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: ktakita@med.hokudai.ac.jp
Accepted for publication: April 8, 2003
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Abstract |
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Methods. The brainstem-spinal cord from 3-day-old Wistar rats was isolated and perfused with artificial cerebrospinal fluid (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 min1) 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 µM, and the selective NOP antagonist CompB 10 µM, were evaluated. Statistical significance was evaluated using ANOVA followed by Dunnetts test (P<0.05).
Results. N/OFQ reduced fR in a concentration-dependent manner. Pretreatment with CompB 10 µM 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 rhythm- generating centre of the medulla oblongata, and this action of N/OFQ is mediated by NOP receptors.
Br J Anaesth 2003; 91: 3859
Keywords: metabolism, respiratory rhythm; pharmacology, agonists opioid, nociceptin/orphanin FQ; receptors, opioid; spinal chord, brainstem
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Introduction |
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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.810 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 direct effects of neuroactive substances and drugs on the structures that generate basal respiratory rhythm can be observed. In addition, this environment precludes the influence 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.
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Methods |
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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 amplified and band-pass filtered (50 Hz to 3 kHz; Fig. 1).
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Respiratory frequency (fR) was measured with firing 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 significance was evaluated using the Students t-test and analysis of variance (ANOVA) followed by Dunnetts test. A value of P<0.05 was considered statistically significant.
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Results |
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Discussion |
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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 1114 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. [Phe1(CH2-NH)Gly2]-N/OFQ (113)-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,1719 although the reason for the differences in its pharmacodynamic profile is unclear. The diversity of this agents actions on the NOP receptor limits its utility as a pharmacological tool for manipulating the NOP receptor. The present study showed that [Phe1
(CH2-NH)Gly2]-N/OFQ (113)-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 findings in our previous study showed that naloxone can completely reverse the fR reduction produced by µ-,
-, and
-opioid receptor agonists in this preparation.20 These findings 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-Bötzinger 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 to high density of NOP receptors in the respiratory-related regions of the rostral ventrolateral medulla.6 7 These findings 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 rhythm-generation centre of the medulla oblongata, and that this action of N/OFQ is mediated by NOP receptors in the ventral portion of this structure.
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Acknowledgements |
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References |
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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: 426[CrossRef][ISI][Medline]
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: 5325[CrossRef][ISI][Medline]
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: 7924[Abstract]
5 Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand-nociceptin/orphanin FQ. Trends Pharmacol Sci 1997; 18: 293300[CrossRef][ISI][Medline]
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: 563605[CrossRef][ISI][Medline]
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: 22951[CrossRef][ISI][Medline]
8 Suzue T. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J Physiol 1984; 354: 17383[Abstract]
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: 114969
10 Ballanyi K, Onimaru H, Homma I. Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog Neurobiol 1999; 59: 583634[CrossRef][ISI][Medline]
11 Rossi GC, Perlmutter M, Leventhal L, Talatti A, Pasternak GW. Orphanin FQ/nociceptin analgesia in the rat. Brain Res 1998; 792: 32730[CrossRef][ISI][Medline]
12 Sandin J, Georgieva J, Schött PA, Ögren SO, Terenius L. Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci 1997; 9: 1947[ISI][Medline]
13 Mao L, Wang JQ. Microinjection of nociceptin (Orphanin FQ) into nucleus tractus solitarii elevates blood pressure and heart rate in both anesthetized and conscious rats. J Pharmacol Exp Ther 2000; 294: 25562
14 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: 11758
15 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: 4553[CrossRef][ISI][Medline]
16 Guerrini R, Calo G, Rizzi A, et al. A new selective antagonist of the nociceptin receptor. Br J Pharmacolol 1998; 123: 1635[Abstract]
17 Candeletti S, Guerrini R, Calo G, Romualdi P, Ferri S. Supraspinal and spinal effects of [Phe1(CH2-NH)Gly2]-nociceptin (113)-NH2 on nociception in the rat. Life Sci 2000; 66: 25764[CrossRef][ISI][Medline]
18 Chiou LC. [Phe1(CH2-NH)Gly2]-nociceptin (113)-NH2 activation of an inward rectifier as a partial agonist of ORL1 receptors in rat periaqueductal gray. Br J Pharmocol 1999; 128: 1037
19 Menzies JRW, Glen T, Davies MRP, Paterson SJ, Corbett AD. In vitro agonist effects of nociceptin and [Phe1(CH2-NH)Gly2]-nociceptin (113)-NH2 in the mouse and rat colon and mouse vas deferens. Eur J Pharmacol 1999; 385: 21723[CrossRef][ISI][Medline]
20 Takita K, Herlenius EAP, Lindahl SGE, Yamamoto Y. Actions of opioids on respiratory activity via activation of brainstem µ-, - and
-receptors; an in vitro study. Brain Res 1997; 778: 23341[CrossRef][ISI][Medline]
21 Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL. Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 1991; 254: 7269[ISI][Medline]
22 Koshiya N, Smith JC. Neuronal pacemaker for breathing visualized in vitro. Nature 1999; 400: 3603[CrossRef][ISI][Medline]