonist OFQ/N (EC50 = 0.3 ± 0.1 nM, n = 12), Ro 64-6198 was a potent and full agonist (EC50 = 0.26 ± 0.08 nM, n = 10) at the ORL1 receptor (Fig. 1C). Both compounds inhibited forskolin-mediated cAMP accumulation by approximately 80 to 90% (Fig. 1C). In contrast to its ability to potently and efficiently inhibit the forskolin response in ORL1-transfected cells, Ro 64-6198 was less potent and efficacious in OP3-transfected CHO cells. While DAMGO (EC50 = 6.3 ± 1.9 nM, n = 4) inhibited cAMP accumulation with high affinity, Ro 64-6198 (EC50 = 89.1 ± 12.4 nM, n = 3) was more than 15-fold less potent (Fig. 1C). Furthermore, Ro 64-6198 was ~350-fold less potent at the OP3 receptor than at the ORL1 receptor. More important, Ro 64-6198 only inhibited forskolin-mediated cAMP inhibition in OP3-expressing cells to a smaller extent (~30%) than DAMGO (Emax ~65%) (Fig. 1C), a well known full agonist at the OP3 receptor (Whistler et al., 1999). Thus, Ro 64-6198 behaves as a partial agonist at the OP3 receptor.
Differential Desensitization of ORL1 by OFQ/N and Ro 64-6198. The ability of OFQ/N and Ro 64-6198 to functionally desensitize the cAMP response, and subsequently, the signaling response of the ORL1 receptor was assessed. In a first set of experiments, the effects of OFQ/N or Ro 64-6198 pre-exposure on forskolin-mediated cAMP accumulation was investigated. After incubation with 100 nM agonist, the cells were washed with either a pH 7.4 or a pH 4.0 buffered PBS solution, to ensure removal of the ligand, and then stimulated with forskolin. Pretreatment of ORL1-expressing cells with either 100 nM OFQ/N or Ro 64-6198 did not render the cells significantly more responsive to forskolin (not shown).
Next, the magnitude of ORL1 desensitization by OFQ/N or Ro 64-6198 was investigated. ORL1-expressing HEK293 cells were preincubated at 37°C for 30 min with vehicle (PBS) or a maximally effective concentration of either 100 nM OFQ/N or Ro 64-6198. After the cells had been washed extensively with a pH 7.4 buffered PBS solution, increasing doses (1 pM to 100 nM) of OFQ/N or Ro 64-6198 were incubated in the presence of forskolin (1 µM), and the cAMP content of the cells was determined. In control cells exposed to vehicle, OFQ/N and Ro 64-6198 potently inhibited forskolin-mediated cAMP accumulation (Fig. 2A) at low agonist concentrations (OFQ/n = 0.19 ± 0.08 nM, n = 3; Ro 64-6198 = 0.22 ± 0.06, n = 3). Furthermore, OFQ/N and Ro 64-6198 produced maximal inhibitions of forskolin-mediated cAMP production by 81 ± 3% (OFQ/N) and 84 ± 4% (Ro 64-6198) that did not differ significantly from one another. In contrast, pre-exposure of ORL1-transfected cells with either OFQ/N or Ro 64-6198 significantly shifted the dose-response curves for both agonists to the right (p < 0.01) and in addition, desensitized their maximal response by 44 ± 3% (OFQ/N) and 50 ± 6% (Ro 64-6198) (p < 0.0001).
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; Smalley et al., 2001). After preincubation with OFQ/N or Ro
64-6198, cells were washed either with pH 7.4 or pH 4 buffer and
finally, binding of [3H]OFQ/N to the intact
cells was determined (Fig. 3). Only in
ORL1-expressing cells that had been desensitized with OFQ/N and washed
with pH 7.4 buffer was a reduced number of binding sites and a
rightward shift of the binding curve observed. However,
OFQ/N-desensitized cells that had been washed with pH 4 buffer or that
had been incubated in the presence of high sucrose concentrations
showed a binding profile similar to that of the control cells (Fig.
3A). In contrast, a pre-exposure with Ro 64-6198 followed by pH 7.4 or
pH 4 washes reduced the number of cell-surface binding sites as well as
the affinity of the ORL1 receptor (pH 7.4 wash:
IC50 ~15 nM; pH 4 wash:
IC50 ~17 nM). However, a full restoration of
the maximal binding capacities and affinity for OFQ/N ligands
(IC50 ~2.5 nM) was observed when the cells were
pretreated with Ro 64-6198 in the presence of high sucrose
concentrations. Thus, we conclude that the reduced binding sites of
ORL1 cells after Ro 64-6198 treatment reflect an internalization of the
ORL1 receptor rather than residual receptor occupancy.
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In Vivo Effects Following Acute or Chronic Exposure of Rats to Ro
64-6198.
