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 Previous Article
The Journal of Neuroscience, January 1, 1999, 19(1):503-510
Identification of the Receptor Subtype Involved in the Analgesic
Effect of Neurotensin
Isabelle
Dubuc1,
Philippe
Sarret2,
Catherine
Labbé-Jullié2,
Jean-Marie
Botto2,
Eric
Honoré2,
Elisabeth
Bourdel3,
Jean
Martinez3,
Jean
Costentin1,
Jean-Pierre
Vincent2,
Patrick
Kitabgi2, and
Jean
Mazella2
1 Unité de Neuropsychopharmacologie
Expérimentale, Centre National de la Recherche Scientifique
(CNRS), 76803 Saint Etienne du Rouvray, France, 2 Institut
de Pharmacologie Moléculaire et Cellulaire, CNRS, 06560 Valbonne,
France, and 3 Laboratoire des Aminoacides, Peptides et
Protéines, ESA CNRS, Faculté de Pharmacie,
Universités de Montpellier 1 et 2, 34060 Montpellier,
France.
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ABSTRACT |
The neuropeptide neurotensin (NT) elicits hypothermic and
naloxone-insensitive analgesic responses after brain injection. Recent
pharmacological evidence obtained with NT agonists and antagonists
suggests that these effects are mediated by a receptor distinct from
the initially cloned high-affinity NT receptor (NTR1). The recent
cloning of a second NT receptor (NTR2) prompted us to evaluate its role
in NT-induced analgesia. Intracerebroventricular injections in mice of
two different antisense oligodeoxynucleotides from the NTR2 markedly
decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This
effect was specific, because NTR1 levels were unaffected, and sense or
scramble oligodeoxynucleotides had no effect. Structure-activity
studies revealed a close correlation between the analgesic potency of
NT analogs and their affinity for the NTR2 and disclosed potent and
selective agonists of this receptor. These data confirm that NTR1 is
involved in the NT-elicited turning behavior and demonstrate that the
NTR2 mediates NT-induced analgesia.
Key words:
analgesia; neurotensin; receptor; levocabastine-sensitive; oligodeoxynucleotide; antisense
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INTRODUCTION |
The neuropeptide neurotensin (NT)
exerts important central functions, including hypothermic and
naloxone-insensitive analgesic responses after brain injection
(Al-Rhodan et al., 1991 ; for review, see Vincent, 1995). NT interacts
with two recently cloned receptors that were originally differentiated
on the basis of their affinity for NT and their sensitivity to the
antihistaminic drug levocabastine (Schotte et al., 1986). The
high-affinity, levocabastine-insensitive NT receptor (NTR1) was cloned
first (Tanaka et al., 1990; Vita et al., 1993) and shown to mediate a
number of peripheral and central NT responses, including the
neuroleptic-like effects of the peptide (Labbé-Jullié et
al., 1994).
In contrast, the functional relevance of the lower-affinity,
levocabastine-sensitive NT receptor (NTR2) cloned later (Chalon et al.,
1996; Mazella et al., 1996) remains to be established. Pharmacological
evidence suggested that two of the most prominent effects of centrally
injected NT i.e., analgesia (Clineschmidt et al., 1979; Nemeroff et
al., 1979; Martin and Naruse, 1982; Behbehani and Pert, 1984; Coquerel
et al., 1988) and hypothermia (Bissette et al., 1976), were not
mediated by the NTR1. Thus, the recently developed nonpeptide NT
antagonist SR 48692 (Gully et al., 1993), which preferentially
recognizes the NTR1, did not antagonize these effects (Dubuc et al.,
1994). Furthermore, a number of metabolically stable peptide and
pseudopeptide NT analogs exhibited analgesic and hypothermic potencies
that did not correlate with their binding affinity for the NTR1
(Labbé-Jullié et al., 1994).
