- Email: [email protected]
orphanin FQ in the rat formalin test
Regulatory Peptides 79 (1999) 159–163
Distinct effect of intracerebroventricular and intrathecal injections of nociceptin / orphanin FQ in the rat formalin test Jin-Lan Wang, Chong-Bin Zhu, Xiao-Ding Cao, Gen-Cheng Wu* State Key Laboratory of Medical Neurobiology, Department of Neurobiology, Shanghai Medical University, 138 Yi Xue Yuan Road, Shanghai 200032, People’ s Republic of China Received 3 June 1998; received in revised form 10 November 1998; accepted 30 November 1998
Abstract Nociceptin / orphanin FQ (nociceptin / OFQ), a newly discovered heptadecapeptide has been regarded as an endogenous ligand for orphan opioid receptor. The present study was designed to investigate the effect of nociceptin / OFQ on pain response and opioid analgesia in the rat formalin test. The results showed that intracerebroventricular injection of 1 mg nociceptin / OFQ enhanced the pain response, and 0.1 or 0.5 mg nociceptin / OFQ had no effect on formalin-induced pain. When 0.1 or 1 mg nociceptin / OFQ were used together with m-, d-, or k-opioid receptor agonists, endomorphin-1, DSLET or U50488H, respectively, it attenuated m- and k- but not d-receptor mediated analgesia. On the other hand, intrathecal injection of nociceptin / OFQ (0.1, 1 and 5 mg) reduced the pain response in the formalin test. In conclusion, nociceptin / OFQ potentiated formalin-induced pain response and antagonized opioid analgesia in the rat brain but inhibited pain response in the spinal cord. 1999 Elsevier Science B.V. All rights reserved. Keywords: Nociceptin / orphanin FQ; Formalin test; Opioid analgesia
1. Introduction Nociceptin / orphanin FQ (nociceptin / OFQ) is a recently discovered peptide, which has been identified as an endogenous ligand of orphan opioid receptor LC132 or ORL1. The discovery of nociceptin / OFQ has resulted in a series of researches with regard to its structure, distribution and physiological functions [1]. This 17-amino acid peptide (FGGFTGARKSARKLANQ) shares sequence homology with other endogenous opioid peptides (especially dynorphin A), but possesses distinct pharmacological characteristics [2,3]. The distribution of nociceptin / OFQ and its receptor is parallel to the endogenous opioid peptides and their receptors [4,5], indicating that there is a close relationship between nociceptin / OFQ and the opioid
*Corresponding author. Tel.: 1 86 21 64041900, ext 2398; fax: 1 86 21 64174579; e-mail: [email protected]
peptidergic system. Just like opioid peptides, nociceptin / OFQ also has a great number of peripheral actions. For example, nociceptin / OFQ can decrease cardiac output, total peripheral resistance [6], and heart rate or blood pressure in the rat [7]. As far as the role of pain perception / modulation is concerned, it becomes one of the most interesting issues. The initial report claimed that central nociceptin / OFQ injection produced an increase in nociceptive responsiveness in mice [3], which was distinct to the effect of opioid peptides. But following researchers failed to see any hyperalgesic effect of nociceptin / OFQ but an anti-opioid effect [8], and the another group reported that nociceptin / OFQ produced hyperalgesia with delayed analgesia when given supraspinally [9]. So far, the results concerning the role of nociceptin / OFQ in pain modulation were in conflict, and most of the experiments were conducted in a variety of thermal or non-thermal pain tests. The purpose of this study was to further explore the effect of nociceptin / OFQ in the central nervous system on
0167-0115 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00161-X
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pain modulation using rat formalin test, so as to get a better understanding of the role of nociceptin / OFQ.
2. Materials and methods
rats’ skulls stereotaxically (P 1 mm, R 2 mm, H 4 mm). The experiment with i.c.v. injection was started 3–4 days after the operation. Drugs were dissolved in sterilized normal saline (NS). The total injection solution 20 ml (10 ml drug solution followed by a saline flush of 10 ml) was pushed into the cerebroventricle smoothly within 1 min.
2.1. Animals Male Sprague–Dawley rats (180–230 g) were supplied by the Experimental Animal Center, Shanghai Medical University. The treatment of the animals conformed to the guidelines of the International Association for the Study of Pain [10].