Average body weight gain and rectal temperatures were not
significantly different among the three treatment groups (single-factor ANOVA, p > 0.05 in all cases, data not shown). The
7-day interim analysis of muscular strength, motor performances, and
nociceptive reactivity revealed normal sensorimotor functions at day 7 in Ro 64-6198-treated compared with vehicle-treated animals (unpaired Student's t test, p > 0.05). In the
elevated plus-maze test on day 15, Ro 64-6198 elicited significant
anxiolytic-like effects as measured in time spent and transitions to
the open arms (Fig. 4) in both groups
exposed to the drug (post hoc Bonferroni, p < 0.01 in
Veh-Ro and p < 0.001 in Ro-Ro versus Veh-Veh group). Subsequent analysis of muscular strength, motor performances, and
nociceptive reactivity revealed no differences in sensorimotor functions at day 15 following Ro 64-6198 or vehicle treatment (single-factor ANOVA, p > 0.05 in all cases; Fig.
5). Liver weights and cytochrome P450
enzyme activity was not different in Ro 64-6198-treated compared with
vehicle-treated animals (1.56 nmol of cytochrome P450/mg of
protein for vehicle and 1.26 for Ro 64-6198, unpaired Student's
t test, p > 0.05).
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Transient Loss of ORL1 Binding Sites in Acutely and Chronically Ro 64-6198-Treated Rats. Membrane homogenates from the brains (n = 2-3/time point) of rats treated acutely (1 dose) or chronically (1 dose/day for 25 days) with Ro 64-6198, and brains from the control groups, were prepared and tested for their affinity to bind [3H]OFQ/N. To eliminate unspecific binding of Ro 64-6198 to the brains, the homogenates were washed once with acidified PBS solution (pH 4.0).
Vehicle-treated animals showed no alterations of brain ORL1 binding sites across all time points analyzed (Fig. 6; Table 2). Marked differences to the vehicle group, however, were observed when rat brain homogenates from animals that had been treated with Ro 64-6198 acutely or chronically (25 days) were assessed for their ORL1 binding sites. In both groups similar amounts of ORL1 receptors (~150 fmol/mg) were calculated before Ro 64-6198 injection, and no significant differences were observed 10 min after injection (Table 2). The number of binding sites in the two Ro 64-6198 treatment groups also did not differ from the values of the control group (Fig. 6; Table 2). However, 30 min post-Ro 64-6198 injection, a dramatic loss of ORL1 binding sites was detected (77% in the acute group and 71% in the chronic group) (Fig. 6; Table 2). The number of binding sites remained strongly reduced (71% in the acute group and 66% in the chronic group) after 60 min and then slowly started to recover (Fig. 6). Three hours after Ro 64-6198 injection, the number of binding sites was back to 47 to 55% of the original number, and after 8 h, a recovery to >70% was detected. Finally, 24 h after Ro 64-6198 injection, a full recovery of the ORL1 binding sites in both treatment groups was noted (Fig. 6; Table 2). For both treatment groups a t1/2 of ~5.5 h for the recovery of ORL1 binding sites was calculated.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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This study provides a full characterization of the novel
nonpeptide OFQ/N agonist Ro 64-6198, a ligand binding the ORL1 receptor with subnanomolar affinity but showing considerably lower affinity to
the OP3 (130-fold), OP2
(250-fold), and OP1 (3500-fold) receptors. Thus,
Ro 64-6198 displays a selectivity profile similar to that of OFQ/N
(Meunier et al., 1995; Reinscheid et al., 1995). Because of the high
sequence homology (~50%) between the ORL1 and the opiate receptors
(Meunier et al., 2000), such a selectivity profile is important for an
ORL1 ligand to avoid undesired side effects mediated by the activation
of opiate receptors. Besides their importance in pain transmission,
opiate receptors, especially the OP3 receptor,
are responsible for many features of drug addiction (Nestler, 1996).
Despite the high sequence similarity, the physiological function of
OFQ/N and its receptor differs considerably from that of the opiate
ligands and receptors. OFQ/N has been reported to facilitate pain
transmission, but the precise effects of OFQ/N on nociceptive
sensitivity remain unclear (reviewed in Meunier et al., 2000). On the
other hand, OFQ/N has been shown to be a potent anxiolytic in vivo
(Jenck et al., 1997) but also promotes memory impairments (Manabe et
al., 1998). Thus, the development of nonpeptide antagonists of the ORL1
receptor have a good clinical potential for the treatment of
Alzheimer's disease, while OFQ/N agonists are beneficial for the
treatment of stress disorders and depression (Jenck et al., 2000;
Wichmann et al., 2000).