To investigate a possible role of the NTR2 in mediating NT-induced
hypothermia and analgesia, we attempted to selectively block its
central expression using in vivo antisense strategies (for
review, see Akhtar and Agrawal, 1997). The amount of
levocabastine-sensitive NT binding sites and the resulting responses to
NT-induced analgesia were specifically decreased in mice
intracerebroventricularly injected with specific NTR2 antisense
oligodeoxynucleotides (ODNs) compared with control animals injected
with solvent, NTR2 sense and scramble ODNs, or with antisense NTR1
ODNs. These results, added to structure-activity studies, identify
NTR2 as the receptor that mediates NT-induced analgesia.
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MATERIALS AND METHODS |
ODN administration. A first pair of sense and
antisense oligodeoxynucleotides (ODN1) was chosen to span the
translation start site of the mouse NTR2 (Mazella et al., 1996). Their
sequences are as follows: sense, 5'-ATG-GAG-ACC-AGC-AGC-CTG-3'; and
antisense, 5'-CAG-GCT-GCT-GGT-CTC-CAT-3'. A second pair of
phosphorothioate antisense and scramble ODN2 corresponded to
nucleotides 30-47 in the coding sequence of NTR2: antisense,
5'-CCT-GCA-CTG-GGG-CTG-GGC-3'; and scramble,
5'-CTC-TCC-GAG-CGG-GTG-GCG-3'. We also synthesized phosphorothioate
ODNs spanning the translation start site of the rat NTR1 (Tanaka et
al., 1990): antisense, 5'-GGA-GCT-GTT-GAG-GTG-CAT-3'; and scramble,
5'-GAG-GTC-TTG-GAG-TGG-ACT-3'. All these ODNs, synthesized by
Eurogentec and analyzed by OLIGO 4.0 (National Biosciences, Inc.), were devoid of internal hair loops and putative cross-reactions. In preliminary experiments with ODN1, mice were treated with either a
single ODN injection per day for 4 d or with two ODN injections per day for 3 d, and behavioral tests were performed on the fifth day. There was no change in the hypothermic and analgesic responses to
NT in antisense ODN-treated animals compared with control animals. The
working conditions then selected for ODN1 were two
intracerebroventricular injections (9:00 A.M. and 6:00 P.M.) for 4 consecutive days, followed by behavioral tests on the fifth day.
When unprotected ODN2 (scramble or antisense) was injected according to
the latter protocol, no effect on NT-induced responses was observed. It
was checked in this case that ODN treatment did not modify the binding
of NT to either NTR1 or NTR2. The use of phosphorothioate ODN2
(scramble and antisense) following the same protocol showed some
toxicity in the treated mice. The final selected conditions for
phosphorothioate ODN2 treatment were two intracerebroventricular
injections per day for 3 consecutive days, followed by behavioral tests
on the fourth day.
Measurements of biological effects. All drugs were
intracerebroventricularly injected in male Swiss CD1 mice
(22-24 gm; Charles River, Saint-Aubin-lès Elbeuf, France)
according to the method of Haley and Mc Cormick (1957). All ODNs were
injected at 10 µl at a dose of 20 µg (3.6 nmol). All experimental
groups of mice consisted of 10-12 animals. On the test day, NT (10 ng = 6.7 pmols or 100 ng = 67 pmols) or solvent (7% saline,
10 µl) was intracerebroventricularly injected. Colonic temperature
was measured with a thermistor probe (Thermalert TH5; Physitemp,
Clifton, NJ) introduced at a depth of 2 cm into the rectum, immediately
before and 20 min after the intracerebroventricular injection.
Antinociception was determined by the writhing test according to Koster
et al. (1959). Writhes were counted over a 15 min period starting from
the fifth minute after intraperitoneal injection of a 0.5% acetic acid
solution. The turning behavior was assessed by measuring the number of
contralateral rotations induced by 10 pg of unilateral intrastriatal
injections of NT as described previously (Gully et al., 1993).
Statistical analyses were performed using two-way ANOVA,
followed by, when the interaction factor between ODN treatment and NT
effect was significant, Dunnett's t tests to assess the
significance of the difference between two groups of mice. All
procedures were done according to the animal care and handling protocol
approved by the Unité de Neuropsychopharmacologie
Expérimentale.