2.2. Drugs Heptadecapeptide nociceptin / OFQ was synthesized and purified in Shanghai Institute of Biochemistry, Chinese Academy of Sciences (Applied Biosystems 430A peptide synthesizer and BOC–Gln–PAM–resin were used). The crude peptide was sequentially purified on columns of Sephadex G-10 and HPLC. Its amino acid composition was consistent with theoretical values). Endomorphin-1 (EM-1, a newly discovered 4-amino-acid peptide), a selective m-opioid receptor agonist [11], was obtained from the same institute. trans-(6)-3,4-Dichloro-N-methylN-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50488H), a k-receptor agonist, was purchased from Sigma (St. Louis, MO, USA), [D-Ser2]–leu–Enkephalin–Thr (DSLET), a d-receptor agonist, was the product of Peninsula Laboratories (USA).
2.4. Implantation of intrathecal ( i.t.) catheters Rats were anaesthetized with sodium pentobarbital (35 mg / kg, i.p.). PE-10 tube was implanted through the gap between the 2nd and 3rd or 3rd and 4th lumbar spines (L2–3 or L3–4) to the subarachnoid space. Experiments with i.t. injection was started 3–4 days after the operation. The drug preparation and injection volume were the same as for the i.c.v. injection.
2.5. Formalin test Experiments were performed in a quiet environment (24618C). After i.c.v. or i.t. injection of drugs, formalin (150 ml, 5%) was injected subcutaneously (s.c.) into the plantar region of the right hindpaw, and the rat was immediately placed into a transparent plastic enclosure (35 3 30 3 30 cm) for 60 min behavioral observation. Pain response was observed every 5 min successively for the first 30 min, and then at 5-min intervals for the remaining 30 min. Pain intensity scoring was calculated as reported previously [12,13].
2.3. Implantation of intracerebroventricular ( i.c.v.) cannulae
2.6. Data analysis
Under pentobarbital anesthesia (35 mg / kg, i.p.), a stainless steel cannula of 0.8 mm diameter was fixed on the
Data were expressed as mean6S.E. One-way ANOVA and Q-test were used to analyze group differences.
Fig. 1. Effect of nociceptin / OFQ (i.c.v.) on formalin-induced pain response. 0.1 or 0.5 mg nociceptin / OFQ had no effect on pain response, 1 mg nociceptin / OFQ significantly potentiated formalin pain. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. OFQ 1 mg.
J.-L. Wang et al. / Regulatory Peptides 79 (1999) 159 – 163
161
Fig. 2. (a) Effect of nociceptin / OFQ (i.c.v.) on endomorphin-1 (EM-1) analgesia in formalin test. EM-1, a m-receptor selective agonist, produced a profound inhibition on formalin-induced pain, which was antagonized by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. EM-1; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 EM-1. (b) Effect of nociceptin / OFQ (i.c.v.) on U50488H analgesia in formalin test. The analgesic effect of U50488H, a k-receptor selective agonist, was attenuated by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. U50488H; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 U50488H. (c) Effect of nociceptin / OFQ (i.c.v.) on DSLET analgesia in formalin test. DSLET (a d-receptor selective agonist) induced analgesia was not affected by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. DSLET; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 DSLET.
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3. Results
mg injection caused a strong inhibitory effect (P , 0.01 vs. NS), the effect being dose dependent (P , 0.05, Fig. 3).
3.1. Effect of nociceptin /OFQ on formalin-induced pain response As reported previously [12,13], formalin induced a twophase response. Phase 1, which appeared immediately after the injection of formalin and lasted 5–10 min, was due to the direct effect of formalin; phase 2, which appeared about 20 min after the injection, was related to the subsequent development of inflammation. Compared with the NS control group, 0.1 or 0.5 mg nociceptin / OFQ had no effect on pain response in either phase (P . 0.05), and 1 mg nociceptin / OFQ potentiated the pain response in phase 2 (P , 0.01) but not phase 1 (P . 0.05, Fig. 1).
