- Email: [email protected]
orphanin FQ peptide receptor in respiratory rhythm generation in the medulla oblongata: an in vitro study
British Journal of Anaesthesia 91 (3): 385±9 (2003)
DOI: 10.1093/bja/aeg191
Roles of nociceptin/orphanin FQ and nociceptin/orphanin FQ peptide receptor in respiratory rhythm generation in the medulla oblongata: an in vitro study K. Takita*, Y. Morimoto and O. Kemmotsu Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan *Corresponding author. E-mail: [email protected]
Methods. The brainstem-spinal cord from 3-day-old Wistar rats was isolated and perfused with arti®cial cerebrospinal ¯uid (27.5°C) equilibrated with oxygen 95% and carbon dioxide 5% at pH 7.4. Respiratory activity was recorded from the C4/C5 ventral roots. The effects of N/OFQ (10 nM, 30 nM, 100 nM) on respiratory frequency (fR) (bursts min±1) was measured. Drugs were administered through the recording chamber by means of a perfusion system. In addition, the effects of pretreatment with the classical non-selective opioid receptor antagonist naloxone 1 mM, and the selective NOP antagonist CompB 10 mM, were evaluated. Statistical signi®cance was evaluated using ANOVA followed by Dunnett's test (P<0.05). Results. N/OFQ reduced fR in a concentration-dependent manner. Pretreatment with CompB 10 mM prevented the N/OFQ 10 nM-induced fR reduction, whereas CompB itself was inactive. Pretreatment with naloxone did not prevent the N/OFQ-induced fR reduction. Conclusion. N/OFQ acts as a neuromodulator to reduce fR in the respiratory rhythmgenerating centre of the medulla oblongata, and this action of N/OFQ is mediated by NOP receptors. Br J Anaesth 2003; 91: 385±9 Keywords: metabolism, respiratory rhythm; pharmacology, agonists opioid, nociceptin/ orphanin FQ; receptors, opioid; spinal chord, brainstem Accepted for publication: April 8, 2003
In addition to the three classical opioid receptors (m, d, and k) a new member of the opioid receptor family, nociceptin/ orphanin FQ (N/OFQ) peptide (NOP) receptor (previously termed ORL1 receptor), was cloned in 1994.1 2 Although structurally related to other members of the opioid receptor family, this receptor was found to bind to classical m-, d-, and k-opioid ligands with very low af®nity. In 1995, N/OFQ was isolated and identi®ed as the endogenous ligand for the NOP receptor.3 4 NOP receptor and N/OFQ are involved in many physiological functions.5 Whilst abundance of the NOP receptor in the medulla oblongata has been noted,6 7 the roles of the NOP receptor and N/OFQ speci®cally in the
respiratory centre of the medulla oblongata have not yet been described. In vitro brainstem-spinal cord preparation from newborn rat has been introduced as an in vitro model for the study of the mammalian respiratory centre.8 Cumulative evidence has shown that this model is well suited to the analysis of primary respiratory rhythm generation and modulation.8±10 A considerable number of studies examining the effects of neuroactive substances on the respiratory centre have been conducted using this in vitro preparation10 for which there are several advantages. Most notably, because this preparation can be maintained in an anaesthetic-free condition, the
Ó The Board of Management and Trustees of the British Journal of Anaesthesia 2003
Downloaded from http://bja.oxfordjournals.org/ by guest on March 31, 2015
Background. Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the orphan opioid receptor-like receptor (NOP receptor, previously termed ORL1), a novel member of the opioid receptor family. The aim of the present study, using in vitro newborn rat preparations, was to elucidate the roles N/OFQ and the NOP receptor play in medullary generation of respiratory rhythm.
Takita et al.
direct effects of neuroactive substances and drugs on the structures that generate basal respiratory rhythm can be observed. In addition, this environment precludes the in¯uence of peripheral chemosensors and suprabulbar structures. The aim of the present study was, using the above model, to elucidate the roles of N/OFQ and the NOP receptor in respiratory rhythm generation in the medulla oblongata.