ODN treatment of cells. Chinese hamster ovary (CHO) cells
stably transfected with the NTR2 as described below were washed twice
and incubated in 5 ml of Opti-MEM. ODNs were mixed for 20 min
with DAC-30 (10 µg; Eurogentic) in 1 ml of Opti-MEM (Life Technologies, Gaithersburg, MD) and then added drop by drop to cells to
give a final concentration of 500 nM. Cells were incubated for 3 hr at 37°C, and the medium was removed and replaced by the appropriate cell growth medium. ODNs efficiently inhibited NTR2 expression when this treatment was repeated for 3 d, with binding experiments being performed on the fourth day. One day treatment or
repeated treatments in the absence of DAC-30 was unable to decrease the
amount of bound iodinated NT to membranes from CHO cells expressing the NTR2.
Transfection of cDNA encoding the mNTR2 and binding
experiments. The eukaryotic expression vector (pcDNA3) containing
the 1.6 kb EcoRI-ApaI fragment of the NTR2 cDNA
was used to transiently transfect COS-7 cells by the DEAE-dextran
precipitation method (Cullen, 1987). Stable expression of the NTR2 into
CHO cells was obtained by transfection with 5 µg of recombinant
pcDNA3 vector using 25 µg of the cationic liposome DAC-30
(Eurogentec) according to the manufacturer's recommendations. Clonal
cell lines were selected by Geneticin (Sigma) (0.5 mg/ml) and isolated
before determination of expression level. The clonal cell line
expressing the highest level of NTR2 was selected and used for in
vitro experiments.
Binding experiments were performed as described previously (Mazella et
al., 1996) using either membranes prepared from brains treated with
solvent or ODNs, or homogenates freshly prepared from cells transiently
(COS-7) or stably (CHO) transfected with the NTR2. Spinal cord was not
analyzed, because this organ primarily contains the inactive variant of
NTR2 (Botto et al., 1997b) and because NT analgesic effects are known
to be of supraspinal origin (Martin and Naruse, 1982). Homogenates, 50 µg of protein from treated cells or 100 µg of protein from
treated brains, were incubated for 30 min at 25°C with increasing
concentrations of 125I-Tyr3-NT (100 Ci/mmol, 0.5-15 nM) (Sadoul et al., 1984) in the absence or presence of 1 µM levocabastine, which selectively
inhibits NT binding to the NTR2 (Schotte et al., 1986; Kitabgi et al., 1987).
Electrophysiological measurements. The pcDNA3 vector
containing either the cDNA of the mouse NTR2 or the cDNA of the rat
NTR1 served as a template to prepare cRNAs using the in
vitro transcription kit from Stratagene (La Jolla, CA).
cRNAs (10 ng) were injected into Xenopus laevis oocytes. The
oocytes were then incubated at 18°C for 2-4 d. Electrophysiological
measurements were performed at 25°C according to the procedure
described previously (Botto et al., 1997a). Drugs were applied rapidly
into the experimental chamber by a puffer pipette (200 µl). Responses
to drugs were recorded under voltage clamp at  60 mV.
In situ hybridization in mouse brain. The
KpnI-BamHI cDNA fragment (493 bp) corresponding
to nucleotides 745-1238 was inserted into the pBluescript KS
cloning vector (Stratagene) by PCR and standard cloning techniques and
used as a template to produce sense and antisense
33P-labeled RNA probes. Mouse brain sections were
post-fixed with 4% paraformaldehyde for 30 min at room temperature.
Sections were incubated in 120 mM phosphate buffer, pH 7.2, containing 50% formamide, 4× SSC, 1× Denhardt's solution, 10%
dextran sulfate, and 0.6% sarcosyl (Fluka) with sense or antisense
33P-labeled RNA probes (3-6× 105
cpm/slice). Slices were then washed and processed as described previously (Sarret et al., 1998).
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RESULTS |
Effects of ODNs on NT binding in vitro
The ability of ODN1 and ODN2 to inhibit NTR2 expression was first
estimated in vitro in CHO cells that had been stably
transfected with the cDNA encoding the NTR2. When cells were treated
for 3 d with the various ODNs using assisted delivery techniques,
treatment with antisense ODN1 and ODN2 resulted in a 30-45% decrease
in NTR2 binding sites measured with
125I-Tyr3-NT (Fig.