3.2. Effect of nociceptin /OFQ on opioid analgesia EM-1 (2 mg), U50488H (50 mg) or DSLET (5 mg), injected by i.c.v. into the rat brain, displayed analgesic effect on formalin-induced pain response in both phases (P , 0.01 vs. NS, Fig. 2). When the drug cocktail containing nociceptin / OFQ (0.1 or 1 mg) and one of the above opioid receptor agonists was injected into the rat brain, the analgesic effect of EM-1 or U50488H was significantly antagonized (P , 0.01 vs. EM-1 or U50488H, Fig. 2a and b), but the effect of DSLET remained unchanged (P . 0.05, Fig. 2c).
3.3. Effect of nociceptin /OFQ ( i.t.) on formalin-induced pain responsiveness In contrast with the above results, i.t. administration of 0.1 or 1 mg nociceptin / OFQ produced a significant analgesic effect in both phases of the formalin test, and 5
4. Discussion In the present study, we compared the effect of nociceptin / OFQ administered intracerebroventricularly and intrathecally on formalin-induced pain response, and found that the effect of nociceptin / OFQ in the brain was opposite to that in the spinal cord. In the brain nociceptin / OFQ potentiated pain response, which was consistent with our previous result [12]; while in the spinal cord, nociceptin / OFQ obviously inhibited formalin-induced pain response. Different mechanisms may contribute to the distinct effect of nociceptin / OFQ in the brain and spinal cord. Being a noxious stimulus, formalin could activate the endogenous pain controlling system, especially opioid– peptidergic system in the brain [14,15]. In this study, we observed that 1 mg nociceptin / OFQ potentiated formalininduced pain response and 0.1 mg nociceptin / OFQ had no effect on it. While nociceptin / OFQ (0.1 or 1 mg) was used with m-, d- or k-receptor agonist, it could reverse the analgesic effect of the m- or k- but not the d-receptor agonist. Therefore, it was conjectured that nociceptin / OFQ potentiated formalin-induced pain response by antagonizing the action of m- or k- opioid receptor. In this study, we did not find an interaction between nociceptin / OFQ and d-receptor, this might indicate that the hyperalgesic role of nociceptin / OFQ was not related to the d-opioid receptor in the formalin test. Mogil et al. observed that nociceptin / OFQ could not only reverse the analgesia effect of m- and k- receptor agonists, but also reverse the effect of the d-receptor agonist DPDPE on the tail-flick test in mice [8], the result being different from ours. Animal species, pain
Fig. 3. Effect of nociceptin / OFQ (i.t.) on formalin-induced pain response. Contrary to that in the brain, nociceptin / OFQ injected into spinal cord produced a significant inhibition on formalin pain response. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. 5 mg OFQ; e P , 0.05, f P , 0.01 vs. 1 mg OFQ.
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model, different drug and dose may be the reasons for the discrepancy. On the contrary, in this study, we observed that i.t. injection of nociceptin / OFQ attenuated the pain response in a dose-dependent manner. Similarly, Erb et al. also found the analgesic effect of nociceptin / OFQ at spinal level using larger doses [16]. It was reported that nociceptin / OFQ inhibited the release of SP and CGRP from sensory nerve terminals [17], depressed the glutamatergic transmission in the spinal cord [18], and inhibited excitatory synaptic transmission in the superficial layers of the dorsal horn [19]. These data suggested that the analgesic effect of nociceptin / OFQ was from the inhibition of the pain transmission. The effect of nociceptin / OFQ in the spinal cord was not reversed by naloxone or antagonists of the a 2 -adrenergic and GABA receptor [20]. In the rat formalin test, there are two phases of response [13], i.c.v. injection of nociceptin / OFQ enhanced the formalin-induced pain response only in phase 2 but not in phase 1. The results indicates that nociceptin / OFQ might play a more important role in formalin induced inflammation (phase 2) than in direct formalin stimulation (phase 1), or, nociceptin / OFQ needs a long latency (5–10 min after i.c.v. injection) to exert its action. The latter further suggests that the hyperalgesic effect of nociceptin / OFQ is not a direct action but through antagonizing the effect of opioid peptides. Injection by i.t. of nociceptin / OFQ reduced the formalin-induced pain response in both phases, similar to the effect of m-, k-, or d-receptor agonists, implying that spinally delivered nociceptin / OFQ might produce analgesic effect directly through its receptor [20]. Taken together, nociceptin / OFQ may act as a neuromodulator in the central nervous system, and plays distinct roles in different regions.
