The Journal of Neuroscience, January 1, 1999, 19(1):503–510 Identification of the Receptor Subtype Involved in the Analgesic Effect of Neurotensin Isabelle Dubuc,1 Philippe Sarret,2 Catherine Labbe´ -Jullie´,2 Jean-Marie Botto,2 Eric Honore´,2 Elisabeth Bourdel,3 Jean Martinez,3 Jean Costentin,1 Jean-Pierre Vincent,2 Patrick Kitabgi,2 and Jean Mazella2 1Unite´ de Neuropsychopharmacologie Expe´ rimentale, Centre National de la Recherche Scientifique (CNRS), 76803 Saint Etienne du Rouvray, France, 2Institut de Pharmacologie Mole´ culaire et Cellulaire, CNRS, 06560 Valbonne, France, and 3Laboratoire des Aminoacides, Peptides et Prote´ ines, ESA CNRS, Faculte´ de Pharmacie, Universite´ s de Montpellier 1 et 2, 34060 Montpellier, France. The neuropeptide neurotensin (NT) elicits hypothermic and This effect was specific, because NTR1 levels were unaffected, naloxone-insensitive analgesic responses after brain injection. and sense or scramble oligodeoxynucleotides had no effect. Recent pharmacological evidence obtained with NT agonists Structure–activity studies revealed a close correlation between and antagonists suggests that these effects are mediated by a the analgesic potency of NT analogs and their affinity for the receptor distinct from the initially cloned high-affinity NT recep- NTR2 and disclosed potent and selective agonists of this re- tor (NTR1). The recent cloning of a second NT receptor (NTR2) ceptor. These data confirm that NTR1 is involved in the NT- prompted us to evaluate its role in NT-induced analgesia. Intra- elicited turning behavior and demonstrate that the NTR2 me- cerebroventricular injections in mice of two different antisense diates NT-induced analgesia. oligodeoxynucleotides from the NTR2 markedly decreased Key words: analgesia; neurotensin; receptor; levocabastine- NTR2 mRNA and protein and reduced NT-induced analgesia. sensitive; oligodeoxynucleotide; antisense The neuropeptide neurotensin (NT) exerts important central not correlate with their binding affinity for the NTR1 (Labbe´- functions, including hypothermic and naloxone-insensitive anal- Jullie´ et al., 1994). gesic responses after brain injection (Al-Rhodan et al., 1991; for To investigate a possible role of the NTR2 in mediating NT- review, see Vincent, 1995). NT interacts with two recently cloned induced hypothermia and analgesia, we attempted to selectively receptors that were originally differentiated on the basis of their block its central expression using in vivo antisense strategies (for affinity for NT and their sensitivity to the antihistaminic drug review, see Akhtar and Agrawal, 1997). The amount of levocabastine (Schotte et al., 1986). The high-affinity, levocabastine-sensitive NT binding sites and the resulting re- levocabastine-insensitive NT receptor (NTR1) was cloned first sponses to NT-induced analgesia were specifically decreased in (Tanaka et al., 1990; Vita et al., 1993) and shown to mediate a mice intracerebroventricularly injected with specific NTR2 anti- number of peripheral and central NT responses, including the sense oligodeoxynucleotides (ODNs) compared with control an- neuroleptic-like effects of the peptide (Labbe´-Jullie´ et al., 1994). imals injected with solvent, NTR2 sense and scramble ODNs, or In contrast, the functional relevance of the lower-affinity, with antisense NTR1 ODNs. These results, added to structure– levocabastine-sensitive NT receptor (NTR2) cloned later (Cha- activity studies, identify NTR2 as the receptor that mediates lon et al., 1996; Mazella et al., 1996) remains to be established. NT-induced analgesia. Pharmacological evidence suggested that two of the most prom- inent effects of centrally injected NT i.e., analgesia (Clineschmidt MATERIALS AND METHODS et al., 1979; Nemeroff et al., 1979; Martin and Naruse, 1982; ODN administration. A first pair of sense and antisense oligodeoxynucle- Behbehani and Pert, 1984; Coquerel et al., 1988) and hypother- otides (ODN1) was chosen to span the translation start site of the mouse NTR2 (Mazella et al., 1996). Their sequences are as follows: sense, mia (Bissette et al., 1976), were not mediated by the NTR1. Thus, 59-ATG-GAG-ACC-AGC-AGC-CTG-39; and antisense, 59-CAG-GCT- the recently developed nonpeptide NT antagonist SR 48692 (Gul- GCT-GGT-CTC-CAT-39. A second pair of phosphorothioate antisense ly et al., 1993), which preferentially recognizes the NTR1, did not and scramble ODN2 corresponded to nucleotides 30–47 in the coding antagonize these effects (Dubuc et al., 1994). Furthermore, a sequence of NTR2: antisense, 59-CCT-GCA-CTG-GGG-CTG-GGC-39; 9 9 number of metabolically stable peptide and pseudopeptide NT and scramble, 5 -CTC-TCC-GAG-CGG-GTG-GCG-3 . We also synthe- sized phosphorothioate ODNs spanning the translation start site of the analogs exhibited analgesic and hypothermic potencies that did rat NTR1 (Tanaka et al., 1990): antisense, 59-GGA-GCT-GTT-GAG- GTG-CAT-39; and scramble, 59-GAG-GTC-TTG-GAG-TGG-ACT-39. All these ODNs, synthesized by Eurogentec and analyzed by OLIGO 4.0 Received July 7, 1998; revised Oct. 15, 1998; accepted Oct. 22, 1998. (National Biosciences, Inc.), were devoid of internal hair loops and putative cross-reactions. In preliminary experiments with ODN1, mice 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 were treated with either a single ODN injection per day for4dorwith this manuscript. two ODN injections per day for 3 d, and behavioral tests were performed Correspondence should be addressed to Jean Mazella, Institut de Pharmacologie on the fifth day. There was no change in the hypothermic and analgesic Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Unite´ responses to NT in antisense ODN-treated animals compared with Propre de Recherche 411, 660 Route des Lucioles, 06560 Valbonne, France. control animals. The working conditions then selected for ODN1 were Copyright © 1998 Society for Neuroscience 0270-6474/98/190503-08$05.00/0 two intracerebroventricular injections (9:00 A.M. and 6:00 P.M.) for 4 504 J. Neurosci., January 1, 1999, 19(1):503–510 Dubuc et al. • Neurotensin Receptors and Analgesia 125 3 125 3 Figure 1. I-Tyr -NT binding to homogenates of CHO cells or mouse brains treated with solvent or ODNs. A, Densities (Bmax )of I-Tyr -NT binding to membranes from NTR2-expressing CHO cells treated for3dwithsolvent (Svt), sense (S), scramble (Scr), or antisense (AS) ODN1 NTR2 6 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 5 (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 6 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. consecutive days, followed by behavioral tests on the fifth day. of DAC-30 was unable to decrease the amount of bound iodinated NT to When unprotected ODN2 (scramble or antisense) was injected according membranes from CHO cells expressing the NTR2. to the latter protocol, no effect on NT-induced responses was observed. Transfection of cDNA encoding the mNTR2 and binding experiments. It was checked in this case that ODN treatment did not modify the The eukaryotic expression vector (pcDNA3) containing the 1.6 kb binding of NT to either NTR1 or NTR2. The use of phosphorothioate EcoRI-ApaI fragment of the NTR2 cDNA was used to transiently trans- ODN2 (scramble and antisense) following the same protocol showed fect COS-7 cells by the DEAE–dextran precipitation method (Cullen, some toxicity in the treated mice. The final selected conditions for 1987). Stable expression of the NTR2 into CHO cells was obtained by phosphorothioate ODN2 treatment were two intracerebroventricular transfection with 5 mg of recombinant pcDNA3 vector using 25 mgofthe injections per day for 3 consecutive days, followed by behavioral tests on cationic liposome DAC-30 (Eurogentec) according to the manufacturer’s the fourth day. recommendations. Clonal cell lines were selected by Geneticin (Sigma) Measurements of biological effects. All drugs were intracerebroventricu- (0.5 mg/ml) and isolated before determination of expression level. The larly injected in male Swiss CD1 mice (22–24 gm; Charles River, Saint- clonal cell line expressing the highest level of NTR2 was selected and Aubin-le`s Elbeuf, France) according to the method of Haley and Mc used for in vitro experiments. Cormick (1957). All ODNs were injected at 10 ml at a dose of 20 mg (3.6 Binding experiments were performed as described previously (Ma- nmol). All experimental groups of mice consisted of 10–12 animals. On zella et al., 1996) using either membranes prepared from brains treated the test day, NT (10 ng 5 6.7 pmols or 100 ng 5 67 pmols) or solvent (7% with solvent or ODNs, or homogenates freshly prepared from cells saline, 10 ml) was intracerebroventricularly injected. Colonic tempera- transiently (COS-7) or stably (CHO) transfected with the NTR2. Spinal ture was measured with a thermistor probe (Thermalert TH5; Physitemp, cord was not analyzed, because this organ primarily contains the inactive Clifton, NJ) introduced at a depth of 2 cm into the rectum, immediately variant of NTR2 (Botto et al., 1997b) and because NT analgesic effects before and 20 min after the intracerebroventricular injection. Antinoci- are known to be of supraspinal origin (Martin and Naruse, 1982). ception was determined by the writhing test according to Koster et al. Homogenates, 50 mg of protein from treated cells or 100 mg of protein (1959).