1A). In contrast,
neither sense ODN1 nor scramble ODN2 were able to modify NTR2 binding
capacity.
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Figure 1.
125I-Tyr3-NT
binding to homogenates of CHO cells or mouse brains treated with
solvent or ODNs. A, Densities
(Bmax) of
125I-Tyr3-NT binding to membranes from
NTR2-expressing CHO cells treated for 3 d with solvent
(Svt), sense (S), scramble
(Scr), or antisense (AS) ODN1 NTR2 or
ODN2 NTR2. Results are expressed as percentage of
Bmax values obtained from cells treated with
DAC-30 alone (Svt): 290 ± 25 fmol/mg protein
(n = 4). B, C,
Densities (Bmax) of
levocabastine-insensitive (NTR1) and levocabastine-sensitive (NTR2) NT
receptors in mouse brain homogenates treated with ODN1 NTR2
(B) or ODN2 NTR2 (C). The
total and NTR1-specific amount of NT binding sites determined in
solvent (Svt), sense (S), or
scramble (Scr) ODN-treated mice were similar to those
reported previously in mouse brain (Mazella et al., 1988).
Bmax values were extrapolated from
saturation curves fitted with the LIGAND program. Values are
mean ± SEM from two (A) and three
(B, C) independent experiments. Data were
tested for significance with a Student's t test;
*p < 0.01 compared with solvent-injected
mice.
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Effect of ODNs on NT binding in vivo
The two different antisense ODNs and their corresponding sense or
scramble ODNs were intracerebroventricularly injected in mice. On the
day after the last ODN injection, the density of functional NT
receptors was determined on treated brains by measuring 125I-Tyr3-NT binding (Sadoul et al.,
1984). Densities of NTR1, corresponding to Bmax
values of bound 125I-Tyr3-NT in the
presence of levocabastine, were similar in brain homogenates from all
groups of mice (Fig. 1B,C). Binding
to NTR2, defined as the difference between Bmax
values obtained in the absence and presence of levocabastine, was
reduced by >45% for antisense-treated mouse brains (Fig.
1B,C). The affinity of the ligand
for both sites was not significantly affected in these different
conditions (data not shown). Thus, NTR2 antisense ODNs specifically
decreased brain NTR2 expression without modifying NTR1 density (Fig.
1B,C).
Effects of antisense ODNs on NTR2 mRNA expression
Next, we investigated the effect of ODN injections on NTR2 mRNA
expression by in situ hybridization. Sagittal or coronal
slices from brains treated with sense or antisense ODN1 showed labeling of identical regions, including cerebellum, hippocampus, thalamus, neocortex, and piriform cortex (Fig.
2A-D). However, the
labeling observed on slices from brains treated with antisense ODN1
(Fig. 2B,D) was significantly
reduced compared with the labeling detected on sense ODN1-treated brain
slices (Fig. 2A,C). Most of the
regions expressing NTR2 mRNA, including thalamic areas, periaqueductal gray, medial habenular nucleus, and bed nucleus of the stria
terminalis, displayed a significant reduction in labeling intensity,
ranging from 37 to 47% of the control value (Fig.
2E). In the cerebellum, the decrease of labeling was
much less pronounced (11%).
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Figure 2.
Localization of NTR2 mRNAs in mouse brains treated
with sense (A, C) or antisense
(B, D) ODN1 NTR2. Sagittal
(A, B) or coronal (C,
D) sections were hybridized with an antisense
33P-labeled cRNA probe corresponding to nucleotides
745-1238 of the mouse NTR2 cDNA (Mazella et al., 1996). Scale bar, 2 mm. E, The decrease in the labeling intensities between
sense and antisense ODN-treated mice was measured in various brain
areas. Values are mean ± SEM of optical densities measured on
four to six sections from three independent experiments and are
expressed as the percentage of decrease between densities measured in
brains treated with sense and antisense ODNs. Statistical data were
analyzed with the Student's t test. Differences in
labeling measured between sense and antisense ODN-treated brains are
significant in all regions analyzed; p < 0.001. Another series of brain sections treated with ODN1 NTR2 were hybridized
with 33P-labeled ODNs specific of the rat NTR1 (Nicot et
al., 1994). No difference was observed in the labeling of both sense or
antisense ODN-treated sections (data not shown), indicating that ODN1
treatment specifically decreased the level of NTR2 messengers.