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
Acknowledgements [16]
This study was supported by grants from the National Natural Science Foundation of China (39670901) and Scientific and Technological Foundation of Shanghai (97QB14018).
[17]
[18]
References [19] [1] Henderson G, Mcknight AT. The orphan opioid receptor and its endogenous ligand – nociceptin / orphanin FQ. Trends Pharmacol Sci 1997;18:293–300. [2] Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, Mazargull H, Vassart G, Parmentier M, Costentin J. Isolation and
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structure of the endogenous agonist of opioid receptor-like ORLl receptor. Nature 1995;377:532–5. Reinscheid RK, Nothacker HP, Bourson A, Ardait AA, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Civelli O. Orphanin FQ: a neuropeptide that activates an opioid like G proteincoupled receptor. Science 1995;270:792–4. Riedl M, Shuster S, Vulchanova L, Wang JL, Loh HH, Elde R. Orphanin FQ / nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord. NeuroReport 1996;7:1369–72. Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a m-, d- or k-opioid receptor type. FEBS Lett 1994;347:284–8. Champion HC, Czapla MA, Kadowitz PJ. Nociceptin, an endogenous ligand for the ORL1 receptor, decrease cardiac output and total peripheral resistance in rat. Peptides 1997;18:729–32. Giuliani S, Tramontana M, Lecci A, Maggi CA. Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur J Pharmacol 1997;333:177–9. Mogil JS, Grisel JE, Zhangs G, Belknap JK, Grandy Dk. Functional antagonist of m-, d- and k- opioid antinociception by orphanin FQ. Neurosci Lett 1996;214:131–4. Rossi GC, Leventhal L, Pasternak GW. Naloxone sensitive orphanin FQ-induced analgesia in mice. Eur J Pharmacol 1996;311:R7–8. Zimmermann M. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 1983;16:109–10. Zadina JE, Hackler L, Ge LJ, Kastin AJ. A potent and selective endogenous agonist for the m-opioid receptor. Nature 1997;386:499–502. Zhu CB, Cao XD, Xu SF, Wu GC. Orphanin FQ potentiates formalin-induced pain behavior and antagonizes morphine analgesia in rats. Neurosci Lett 1997;235:37–40. Dubuisson D, Dennis SG. The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 1977;4:161–74. Du JH, He LF. Alteration of spinal dorsal horn substance P following electroacupuncture analgesia – a study of the formalin test with immunohistochemistry and densitometry. Acupunc ElectroTherapeut Res Int J 1992;17:1–6. Sugimoto M, Kuraishi Y, Satoh M, Takagi H. Involvement of medullary opioid–peptidergic and spinal systems in the regulation of formalin-induced persistent pain. Neuropharmacology 1986;25:481– 5. Erb K, Liebel JT, Tegeder I, Zeilhofer HU, Brune K, Geisslinger G. Spinally delivered nociceptin / orphanin FQ reduces flinching behavior in the rat formalin test. NeuroReport 1997;8:1967–70. Helyes Z, Nemeth J, Pinter E, Szolcsanyi J. Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals. Br J Pharmacol 1997;121:613–5. Faber ESL, Chambers JP, Evans RH, Henderson G. Depression of glutamatergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro. Br J Pharmacol 1996;119:189–90. Liebel JT, Swandulla D, Zeilhofer HU. Modulation of excitatory synaptic transmission by nociceptin in superficial dorsal horn neurons of the neonatal rat spinal cord. Br J Pharmacol 1997;121:425–32. Xu XJ, Hao JX, Wiesenfeld-Hallin Z. Nociceptin or antinociceptin: potent spinal antinociceptive effect of orphanin FQ / nociceptin in the rat. NeuroReport 1996;7:2092–4.