In the cAMP inhibition assay, Ro 64-6198 was a full agonist at the ORL1
receptor, and its potency did not differ from that of the natural
ligand OFQ/N. As expected from the strong differences in the binding
assays, Ro 64-6198 was >300-fold less potent at inhibiting
forskolin-mediated cAMP inhibition in OP3- than
in ORL1-expressing cells. Surprisingly, Ro 64-6198 was only a partial agonist at the OP3 receptor, displaying less than
50% of the efficacy of DAMGO, a full agonist at this receptor. This
result contrasts with our original finding that Ro 64-6198 is a full
agonist at the OP3 receptor in the GTP
S
binding assay (Jenck et al., 2000), a functional assay that is
conducted with membrane preparations (Röver et al., 2000).
However, in this study we used cells expressing significantly lower
amounts of the OP3 receptor (200 fmol/mg versus >2 pmol/mg) than in our previous studies (Wichmann et al., 1999, 2000;
Röver et al., 2000). Furthermore, because the cAMP assay is
performed with intact cells, it may be more predictive for the
functional properties of a ligand than an assay conducted with isolated
membranes. Indeed, the central nervous system effects elicited by Ro
64-6198 cannot be blocked by the high-affinity OP3 receptor antagonist naloxone (unpublished
observations). Thus, it is likely that Ro 64-6198 does not
activate the OP3 receptor in vivo.
To gain more insights into the functional properties of Ro 64-6198, we performed a series of desensitization experiments to study the functional regulation of the ORL1 receptor by this compound. We compared the following parameters: the effect of agonist activation of the ORL1 receptor on maximal forskolin-mediated cAMP production, the effects of either physiological (pH 7.4) or acidic (pH 4.0) pH washes on OFQ/N- or Ro 64-6198-mediated functional desensitization of the ORL1 receptor, and finally, the effects of physiological or acidic pH washes on the numbers of ORL1 binding sites.
In the recombinant expression system, the ORL1 receptor, like the
opiate receptor, inhibits cAMP production (Reinscheid et al., 1995;
Blake et al., 1997). Thus, cAMP inhibition assays are performed
in the presence of forskolin, a direct activator of adenylyl cyclase
(Hauger et al., 1997). Because none of the pretreatments produced
elevated cAMP levels, we assumed that the different magnitudes of
agonist-induced cAMP inhibition represented true desensitization of the
signaling responses.
Striking differences in the desensitization potencies of OFQ/N and Ro
64-6198 were observed when ORL1-expressing cells were washed with
either pH 7.4 or pH 4.0 buffer. No differences in Ro 64-6198's
desensitizing properties were observed with both buffers, and Ro
64-6198 potently desensitized the maximal responses of the ORL1
receptor and significantly shifted the agonist-dose response curves to
the right. In agreement with the functional studies, we observed a
strong reduction of ORL1 binding sites after pre-exposure with Ro
64-6198, and this reduction was also independent of the washing
conditions. Furthermore, when the desensitization experiments with Ro
64-6198 were conducted in the presence of high sucrose concentrations,
no reduction of ORL1 binding sites after acidic washes was observed.
Since high sucrose concentrations prevent receptor internalization
(Keith et al., 1996; Smalley et al., 2001), we conclude that acidic
washes quantitatively remove the nonpeptide agonist from its receptor,
and that the reduced responsiveness of the ORL1 receptor after Ro
64-6198 challenge reflects desensitization of the second messenger
cascade and receptor internalization. Surprisingly, this seems not to
be the case for OFQ/N. We observed a functional desensitization of the
ORL1 receptor and a reduction of receptor sites after OFQ/N exposure
only when the cells had been washed with pH 7.4 buffer. However, after
acidic washes of OFQ/N-exposed cells, the functional response of the ORL1 receptor was completely restored, and a full recovery of the
number of OFQ/N binding sites was observed. These results indicate that
OFQ/N occupies the ORL1 receptor and thereby blocks it from being
stimulated again, without promoting receptor sequestration and
internalization (Freedman and Lefkowitz, 1996; Carman and Benovic,
1998). Future experiments using a tagged ORL1 receptor should further
aid insight into the differential regulation of the ORL1 receptor by
its natural ligand OFQ/N or by the synthetic compound Ro 64-6198.
Another important part of our study addressed the question whether
chronic Ro 64-6198 administration induces tolerance in vivo (Matthes et
al., 1996). Tolerance is characterized as a loss of efficacy of a drug
during chronic treatment (Harrison et al., 1998). This is exemplified
by morphine, a high-affinity agonist at the OP3
receptor (Whistler et al., 1999). Usually during chronic treatment,
morphine doses have to be gradually increased to maintain its analgesic
properties (Mestek et al., 1996; Raynor et al., 1996). Similarly, other
opiate alkaloids and drugs of abuse, like heroin, also cause tolerance
in drug addicts (Mestek et al., 1996; Nestler, 1996; Raznor et al.,
1996). While in recombinant in vitro systems morphine seems not to
mediate OP3 receptor internalization (Whistler
and von Zastrow, 1998; Zhang et al., 1998; Whistler et al., 1999), it
has been speculated that the lack of internalization might account for
maladaptive processes which finally might cause tolerance in vivo.