Methods This study was approved by the Animal Care Committee of the Hokkaido University Graduate School of Medicine. The head and upper thorax of Wistar rats (3 days old, n=48) were dissected under deep ether anaesthesia, and the brainstem and spinal cord were isolated. The brainstem was rostrally decerebrated between the VIth cranial nerve roots and the lower border of the trapezoid body. The preparation was placed in a small chamber (volume 2.5 ml) and continuously perfused at a rate of 5.0±6.0 ml min±1 with arti®cial cerebrospinal ¯uid (aCSF) (mM): NaCl, 124; KCl, 5.0; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 30. The solution was equilibrated with oxygen 95% and carbon dioxide 5% to pH 7.4 at 27.5°C. The reservoir containing the aCSF, the perfusion system, and the
recording chamber was heated using a water bath to 27.5 (SD 0.3)°C. The pH and temperature of the aCSF in the reservoir, and the temperature of the superfusate in the recording chamber, were continuously monitored. Respiratory activity corresponding to inspiration was monitored at the C4 or C5 ventral root using suction electrodes; this activity is known to synchronize with phrenic nerve discharges and with contraction of the inspiratory intercostal muscles.8 10 Recording signals were ampli®ed and band-pass ®ltered (50 Hz to 3 kHz; Fig. 1). More than 20 min after the recording of respiratory activity was established, drugs were administered through the recording chamber via the perfusion system. N/OFQ 10 nM, 30 nM, or 100 nM was applied for 20 min with each preparation being exposed once to a single concentration of N/OFQ. In the ®ve preparations used as non-treated controls, no drug was applied. Naloxone 1 mM, the putative NOP antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol2-one (CompB) 10 mM or [Phe1y (CH2-NH)Gly2]-N/OFQ (1±13)-NH2 1±3 mM were applied 20 min before coapplication with N/OFQ 10 nM for a further 20 min. In some experiments, after 20 min exposure to N/OFQ 10 nM, the combination of N/OFQ and CompB 10 mM was applied. N/ OFQ, naloxone hydrochloride, and [Phe1y(CH2-NH)Gly2]-
386
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Fig 1 Schematic diagram of the newborn rat brainstem-spinal cord preparation ventral surface. Respiratory motoneuron activity corresponding to inspiration was recorded from the C4/C5 ventral root through a glass suction electrode. AICA=anterior inferior cerebellar artery; IX±XII=cranial nerves; and C1±C4=cervical ventral nerves.
N/OFQ and the respiratory centre
nociceptin (1±13)-NH2 were obtained from Sigma (St Louis, MO, USA). CompB was a generous gift from Banyu Pharmaceutical Company (Tokyo, Japan). Respiratory frequency (fR) was measured with ®ring frequency of C4 activity regarded as fR. Average values were calculated from the number of bursts recorded over a 3 min period. All data are presented as mean (SD). Statistical Table 1 Effects of nociceptin/orphanin FQ (N/OFQ) on fR. Values are means (SD). *P<0.05, **P<0.01 vs baseline; ²P<0.01 vs control Concentration (nM)
0 (control) 10
30
100
n 5 5 6 4 fR (bursts min±1) Baseline 9.7 (1.6) 10.3 (2.8) 10.0 (1.5) 10.4 (2.9) 20 min after N/OFQ 9.4 (0.5) 5.6 (0.5)* 3.5 (1.9)** 1.0 (0.4)** % of baseline value 98.3 (12.2) 59.3 (16.3)² 36.5 (23.3)² 8.4 (2.5)²
Pretreatment Naloxone 1 mM CompB 10 mM Phe1Y 1 mM 3 mM
n 5 6 4 4
fR (bursts min±1) Baseline 8.7 (2.1) Baseline 10.9 (0.8) Baseline 10.5 (2.1) 8.4 (1.7)
Naloxone 8.4 (2.7) CompB 11.4 (0.6) Phe1Y 10.3 (1.7) 5.4 (1.5)§
Naloxone + N/OFQ 4.3 (1.5)* CompB + N/OFQ 10.9 (1.9) Phe1Y + N/OFQ 5.5 (2.9)² 3.8 (1.8)§
Results N/OFQ reduced fR in a concentration-dependent manner (Table 1). The effect of N/OFQ lasted 30±60 min after peptide removal. In the preparation in which the dorsal half of the brainstem was removed (n=4), N/OFQ 10 nM also caused an fR reduction [8.3 (2.5) bursts min±1 to 4.3 (3.0) bursts min±1, P<0.05]. The effects of pretreatment with naloxone, CompB and [Phe1y(CH2-NH)Gly2]-N/OFQ (1± 13)-NH2, on the actions of N/OFQ are shown in Table 2. Naloxone 1 mM pretreatment did not prevent N/OFQ 10 nMinduced fR reduction. CompB 10 mM pretreatment prevented N/OFQ 10 nM-induced fR reduction, and had no signi®cant effects on fR alone. CompB 10 mM also reversed N/OFQ 10 nM-induced fR reduction [6.6 (2.7) bursts min±1 to 9.8 (2.3) bursts min±1; P<0.01, n=5; Fig. 2]. [Phe1y(CH2NH)Gly2]-N/OFQ (1±13)-NH2 1 mM pretreatment did not prevent N/OFQ 10 nM-induced fR reduction, but higher concentrations of [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)NH2, 3 mM caused fR reduction per se.