Distinct effect of intracerebroventricular and intrathecal injections of nociceptin / orphanin FQ in the rat formalin test Jin-Lan Wang, Chong-Bin Zhu, Xiao-Ding Cao, Gen-Cheng Wu* State Key Laboratory of Medical Neurobiology, Department of Neurobiology, Shanghai Medical University, 138 Yi Xue Yuan Road, Shanghai 200032, People’ s Republic of China Received 3 June 1998; received in revised form 10 November 1998; accepted 30 November 1998
Abstract Nociceptin / orphanin FQ (nociceptin / OFQ), a newly discovered heptadecapeptide has been regarded as an endogenous ligand for orphan opioid receptor. The present study was designed to investigate the effect of nociceptin / OFQ on pain response and opioid analgesia in the rat formalin test. The results showed that intracerebroventricular injection of 1 mg nociceptin / OFQ enhanced the pain response, and 0.1 or 0.5 mg nociceptin / OFQ had no effect on formalin-induced pain. When 0.1 or 1 mg nociceptin / OFQ were used together with m-, d-, or k-opioid receptor agonists, endomorphin-1, DSLET or U50488H, respectively, it attenuated m- and k- but not d-receptor mediated analgesia. On the other hand, intrathecal injection of nociceptin / OFQ (0.1, 1 and 5 mg) reduced the pain response in the formalin test. In conclusion, nociceptin / OFQ potentiated formalin-induced pain response and antagonized opioid analgesia in the rat brain but inhibited pain response in the spinal cord. 1999 Elsevier Science B.V. All rights reserved. Keywords: Nociceptin / orphanin FQ; Formalin test; Opioid analgesia
1. Introduction Nociceptin / orphanin FQ (nociceptin / OFQ) is a recently discovered peptide, which has been identified as an endogenous ligand of orphan opioid receptor LC132 or ORL1. The discovery of nociceptin / OFQ has resulted in a series of researches with regard to its structure, distribution and physiological functions [1]. This 17-amino acid peptide (FGGFTGARKSARKLANQ) shares sequence homology with other endogenous opioid peptides (especially dynorphin A), but possesses distinct pharmacological characteristics [2,3]. The distribution of nociceptin / OFQ and its receptor is parallel to the endogenous opioid peptides and their receptors [4,5], indicating that there is a close relationship between nociceptin / OFQ and the opioid
*Corresponding author. Tel.: 1 86 21 64041900, ext 2398; fax: 1 86 21 64174579; e-mail: [email protected]
peptidergic system. Just like opioid peptides, nociceptin / OFQ also has a great number of peripheral actions. For example, nociceptin / OFQ can decrease cardiac output, total peripheral resistance [6], and heart rate or blood pressure in the rat [7]. As far as the role of pain perception / modulation is concerned, it becomes one of the most interesting issues. The initial report claimed that central nociceptin / OFQ injection produced an increase in nociceptive responsiveness in mice [3], which was distinct to the effect of opioid peptides. But following researchers failed to see any hyperalgesic effect of nociceptin / OFQ but an anti-opioid effect [8], and the another group reported that nociceptin / OFQ produced hyperalgesia with delayed analgesia when given supraspinally [9]. So far, the results concerning the role of nociceptin / OFQ in pain modulation were in conflict, and most of the experiments were conducted in a variety of thermal or non-thermal pain tests. The purpose of this study was to further explore the effect of nociceptin / OFQ in the central nervous system on
0167-0115 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00161-X
160
J.-L. Wang et al. / Regulatory Peptides 79 (1999) 159 – 163
pain modulation using rat formalin test, so as to get a better understanding of the role of nociceptin / OFQ.
2. Materials and methods
rats’ skulls stereotaxically (P 1 mm, R 2 mm, H 4 mm). The experiment with i.c.v. injection was started 3–4 days after the operation. Drugs were dissolved in sterilized normal saline (NS). The total injection solution 20 ml (10 ml drug solution followed by a saline flush of 10 ml) was pushed into the cerebroventricle smoothly within 1 min.
2.1. Animals Male Sprague–Dawley rats (180–230 g) were supplied by the Experimental Animal Center, Shanghai Medical University. The treatment of the animals conformed to the guidelines of the International Association for the Study of Pain [10].
2.2. Drugs Heptadecapeptide nociceptin / OFQ was synthesized and purified in Shanghai Institute of Biochemistry, Chinese Academy of Sciences (Applied Biosystems 430A peptide synthesizer and BOC–Gln–PAM–resin were used). The crude peptide was sequentially purified on columns of Sephadex G-10 and HPLC. Its amino acid composition was consistent with theoretical values). Endomorphin-1 (EM-1, a newly discovered 4-amino-acid peptide), a selective m-opioid receptor agonist [11], was obtained from the same institute. trans-(6)-3,4-Dichloro-N-methylN-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50488H), a k-receptor agonist, was purchased from Sigma (St. Louis, MO, USA), [D-Ser2]–leu–Enkephalin–Thr (DSLET), a d-receptor agonist, was the product of Peninsula Laboratories (USA).