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Effect of antisense ODN injection on NT-induced analgesia
Mice injected with NTR2-derived ODNs were subjected to the
writhing test used as a means to assess analgesia. The number of writhes/15 min in the absence of NT was similar in solvent-, sense ODN1-, and scramble ODN2-treated mice (control groups), as well as in
antisense ODN1- and ODN2-treated mice (Fig.
3). Intracerebroventricular injections of
NT (10 ng) significantly decreased the number of writhes in all three
control groups (Fig. 3). In contrast, the peptide at 10 ng was without
significant effect on the number of writhes in antisense ODN1- and
ODN2-treated animals (Fig. 3). This reversal of the NT response
observed on the first day after antisense ODN treatment was no longer
present on the third day after treatment (data not shown), thus
suggesting a rapid clearance of ODNs in mouse brain. When a dose of 100 ng of NT was tested in antisense ODN2-treated mice, a significant
analgesic response was obtained, although it was reduced compared with
control animals (Fig. 3).
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Figure 3.
Basal and NT-induced analgesic responses of mice
treated with solvent, sense, or antisense ODN1, and scramble or
antisense ODN2. Antinociception was assessed by the writhing test.
Writhes were counted over a 15 min period after intraperitoneal
injection of 0.5% acetic acid after intracerebroventricular injection
of either solvent or 10 or 100 ng of NT. The number of indicated
writhes is the mean ± SEM from groups of 10-12 mice. Two-way
ANOVA revealed a positive interaction between ODN treatment and NT
analgesic effect. *p < 0.05;
**p < 0.01; n.s., not significant
when compared with groups that received saline injections.
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Effect of antisense ODN injection on NT-induced hypothermia
The effects of antisense ODNs of the NTR2 on the NT-induced
hypothermia in mice are shown in Figure
4. Although antisense ODN1 treatment
showed a tendency to decrease the response induced by 10 ng of NT (Fig.
4), statistical analysis of the data indicated that the peptide
response was not significantly reduced. Antisense ODN2 treatment did
not modify the response obtained with 10 ng of NT but decreased,
although not significantly, the hypothermia induced by 100 ng of NT
(Fig. 4).
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Figure 4.
Basal and NT-induced hypothermic responses of mice
treated with solvent, sense, or antisense ODN1, and scramble or
antisense ODN2. Colonic temperature was measured immediately before or
20 min after intracerebroventricular injection of 10 or 100 ng of NT.
Indicated temperatures are mean ± SEM from groups of 10-12 mice.
Two-way ANOVA showed no interaction between ODN treatment and NT
hypothermic effect.
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Effects of antisense ODNs of NTR1
As a further control of the specificity of action of NTR2-derived
antisense ODNs, mice were treated with scramble and antisense ODNs
corresponding to sequence 1-18 of the NTR1 (Tanaka et al., 1990).
Binding studies revealed that the levocabastine-insensitive binding
component (NTR1) was significantly decreased by 30% in brain
homogenates from antisense-treated mice compared with solvent- and
scramble ODN-treated animals, whereas the levels of NTR2 were similar
in all three groups of mice (Fig.