Discussion The current study demonstrated that N/OFQ has an inhibitory effect on the medullary structures involved in
Fig 2 (A) C4 activity recorded from the in vitro preparation in standard solution. (B) 20 min after perfusion with nociceptin/orphanin FQ 10 nM. (C) 15 min after perfusion with nociceptin/orphanin FQ 10 nM and CompB 10 mM.
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Table 2 Effect of pretreatment with naloxone, CompB and [Phe1Y (CH2NH) Gly2]±N/OFQ (1±13)±NH2 (Phe1Y) on the action of nociceptin/ orphanin FQ (N/OFQ, 10 nM). Values are means (SD). *P<0.05 vs baseline, naloxone; ²P<0.05 vs baseline, Phe1Y; §P<0.05 vs baseline
signi®cance was evaluated using the Student's t-test and analysis of variance (ANOVA) followed by Dunnett's test. A value of P<0.05 was considered statistically signi®cant.
Takita et al.
to high density of NOP receptors in the respiratory-related regions of the rostral ventrolateral medulla.6 7 These ®ndings suggest that N/OFQ-induced fR reduction observed here may result from activation of these NOP receptors. We conclude that N/OFQ acts as a neuromodulator to reduce respiratory frequency in the respiratory rhythmgeneration centre of the medulla oblongata, and that this action of N/OFQ is mediated by NOP receptors in the ventral portion of this structure.
Acknowledgements We appreciate the generous supply of CompB given by Banyu Pharmaceutical Company (Tokyo, Japan). We also thank Professor Sten G. E. Lindahl, Department of Anesthesiology and Intensive Care, Karolinska Hospital and Karolinska Institutet (Stockholm, Sweden), for reviewing this manuscript. This work was supported by Grant 13671557 for Scienti®c Research from the Ministry of Education, Science and Culture, Japan.
References
388
1 Bunzow JR, Saez C, Mortrud M, et al. 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 2 Fukuda K, Kato S, Mori K, et al. cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett 1994; 343: 42±6 3 Meunier JC, Mollereau C, Toll L, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 1995; 377: 532±5 4 Reinscheid RK, Nothacker H-P, Bourson A, et al. Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor. Science 1995; 270: 792±4 5 Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand-nociceptin/orphanin FQ. Trends Pharmacol Sci 1997; 18: 293±300 6 Neal CR, Mansour A, Reinscheid R, et al. Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with 125I[14Try]-orphanin FQ binding. J Comp Neurol 1999; 412: 563±605 7 Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol 1996; 368: 229±51 8 Suzue T. Respiratory rhythm generation in the in vitro brain stemspinal cord preparation of the neonatal rat. J Physiol 1984; 354: 173±83 9 Smith JC, Greer JJ, Liu G, Feldman JL. Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J Neurophysiol 1990; 64: 1149±69 10 Ballanyi K, Onimaru H, Homma I. Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog Neurobiol 1999; 59: 583±634 11 Rossi GC, Perlmutter M, Leventhal L, Talatti A, Pasternak GW. Orphanin FQ/nociceptin analgesia in the rat. Brain Res 1998; 792: 327±30 È gren SO, Terenius L. 12 Sandin J, Georgieva J, SchoÈtt PA, O Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci 1997; 9: 194±7 13 Mao L, Wang JQ. Microinjection of nociceptin (Orphanin FQ) into nucleus tractus solitarii elevates blood pressure and heart
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the generation of respiratory rhythm via activation of the NOP receptor, a new member of the opioid receptor family. Since N/OFQ was discovered in 1995, many studies have been performed to elucidate its actions on in vivo activity when injected intracerebroventricularly or microinjected into restricted areas of the central nervous system, of animals including rats and mice. Those studies showed that N/OFQ has many physiological and pharmacological actions in vivo, including nociception; analgesia; effects on locomotion, learning, cardiovascular control and inhibition of cough.3 4 11±14 However, there has been no report of respiratory effects of N/OFQ. This