2.4. Implantation of intrathecal ( i.t.) catheters Rats were anaesthetized with sodium pentobarbital (35 mg / kg, i.p.). PE-10 tube was implanted through the gap between the 2nd and 3rd or 3rd and 4th lumbar spines (L2–3 or L3–4) to the subarachnoid space. Experiments with i.t. injection was started 3–4 days after the operation. The drug preparation and injection volume were the same as for the i.c.v. injection.
2.5. Formalin test Experiments were performed in a quiet environment (24618C). After i.c.v. or i.t. injection of drugs, formalin (150 ml, 5%) was injected subcutaneously (s.c.) into the plantar region of the right hindpaw, and the rat was immediately placed into a transparent plastic enclosure (35 3 30 3 30 cm) for 60 min behavioral observation. Pain response was observed every 5 min successively for the first 30 min, and then at 5-min intervals for the remaining 30 min. Pain intensity scoring was calculated as reported previously [12,13].
2.3. Implantation of intracerebroventricular ( i.c.v.) cannulae
2.6. Data analysis
Under pentobarbital anesthesia (35 mg / kg, i.p.), a stainless steel cannula of 0.8 mm diameter was fixed on the
Data were expressed as mean6S.E. One-way ANOVA and Q-test were used to analyze group differences.
Fig. 1. Effect of nociceptin / OFQ (i.c.v.) on formalin-induced pain response. 0.1 or 0.5 mg nociceptin / OFQ had no effect on pain response, 1 mg nociceptin / OFQ significantly potentiated formalin pain. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. OFQ 1 mg.
J.-L. Wang et al. / Regulatory Peptides 79 (1999) 159 – 163
161
Fig. 2. (a) Effect of nociceptin / OFQ (i.c.v.) on endomorphin-1 (EM-1) analgesia in formalin test. EM-1, a m-receptor selective agonist, produced a profound inhibition on formalin-induced pain, which was antagonized by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. EM-1; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 EM-1. (b) Effect of nociceptin / OFQ (i.c.v.) on U50488H analgesia in formalin test. The analgesic effect of U50488H, a k-receptor selective agonist, was attenuated by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. U50488H; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 U50488H. (c) Effect of nociceptin / OFQ (i.c.v.) on DSLET analgesia in formalin test. DSLET (a d-receptor selective agonist) induced analgesia was not affected by nociceptin / OFQ. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. DSLET; e P , 0.05, f P , 0.01 vs. 1 mg OFQ 1 DSLET.
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3. Results
mg injection caused a strong inhibitory effect (P , 0.01 vs. NS), the effect being dose dependent (P , 0.05, Fig. 3).
3.1. Effect of nociceptin /OFQ on formalin-induced pain response As reported previously [12,13], formalin induced a twophase response. Phase 1, which appeared immediately after the injection of formalin and lasted 5–10 min, was due to the direct effect of formalin; phase 2, which appeared about 20 min after the injection, was related to the subsequent development of inflammation. Compared with the NS control group, 0.1 or 0.5 mg nociceptin / OFQ had no effect on pain response in either phase (P . 0.05), and 1 mg nociceptin / OFQ potentiated the pain response in phase 2 (P , 0.01) but not phase 1 (P . 0.05, Fig. 1).
3.2. Effect of nociceptin /OFQ on opioid analgesia EM-1 (2 mg), U50488H (50 mg) or DSLET (5 mg), injected by i.c.v. into the rat brain, displayed analgesic effect on formalin-induced pain response in both phases (P , 0.01 vs. NS, Fig. 2). When the drug cocktail containing nociceptin / OFQ (0.1 or 1 mg) and one of the above opioid receptor agonists was injected into the rat brain, the analgesic effect of EM-1 or U50488H was significantly antagonized (P , 0.01 vs. EM-1 or U50488H, Fig. 2a and b), but the effect of DSLET remained unchanged (P . 0.05, Fig. 2c).