5A). The functional consequence of decreased NTR1 expression was assessed on the turning behavior induced in mice by unilateral injections of NT, an effect thought to be mediated via the NTR1, because it is blocked by the
selective NTR1 antagonist SR 48692 (Gully et al., 1993; Dubuc et al.,
1994). Figure 5B shows that the number of turns induced by
unilateral injection of 10 pg of NT was reduced by 50% in antisense ODN-treated animals compared with solvent- or scramble ODN-treated mice. This result confirms the involvement of the NTR1 in mediating the
turning behavior elicited by NT in mice. We also verified that the
antisense ODN2 directed against the NTR2, which had no effect on the
amount of NTR1 (Fig. 1), did not affect the turning behavior elicited
by NT (Fig. 5B). Finally, the analgesic and hypothermic
responses to NT (10 ng) of mice treated with the NTR1-derived antisense
were not affected compared with solvent- or scramble ODN-treated
animals (Fig. 6).
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Figure 5.
Brain densities of NT receptors and turning
behavior of mice treated with NTR1- or NTR2-specific ODNs.
A, Densities (Bmax) of
levocabastine-insensitive (NTR1) and levocabastine-sensitive (NTR2) NT
receptors in mouse brain homogenates treated with ODN NTR1.
Bmax values were extrapolated from
saturation curves fitted with the LIGAND program. Values are mean ± SEM from three independent experiments. *p < 0.01 compared with solvent-injected mice. B, The number
of contralateral rotations induced by 10 pg of unilateral intrastriatal
injections of NT was measured in mice treated with solvent, scramble,
or antisense ODNs specific for the NTR1 or NTR2. The number of
indicated turns is the mean ± SEM from groups of 10-12 mice.
Two-way ANOVA revealed a positive interaction between ODN
NTR1-treatment, but not ODN NTR2-treatment, and NT-elicited turns.
*p < 0.05 when compared with groups that received
saline injections. Svt, Solvent; Scr,
scramble; AS, antisense.
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Figure 6.
Basal and NT-induced analgesic and
hypothermic responses of mice treated with solvent, scramble, or
antisense NTR1-specific ODNs. A, Colonic temperature was
measured immediately before or 20 min after intracerebroventricular
injection of 10 ng of NT. Indicated temperatures are mean ± SEM
from groups of 10-12 mice. *p < 0.01. B, Antinociception was assessed by the writhing test.
Writhes were counted over a 15 min period after intraperitoneal
injection of 0.5% acetic acid after intracerebroventricular
injection of solvent (open bars) or 10 ng of NT
(hatched bars). The number of indicated writhes is the
mean ± SEM from groups of 10-12 mice.
*p < 0.01. Two-way ANOVA showed no interaction
between ODN treatment and NT hypothermic and analgesic effects.
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Compared potencies of NT analogs for binding to the NTR2 and for
inducing analgesia and hypothermia
We have reported previously on metabolically stable peptide and
pseudopeptide analogs of NT whose hypothermic and analgesic potencies
did not correlate with their affinity for the NTR1
(Labbé-Jullié et al., 1994). It was therefore of interest
to determine their potency for binding to the NTR2 and to compare it
with that for inducing analgesia and hypothermia (Table
1). A correlation analysis between the
binding and pharmacological potencies of six NT analogs is presented in
Figure 7. There was a highly significant
correlation (r = 0.96; p < 0.005)
between analog potencies for binding to the NTR2 and for inducing
analgesia (Fig. 7A). In contrast, the correlation between
hypothermia and binding was not statistically significant
(r = 0.72) (Fig. 7B).
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Figure 7.
Correlations between binding potencies to the NTR2
and analgesic (A) and hypothermic
(B) potencies of NT analogs. All values are
derived from Table 1. Analogs are numbered according to Table 1. The
insets show correlation coefficients
(r), together with their statistical
significance. n.s., Not significant.
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The structure-activity data in Table 1 provide interesting information
regarding the selectivity of NT analogs for the NTR2 versus the NTR1.
Thus, the potent analgesic analogs Boc-[ 11,12]NT-(8-13) and
Boc-[Lys8-9,D-Nal11]NT-(8-13)
bind with 90 and 25 times greater affinity to the NTR2 than to the
NTR1, respectively. Boc-[11,12]NT-(8-13) appears therefore as a
highly selective (~100-fold) ligand with a relatively good affinity
(Kd, ~40 nM) for the NTR2.