discrepancy between the results of the present in vitro study and those of previous in vivo studies suggests that the effects of N/OFQ on suprabulbar structures may modify medullary NOP receptor-mediated respiratory depressant effect. CompB is reported to be a potent and selective NOP receptor antagonist.15 The results of the current series of experiments using CompB show that N/OFQ in the medullary structures is able to modulate respiratory rhythm and this effect is mediated by NOP receptors. The present study also showed that CompB had no effect on fR generation per se indicating that endogenous N/OFQ does not participate in basal respiratory rhythm generation in the medulla oblongata. [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)NH2 was introduced as a competitive antagonist of the NOP receptor in guinea-pig ileum and mouse vas deferens preparations.16 However, other studies have shown that this peptide acts as an agonist or a partial agonist for the NOP receptor in vitro and in vivo,17±19 although the reason for the differences in its pharmacodynamic pro®le is unclear. The diversity of this agent's actions on the NOP receptor limits its utility as a pharmacological tool for manipulating the NOP receptor. The present study showed that [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)-NH2 acted mainly as an agonist for the NOP receptor. In the present study, pretreatment with naloxone did not affect the N/OFQ-induced fR reduction, whereas ®ndings in our previous study showed that naloxone can completely reverse the fR reduction produced by m-, d-, and k-opioid receptor agonists in this preparation.20 These ®ndings indicate that conventional opioid receptors are not involved in the action of N/OFQ in the medullary respiration-related structures. The present study indicated that the ventral portion of the medulla oblongata is important in N/OFQ-induced fR reduction, given that removal of the dorsal half of the medulla did not cause any changes in the response to N/OFQ application. Previous studies have suggested that the centre or kernel of respiratory rhythm generation may be located in the pre-BoÈtzinger complex, a limited region of the rostroventrolateral medulla that corresponds to the nucleus reticularis rostroventrolateralis.10 21 22 The mechanism of respiratory rhythmogenesis is not yet fully understood. Studies using in situ hybridization and immunohistochemistry show high NOP mRNA expression and moderate
N/OFQ and the respiratory centre
14 15
16 17 18
rate in both anesthetized and conscious rats. J Pharmacol Exp Ther 2000; 294: 255±62 McLeod RL, Parra LE, Mutter JC, et al. Nociceptin inhibits cough in the guinea-pig by activation of ORL1 receptors. Br J Pharmacolol 2001; 132: 1175±8 Ozaki S, Kawamoto H, Itoh Y, et al. In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur J Pharmacol 2000; 402: 45±53 Guerrini R, Calo G, Rizzi A, et al. A new selective antagonist of the nociceptin receptor. Br J Pharmacolol 1998; 123: 163±5 Candeletti S, Guerrini R, Calo G, Romualdi P, Ferri S. Supraspinal and spinal effects of [Phe1y(CH2-NH)Gly2]-nociceptin (1±13)NH2 on nociception in the rat. Life Sci 2000; 66: 257±64 Chiou LC. [Phe1y(CH2-NH)Gly2]-nociceptin (1±13)-NH2
19
20 21 22
activation of an inward recti®er as a partial agonist of ORL1 receptors in rat periaqueductal gray. Br J Pharmocol 1999; 128: 103±7 Menzies JRW, Glen T, Davies MRP, Paterson SJ, Corbett AD. In vitro agonist effects of nociceptin and [Phe1y(CH2-NH)Gly2]nociceptin (1±13)-NH2 in the mouse and rat colon and mouse vas deferens. Eur J Pharmacol 1999; 385: 217±23 Takita K, Herlenius EAP, Lindahl SGE, Yamamoto Y. Actions of opioids on respiratory activity via activation of brainstem m-, dand k-receptors; an in vitro study. Brain Res 1997; 778: 233±41 Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL. Pre-BoÈtzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 1991; 254: 726±9 Koshiya N, Smith JC. Neuronal pacemaker for breathing visualized in vitro. Nature 1999; 400: 360±3