However, in a recent study it was shown that morphine is capable of
mediating OP3 receptor internalization in vivo,
while in animals being deficient for 
-arrestin-2, an important
mediator of G protein-coupled receptor internalization, no
tolerance to morphine developed (Bohn et al., 2000). Thus, it seems
likely that internalization plays an important role in the development
of opiate receptor tolerance and that the potency of an agonist to
mediate internalization in vitro is not predictive for its potential to
cause tolerance in vivo.
However, no tolerance developed to the anxiolytic-like effects of Ro
64-6198 following 2 weeks of daily administration in rats. In addition,
Ro 64-6198 was well tolerated at anxiolytic doses administered
chronically, did not interfere with sensorimotor functions at this
dosage, and did not affect hepatic function as no enzyme induction was
observed in the animals. These results provide further evidence that
the selective ORL1 agonist Ro 64-6198 may prove a useful anxiolytic
compound. Preliminary studies from our laboratory indicate that the in
vivo effects of Ro 64-6198 are exclusively mediated by the ORL1
receptor. In ORL1-deficient animals, the anxiolytic effects of Ro
64-6198 and its sedative side effects are abolished (G. A. Higgins and
A. J. Grottick, unpublished observations). For OFQ/N, tolerance
has been reported to the locomotor-inhibiting effects of OFQ/N given at
high dosage (Devine et al., 1996), to its supraspinal antimorphine
effects (Lutfy et al., 1999), and to its spinal antinociceptive action (Hao et al., 1997). These results suggest that differences exist between the endogenous ligand and exogenous synthetic agonists in their
interactions with ORL1 receptors.
In agreement with the behavioral data, the number of ORL1 binding sites was not altered during chronic treatment with Ro 64-6198. However, we observed a strong and rapid reduction of ORL1 binding sites when Ro 64-6198 was given acutely. These effects were seen in the vehicle group and in animals that had been treated chronically with Ro 64-6198. In both groups a >70% reduction of ORL1 binding sites was monitored as rapidly as 30 min after i.p. injection of the compound. Afterward, the ORL1 receptor slowly reappeared with a t1/2 of 5.5 h, and the number of OFQ/N binding sites was fully restored after 24 h. Thus, we conclude that the reduction of OFQ/N binding sites after acute Ro 64-6198 application represents a temporary and reversible down-regulation of the ORL1 receptor and that a once-daily dosage of a nonpeptide OFQ/N agonist is likely to prevent the development of tolerance.
In conclusion, we have characterized the novel nonpeptide OFQ/N agonist Ro 64-6198 as a selective full agonist at the ORL1 receptor in vitro. Pre-exposure of ORL1-expressing cells with Ro 64-6198 potently desensitized cAMP responses and down-regulated the number of cell-surface ORL1 receptors. In contrast, OFQ/N, despite its potency to desensitize the second messenger responses, was unable to down-regulate its receptor. In rats treated chronically with Ro 64-6198, no signs of tolerance were observed. ORL1 binding sites were unaltered by the chronic treatment, and only a transient decrease of the receptor was observed after acute Ro 64-6198 administration.
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Acknowledgments |
|---|
We thank Ivo Vogel for helping with the membrane preparations, Philipp Oberli for his help in synthesizing Ro 64-6198, and Martine Maco for skillful technical assistance. Finally, we thank Dr. Kenneth Lundstrom for the infection of BHK cells to obtain OP1 and OP2 membranes.
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Footnotes |
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Accepted for publication May 1, 2001.
Received for publication December 21, 2000.
This study was supported by F. Hoffmann-La Roche AG.
Address correspondence to: Dr. Frank M. Dautzenberg, Axovan Ltd. Innovation Center, Gewerbestrasse 16, 4123 Allschwil, Switzerland. E-mail: frank.dautzenberg{at}axovan.com
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Abbreviations |
|---|
ORL1, OFQ/N receptor; HEK293, human embryonic kidney 293; DAMGO, [D-ala2, N-methyl-phe4, glyol5][tyrosyl-3,5]-enkephalin; OFQ/N, orphanin FQ/nociceptin; OP1, delta opiate receptor; OP2, kappa opiate receptor; OP3, mu opiate receptor; Ro 64-6198, {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one}; BSA, bovine serum albumin; BHK, baby hamster kidney; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ANOVA, analysis of variance.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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-arrestin-2 determines morphine tolerance but not dependence.
Nature (Lond)
408:
720-723[Medline].
receptors and the role of position 185 for receptor-ligand selectivity.
Neuropharmacology
39:
1368-1376[Medline].-arrestin.
Proc Natl Acad Sci USA
95:
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