3.3. Effect of nociceptin /OFQ ( i.t.) on formalin-induced pain responsiveness In contrast with the above results, i.t. administration of 0.1 or 1 mg nociceptin / OFQ produced a significant analgesic effect in both phases of the formalin test, and 5
4. Discussion In the present study, we compared the effect of nociceptin / OFQ administered intracerebroventricularly and intrathecally on formalin-induced pain response, and found that the effect of nociceptin / OFQ in the brain was opposite to that in the spinal cord. In the brain nociceptin / OFQ potentiated pain response, which was consistent with our previous result [12]; while in the spinal cord, nociceptin / OFQ obviously inhibited formalin-induced pain response. Different mechanisms may contribute to the distinct effect of nociceptin / OFQ in the brain and spinal cord. Being a noxious stimulus, formalin could activate the endogenous pain controlling system, especially opioid– peptidergic system in the brain [14,15]. In this study, we observed that 1 mg nociceptin / OFQ potentiated formalininduced pain response and 0.1 mg nociceptin / OFQ had no effect on it. While nociceptin / OFQ (0.1 or 1 mg) was used with m-, d- or k-receptor agonist, it could reverse the analgesic effect of the m- or k- but not the d-receptor agonist. Therefore, it was conjectured that nociceptin / OFQ potentiated formalin-induced pain response by antagonizing the action of m- or k- opioid receptor. In this study, we did not find an interaction between nociceptin / OFQ and d-receptor, this might indicate that the hyperalgesic role of nociceptin / OFQ was not related to the d-opioid receptor in the formalin test. Mogil et al. observed that nociceptin / OFQ could not only reverse the analgesia effect of m- and k- receptor agonists, but also reverse the effect of the d-receptor agonist DPDPE on the tail-flick test in mice [8], the result being different from ours. Animal species, pain
Fig. 3. Effect of nociceptin / OFQ (i.t.) on formalin-induced pain response. Contrary to that in the brain, nociceptin / OFQ injected into spinal cord produced a significant inhibition on formalin pain response. a P , 0.05, b P , 0.01 vs. NS; c P , 0.05, d P , 0.01 vs. 5 mg OFQ; e P , 0.05, f P , 0.01 vs. 1 mg OFQ.
J.-L. Wang et al. / Regulatory Peptides 79 (1999) 159 – 163
model, different drug and dose may be the reasons for the discrepancy. On the contrary, in this study, we observed that i.t. injection of nociceptin / OFQ attenuated the pain response in a dose-dependent manner. Similarly, Erb et al. also found the analgesic effect of nociceptin / OFQ at spinal level using larger doses [16]. It was reported that nociceptin / OFQ inhibited the release of SP and CGRP from sensory nerve terminals [17], depressed the glutamatergic transmission in the spinal cord [18], and inhibited excitatory synaptic transmission in the superficial layers of the dorsal horn [19]. These data suggested that the analgesic effect of nociceptin / OFQ was from the inhibition of the pain transmission. The effect of nociceptin / OFQ in the spinal cord was not reversed by naloxone or antagonists of the a 2 -adrenergic and GABA receptor [20]. In the rat formalin test, there are two phases of response [13], i.c.v. injection of nociceptin / OFQ enhanced the formalin-induced pain response only in phase 2 but not in phase 1. The results indicates that nociceptin / OFQ might play a more important role in formalin induced inflammation (phase 2) than in direct formalin stimulation (phase 1), or, nociceptin / OFQ needs a long latency (5–10 min after i.c.v. injection) to exert its action. The latter further suggests that the hyperalgesic effect of nociceptin / OFQ is not a direct action but through antagonizing the effect of opioid peptides. Injection by i.t. of nociceptin / OFQ reduced the formalin-induced pain response in both phases, similar to the effect of m-, k-, or d-receptor agonists, implying that spinally delivered nociceptin / OFQ might produce analgesic effect directly through its receptor [20]. Taken together, nociceptin / OFQ may act as a neuromodulator in the central nervous system, and plays distinct roles in different regions.
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This study was supported by grants from the National Natural Science Foundation of China (39670901) and Scientific and Technological Foundation of Shanghai (97QB14018).
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