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389
DOI: 10.1093/bja/aeg191
Roles of nociceptin/orphanin FQ and nociceptin/orphanin FQ peptide receptor in respiratory rhythm generation in the medulla oblongata: an in vitro study K. Takita*, Y. Morimoto and O. Kemmotsu Department of Anesthesiology and Critical Care Medicine, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan *Corresponding author. E-mail: [email protected]
Methods. The brainstem-spinal cord from 3-day-old Wistar rats was isolated and perfused with arti®cial cerebrospinal ¯uid (27.5°C) equilibrated with oxygen 95% and carbon dioxide 5% at pH 7.4. Respiratory activity was recorded from the C4/C5 ventral roots. The effects of N/OFQ (10 nM, 30 nM, 100 nM) on respiratory frequency (fR) (bursts min±1) was measured. Drugs were administered through the recording chamber by means of a perfusion system. In addition, the effects of pretreatment with the classical non-selective opioid receptor antagonist naloxone 1 mM, and the selective NOP antagonist CompB 10 mM, were evaluated. Statistical signi®cance was evaluated using ANOVA followed by Dunnett's test (P<0.05). Results. N/OFQ reduced fR in a concentration-dependent manner. Pretreatment with CompB 10 mM prevented the N/OFQ 10 nM-induced fR reduction, whereas CompB itself was inactive. Pretreatment with naloxone did not prevent the N/OFQ-induced fR reduction. Conclusion. N/OFQ acts as a neuromodulator to reduce fR in the respiratory rhythmgenerating centre of the medulla oblongata, and this action of N/OFQ is mediated by NOP receptors. Br J Anaesth 2003; 91: 385±9 Keywords: metabolism, respiratory rhythm; pharmacology, agonists opioid, nociceptin/ orphanin FQ; receptors, opioid; spinal chord, brainstem Accepted for publication: April 8, 2003
In addition to the three classical opioid receptors (m, d, and k) a new member of the opioid receptor family, nociceptin/ orphanin FQ (N/OFQ) peptide (NOP) receptor (previously termed ORL1 receptor), was cloned in 1994.1 2 Although structurally related to other members of the opioid receptor family, this receptor was found to bind to classical m-, d-, and k-opioid ligands with very low af®nity. In 1995, N/OFQ was isolated and identi®ed as the endogenous ligand for the NOP receptor.3 4 NOP receptor and N/OFQ are involved in many physiological functions.5 Whilst abundance of the NOP receptor in the medulla oblongata has been noted,6 7 the roles of the NOP receptor and N/OFQ speci®cally in the
respiratory centre of the medulla oblongata have not yet been described. In vitro brainstem-spinal cord preparation from newborn rat has been introduced as an in vitro model for the study of the mammalian respiratory centre.8 Cumulative evidence has shown that this model is well suited to the analysis of primary respiratory rhythm generation and modulation.8±10 A considerable number of studies examining the effects of neuroactive substances on the respiratory centre have been conducted using this in vitro preparation10 for which there are several advantages. Most notably, because this preparation can be maintained in an anaesthetic-free condition, the
Ó The Board of Management and Trustees of the British Journal of Anaesthesia 2003
Downloaded from http://bja.oxfordjournals.org/ by guest on March 31, 2015
Background. Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the orphan opioid receptor-like receptor (NOP receptor, previously termed ORL1), a novel member of the opioid receptor family. The aim of the present study, using in vitro newborn rat preparations, was to elucidate the roles N/OFQ and the NOP receptor play in medullary generation of respiratory rhythm.
Takita et al.
direct effects of neuroactive substances and drugs on the structures that generate basal respiratory rhythm can be observed. In addition, this environment precludes the in¯uence of peripheral chemosensors and suprabulbar structures. The aim of the present study was, using the above model, to elucidate the roles of N/OFQ and the NOP receptor in respiratory rhythm generation in the medulla oblongata.