The selectivity of Boc-[11,12]NT-(8-13) was confirmed by
experiments performed in Xenopus laevis oocytes injected
with either NTR2 or NTR1 cRNAs. Thus, both NT and
Boc-[11,12]NT-(8-13) at 1 µM elicited similar
current responses (250 and 200 nA, respectively) in NTR2-expressing
oocytes (Fig. 8A). In
contrast, at the same concentration, the response measured for the
pseudopeptide NT analog was 100 times smaller than that for NT in
NTR1-expressing oocytes (10 nA for Boc-[11,12]NT-(8-13) compared
with 1 µA for NT) (Fig. 8B).
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Figure 8.
Current traces recorded from
Xenopus oocytes injected with in vitro
synthesized NTR2 (A) or NTR1
(B) mRNA. Downward deflections induced by
application of 1 µM NT or Boc-[11,12]NT-(8-13)
(analog 2 in Table 1) indicate Ca2+-activated
Cl currents.
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DISCUSSION |
In the present study, we provide strong evidence that the NTR2
mediates the central analgesic response to NT. Thus, using in
vivo antisense strategies, we were able to specifically decrease the expression of the NTR2 and to show that this decrease was accompanied by a reduction in the analgesic response elicited by NT. In
addition, we found that the potencies of NT analogs for binding to the
NTR2 correlated well with their potency for inducing analgesia.
Such strategies have been successfully used by many groups to reveal a
variety of receptor functions. For example, intracerebroventricular injections of antisense ODNs directed against the anxiolytic
neuropeptide Y-Y1 receptor selectively reduced its cerebral expression
in the rat and produced anxiogenic-like effects (Wahlestedt et al.,
1993a). An identical approach performed on the NMDA-R1 receptor channel protected cortical neurons from NMDA excitotoxicity and reduced focal
ischemic infarctions (Wahlestedt et al., 1993b).
The validity of ODNs used in this work was first assessed on CHO cells
stably transfected with NTR2 in which repeated treatments for 3 d
with NTR2-specific antisense ODNs led to a 35-40% decrease in
receptor expression. Then, conditions were selected for the in
vivo treatment of mice with ODNs. These conditions led to a selective decrease of NTR2 expression in the brain and to a significant reduction in the analgesic response to NT. It should be noted that
while looking for working conditions for in vivo ODN
treatment, a number of preliminary experiments failed to alter
NT-induced analgesia. In all such experiments, postmortem NT binding
experiments showed no change in the brain levels of NTR2. Conversely,
in all experiments in which antisense ODNs decreased the NT analgesic response there was a concomitant reduction in NTR2 brain levels. The
specificity of action of the chosen antisense ODNs is further supported
by a number of observations. First, the reduction in NTR2 binding sites
and the decrease in NT-induced analgesia were obtained with two
different sets of antisense ODNs against the NTR2, whereas their sense
or scramble counterparts were devoid of effect. Second, the decrease in
NT binding in the brain of treated mice resulted from a selective
decrease in NTR2 binding capacity, with no change in the
capacity of NTR1. Third, ODNs against the NTR1 were without effect on
NTR2 binding capacity and NT-induced analgesia, whereas they decreased
the number of NTR1 binding sites. Finally, the turning behavior
elicited by NT was reduced in NTR1 antisense ODN-treated mice,
whereas it was unaffected in NTR2 antisense ODN2-treated animals. It is
important to stress that although phosphorothioate ODNs often produce
nonspecific effects when injected into the brain of rodents, such
nonspecific effects were not observed in this study, because, for
example, ODNs did not affect body temperature per se.
The loss of NTR2 binding measured after antisense ODN injections could
be the consequence of both the inhibition of mRNA translation by
antisense ODNs and the decrease of NTR2 mRNAs level in brains of
antisense-treated animals (Fig. 2). This latter observation is usually
explained by the action of RNase-H, which degrades the RNA strand of an
RNA-DNA duplex (Phillips and Gyurko, 1995). It is interesting to note
that brain areas identified previously as being involved in NT-induced
analgesia (Behbehani, 1992) correspond to regions, such as the
periaqueductal gray, in which the expression of NTR2 mRNAs is the most
affected by antisense ODN treatment.