Methods This study was approved by the Animal Care Committee of the Hokkaido University Graduate School of Medicine. The head and upper thorax of Wistar rats (3 days old, n=48) were dissected under deep ether anaesthesia, and the brainstem and spinal cord were isolated. The brainstem was rostrally decerebrated between the VIth cranial nerve roots and the lower border of the trapezoid body. The preparation was placed in a small chamber (volume 2.5 ml) and continuously perfused at a rate of 5.0±6.0 ml min±1 with arti®cial cerebrospinal ¯uid (aCSF) (mM): NaCl, 124; KCl, 5.0; KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 30. The solution was equilibrated with oxygen 95% and carbon dioxide 5% to pH 7.4 at 27.5°C. The reservoir containing the aCSF, the perfusion system, and the
recording chamber was heated using a water bath to 27.5 (SD 0.3)°C. The pH and temperature of the aCSF in the reservoir, and the temperature of the superfusate in the recording chamber, were continuously monitored. Respiratory activity corresponding to inspiration was monitored at the C4 or C5 ventral root using suction electrodes; this activity is known to synchronize with phrenic nerve discharges and with contraction of the inspiratory intercostal muscles.8 10 Recording signals were ampli®ed and band-pass ®ltered (50 Hz to 3 kHz; Fig. 1). More than 20 min after the recording of respiratory activity was established, drugs were administered through the recording chamber via the perfusion system. N/OFQ 10 nM, 30 nM, or 100 nM was applied for 20 min with each preparation being exposed once to a single concentration of N/OFQ. In the ®ve preparations used as non-treated controls, no drug was applied. Naloxone 1 mM, the putative NOP antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol2-one (CompB) 10 mM or [Phe1y (CH2-NH)Gly2]-N/OFQ (1±13)-NH2 1±3 mM were applied 20 min before coapplication with N/OFQ 10 nM for a further 20 min. In some experiments, after 20 min exposure to N/OFQ 10 nM, the combination of N/OFQ and CompB 10 mM was applied. N/ OFQ, naloxone hydrochloride, and [Phe1y(CH2-NH)Gly2]-
386
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Fig 1 Schematic diagram of the newborn rat brainstem-spinal cord preparation ventral surface. Respiratory motoneuron activity corresponding to inspiration was recorded from the C4/C5 ventral root through a glass suction electrode. AICA=anterior inferior cerebellar artery; IX±XII=cranial nerves; and C1±C4=cervical ventral nerves.
N/OFQ and the respiratory centre
nociceptin (1±13)-NH2 were obtained from Sigma (St Louis, MO, USA). CompB was a generous gift from Banyu Pharmaceutical Company (Tokyo, Japan). Respiratory frequency (fR) was measured with ®ring frequency of C4 activity regarded as fR. Average values were calculated from the number of bursts recorded over a 3 min period. All data are presented as mean (SD). Statistical Table 1 Effects of nociceptin/orphanin FQ (N/OFQ) on fR. Values are means (SD). *P<0.05, **P<0.01 vs baseline; ²P<0.01 vs control Concentration (nM)
0 (control) 10
30
100
n 5 5 6 4 fR (bursts min±1) Baseline 9.7 (1.6) 10.3 (2.8) 10.0 (1.5) 10.4 (2.9) 20 min after N/OFQ 9.4 (0.5) 5.6 (0.5)* 3.5 (1.9)** 1.0 (0.4)** % of baseline value 98.3 (12.2) 59.3 (16.3)² 36.5 (23.3)² 8.4 (2.5)²
Pretreatment Naloxone 1 mM CompB 10 mM Phe1Y 1 mM 3 mM
n 5 6 4 4
fR (bursts min±1) Baseline 8.7 (2.1) Baseline 10.9 (0.8) Baseline 10.5 (2.1) 8.4 (1.7)
Naloxone 8.4 (2.7) CompB 11.4 (0.6) Phe1Y 10.3 (1.7) 5.4 (1.5)§
Naloxone + N/OFQ 4.3 (1.5)* CompB + N/OFQ 10.9 (1.9) Phe1Y + N/OFQ 5.5 (2.9)² 3.8 (1.8)§
Results N/OFQ reduced fR in a concentration-dependent manner (Table 1). The effect of N/OFQ lasted 30±60 min after peptide removal. In the preparation in which the dorsal half of the brainstem was removed (n=4), N/OFQ 10 nM also caused an fR reduction [8.3 (2.5) bursts min±1 to 4.3 (3.0) bursts min±1, P<0.05]. The effects of pretreatment with naloxone, CompB and [Phe1y(CH2-NH)Gly2]-N/OFQ (1± 13)-NH2, on the actions of N/OFQ are shown in Table 2. Naloxone 1 mM pretreatment did not prevent N/OFQ 10 nMinduced fR reduction. CompB 10 mM pretreatment prevented N/OFQ 10 nM-induced fR reduction, and had no signi®cant effects on fR alone. CompB 10 mM also reversed N/OFQ 10 nM-induced fR reduction [6.6 (2.7) bursts min±1 to 9.8 (2.3) bursts min±1; P<0.01, n=5; Fig. 2]. [Phe1y(CH2NH)Gly2]-N/OFQ (1±13)-NH2 1 mM pretreatment did not prevent N/OFQ 10 nM-induced fR reduction, but higher concentrations of [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)NH2, 3 mM caused fR reduction per se.
Discussion The current study demonstrated that N/OFQ has an inhibitory effect on the medullary structures involved in
Fig 2 (A) C4 activity recorded from the in vitro preparation in standard solution. (B) 20 min after perfusion with nociceptin/orphanin FQ 10 nM. (C) 15 min after perfusion with nociceptin/orphanin FQ 10 nM and CompB 10 mM.
387
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Table 2 Effect of pretreatment with naloxone, CompB and [Phe1Y (CH2NH) Gly2]±N/OFQ (1±13)±NH2 (Phe1Y) on the action of nociceptin/ orphanin FQ (N/OFQ, 10 nM). Values are means (SD). *P<0.05 vs baseline, naloxone; ²P<0.05 vs baseline, Phe1Y; §P<0.05 vs baseline
signi®cance was evaluated using the Student's t-test and analysis of variance (ANOVA) followed by Dunnett's test. A value of P<0.05 was considered statistically signi®cant.
Takita et al.
to high density of NOP receptors in the respiratory-related regions of the rostral ventrolateral medulla.6 7 These ®ndings suggest that N/OFQ-induced fR reduction observed here may result from activation of these NOP receptors. We conclude that N/OFQ acts as a neuromodulator to reduce respiratory frequency in the respiratory rhythmgeneration centre of the medulla oblongata, and that this action of N/OFQ is mediated by NOP receptors in the ventral portion of this structure.
Acknowledgements We appreciate the generous supply of CompB given by Banyu Pharmaceutical Company (Tokyo, Japan). We also thank Professor Sten G. E. Lindahl, Department of Anesthesiology and Intensive Care, Karolinska Hospital and Karolinska Institutet (Stockholm, Sweden), for reviewing this manuscript. This work was supported by Grant 13671557 for Scienti®c Research from the Ministry of Education, Science and Culture, Japan.
References
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1 Bunzow JR, Saez C, Mortrud M, et al. 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 2 Fukuda K, Kato S, Mori K, et al. cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett 1994; 343: 42±6 3 Meunier JC, Mollereau C, Toll L, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 1995; 377: 532±5 4 Reinscheid RK, Nothacker H-P, Bourson A, et al. Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor. Science 1995; 270: 792±4 5 Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand-nociceptin/orphanin FQ. Trends Pharmacol Sci 1997; 18: 293±300 6 Neal CR, Mansour A, Reinscheid R, et al. Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with 125I[14Try]-orphanin FQ binding. J Comp Neurol 1999; 412: 563±605 7 Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol 1996; 368: 229±51 8 Suzue T. Respiratory rhythm generation in the in vitro brain stemspinal cord preparation of the neonatal rat. J Physiol 1984; 354: 173±83 9 Smith JC, Greer JJ, Liu G, Feldman JL. Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J Neurophysiol 1990; 64: 1149±69 10 Ballanyi K, Onimaru H, Homma I. Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog Neurobiol 1999; 59: 583±634 11 Rossi GC, Perlmutter M, Leventhal L, Talatti A, Pasternak GW. Orphanin FQ/nociceptin analgesia in the rat. Brain Res 1998; 792: 327±30 È gren SO, Terenius L. 12 Sandin J, Georgieva J, SchoÈtt PA, O Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci 1997; 9: 194±7 13 Mao L, Wang JQ. Microinjection of nociceptin (Orphanin FQ) into nucleus tractus solitarii elevates blood pressure and heart
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the generation of respiratory rhythm via activation of the NOP receptor, a new member of the opioid receptor family. Since N/OFQ was discovered in 1995, many studies have been performed to elucidate its actions on in vivo activity when injected intracerebroventricularly or microinjected into restricted areas of the central nervous system, of animals including rats and mice. Those studies showed that N/OFQ has many physiological and pharmacological actions in vivo, including nociception; analgesia; effects on locomotion, learning, cardiovascular control and inhibition of cough.3 4 11±14 However, there has been no report of respiratory effects of