NTR2 antisense ODN administration in mice did not totally block the
analgesic response to NT. In particular, the animals were still partly
responsive to a 100 ng dose of peptide. This suggests that the blockade
of NT-induced analgesia was not the consequence of a toxic effect of
the ODNs. Actually, the apparent loss of NT potency is consistent with
the fact that ODN treatment partially decreased the number of NTR2s
available for NT binding in mouse brain, and that, therefore, a higher
dose of peptide was needed for recruiting the number of NTR2s that must
be activated to trigger the analgesic response.
The binding potencies of a series of metabolically stable peptide and
pseudopeptide NT analogs toward the NTR2 correlated well with their
potencies to induce analgesia. These pharmacological data provide
additional evidence that the NTR2 is involved in NT-induced analgesia.
One of the tested pseudopeptide NT analogs, Boc-[11,12]NT-(8-13),
showed a rather good affinity for the NTR2 (Kd, 40 nM) and a high
selectivity (100-fold) for the NTR2 over the NTR1. The selectivity was
confirmed by showing that the pseudopeptide elicited a 25-fold greater
response in NTR2-expressing oocytes than NTR1-expressing oocytes.
Together, these binding and functional data suggest that
Boc-[11,12]NT-(8-13) might represent the starting point for the
development of a selective, high-affinity agonist ligand of the NTR2.
Although NTR2 antisense ODN treatment tended to decrease the NT-induced
hypothermia, statistical analysis of the data revealed that this effect
was not significant. This could suggest either that a NT receptor
different from the NTR1 and NTR2 subserves the hypothermic effect of
the peptide or that ODNs were unable to induce a functionally
significant reduction of NTR2 levels in those brain regions involved in
NT-mediated hypothermia. Structure-activity data indicated that there
was no significant correlation between the binding potency and that for
inducing hypothermia. In particular, two analogs,
Boc-[12,13]NT-(8-13) and
Boc-[Lys8-9,Nal11]NT-(8-13),
which exhibited equipotency for binding to the NTR2 and inducing
analgesia, differed by a factor of 25 in their potencies for
inducing hypothermia (Table 1). This would support the hypothesis that
the NT hypothermic response is mediated through a receptor distinct
from the NTR2. In a recent study, Tyler et al. (1998a) reported on NT
analogs that showed markedly different potencies in their ability to
induce hypothermia and analgesia. This again lends support to the idea
that NT elicits hypothermia through an as yet unidentified receptor
subtype. Interestingly, a third NT receptor whose role is not yet
elucidated has been recently cloned (Mazella et al., 1998).
In conclusion, the antisense ODN strategy used here has proven to be a
useful approach to assess the functional relevance of the NTR2 in the
absence of selective antagonist. Our results strongly argue that the
NTR2 is directly involved in the expression of NT-induced analgesia. In
contrast, the results suggest, in agreement with others (Tyler et al.,
1998b), that NT-induced hypothermia is mediated through an as yet
unidentified NT receptor subtype. Finally, the results identify
agonists that are potent and selective for the NTR2. Altogether, this
should make it feasible, using drug screening and/or rational design
strategies, to develop NTR2 agonists that may pass the blood-brain
barrier and act as potent nonopioid analgesics. If the analgesic
effect of NT is demonstrated in humans and if this effect is
mediated by NTR2, such compounds could be useful for the treatment
of pain.
| |
FOOTNOTES |
Received July 7, 1998; revised Oct. 15, 1998; accepted Oct. 22, 1998.
We thank G. Jarretou and M. Jodar for technical assistance and F. Aguila for photographic work. A special thanks is given to Dr. A. Beaudet for carefully reading this manuscript.
Correspondence should be addressed to Jean Mazella, Institut de
Pharmacologie Moléculaire et Cellulaire, Centre National de la
Recherche Scientifique, Unité Propre de Recherche 411, 660 Route
des Lucioles, 06560 Valbonne, France.
| |
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Top
Abstract
Introduction
Gene