N/OFQ. This discrepancy between the results of the present in vitro study and those of previous in vivo studies suggests that the effects of N/OFQ on suprabulbar structures may modify medullary NOP receptor-mediated respiratory depressant effect. CompB is reported to be a potent and selective NOP receptor antagonist.15 The results of the current series of experiments using CompB show that N/OFQ in the medullary structures is able to modulate respiratory rhythm and this effect is mediated by NOP receptors. The present study also showed that CompB had no effect on fR generation per se indicating that endogenous N/OFQ does not participate in basal respiratory rhythm generation in the medulla oblongata. [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)NH2 was introduced as a competitive antagonist of the NOP receptor in guinea-pig ileum and mouse vas deferens preparations.16 However, other studies have shown that this peptide acts as an agonist or a partial agonist for the NOP receptor in vitro and in vivo,17±19 although the reason for the differences in its pharmacodynamic pro®le is unclear. The diversity of this agent's actions on the NOP receptor limits its utility as a pharmacological tool for manipulating the NOP receptor. The present study showed that [Phe1y(CH2-NH)Gly2]-N/OFQ (1±13)-NH2 acted mainly as an agonist for the NOP receptor. In the present study, pretreatment with naloxone did not affect the N/OFQ-induced fR reduction, whereas ®ndings in our previous study showed that naloxone can completely reverse the fR reduction produced by m-, d-, and k-opioid receptor agonists in this preparation.20 These ®ndings indicate that conventional opioid receptors are not involved in the action of N/OFQ in the medullary respiration-related structures. The present study indicated that the ventral portion of the medulla oblongata is important in N/OFQ-induced fR reduction, given that removal of the dorsal half of the medulla did not cause any changes in the response to N/OFQ application. Previous studies have suggested that the centre or kernel of respiratory rhythm generation may be located in the pre-BoÈtzinger complex, a limited region of the rostroventrolateral medulla that corresponds to the nucleus reticularis rostroventrolateralis.10 21 22 The mechanism of respiratory rhythmogenesis is not yet fully understood. Studies using in situ hybridization and immunohistochemistry show high NOP mRNA expression and moderate
N/OFQ and the respiratory centre
14 15
16 17 18
rate in both anesthetized and conscious rats. J Pharmacol Exp Ther 2000; 294: 255±62 McLeod RL, Parra LE, Mutter JC, et al. Nociceptin inhibits cough in the guinea-pig by activation of ORL1 receptors. Br J Pharmacolol 2001; 132: 1175±8 Ozaki S, Kawamoto H, Itoh Y, et al. In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur J Pharmacol 2000; 402: 45±53 Guerrini R, Calo G, Rizzi A, et al. A new selective antagonist of the nociceptin receptor. Br J Pharmacolol 1998; 123: 163±5 Candeletti S, Guerrini R, Calo G, Romualdi P, Ferri S. Supraspinal and spinal effects of [Phe1y(CH2-NH)Gly2]-nociceptin (1±13)NH2 on nociception in the rat. Life Sci 2000; 66: 257±64 Chiou LC. [Phe1y(CH2-NH)Gly2]-nociceptin (1±13)-NH2
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activation of an inward recti®er as a partial agonist of ORL1 receptors in rat periaqueductal gray. Br J Pharmocol 1999; 128: 103±7 Menzies JRW, Glen T, Davies MRP, Paterson SJ, Corbett AD. In vitro agonist effects of nociceptin and [Phe1y(CH2-NH)Gly2]nociceptin (1±13)-NH2 in the mouse and rat colon and mouse vas deferens. Eur J Pharmacol 1999; 385: 217±23 Takita K, Herlenius EAP, Lindahl SGE, Yamamoto Y. Actions of opioids on respiratory activity via activation of brainstem m-, dand k-receptors; an in vitro study. Brain Res 1997; 778: 233±41 Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL. Pre-BoÈtzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 1991; 254: 726±9 Koshiya N, Smith JC. Neuronal pacemaker for breathing visualized in vitro. Nature 1999; 400: 360±3
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