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orphanin FQ
Neuroscience Letters 288 (2000) 123±126
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Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ Jason J. McDougall a,*, Matthias Pawlak b, Ulrike Hanesch b, Robert F. Schmidt b a
McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, 3330 Hospital Drive NW, Calgary AB., T2N 4N1 Canada b Physiologisches Institut der UniversitaÈt WuÈrzburg, RoÈntgenring 9, D-97070 WuÈrzburg, Germany Received 4 May 2000; received in revised form 22 May 2000; accepted 22 May 2000
Abstract The peripheral effects of nociceptin were examined in normal and acutely in¯amed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 mM) caused a sensitization of normal and in¯amed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no signi®cant effect on afferent discharge rate in normal knees, however, in in¯amed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These ®ndings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the in¯ammatory status of the tissue. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arthritis; In¯ammation; Knee joint; Neuropeptides; Pain
Following the genomic sequencing of the d-, m- and kopioid receptors, a novel receptor was unearthed which possessed 65% homology to the conventional opioid receptors but which did not bind any of the known endorphins [3,5,10,19,24]. Consigned to orphan status, the receptor was termed the opioid receptor-like receptor (ORL-1) and which has been found to be widely distributed throughout the central and peripheral nervous systems [1,7,19]. The endogenous ligand for ORL-1 was later found to be a heptadecapeptide called orphanin FQ [22], also known and hereafter referred to as nociceptin [17]. With neuropeptidergic characteristics, nociceptin has been localized in various regions of the central nervous system [20,21,23] where it is involved in processes such as pain modulation, long term potentiation, and the regulation of locomotor activity [6,8,11,16]. The experimental evidence pertaining to the role of nociceptin in central nociceptive control mechanisms is contentious. Behavioural studies have indicated that intracerebroventricular or intrathecal administration of the peptide can produce either hyperalgesia, have no effect or even cause analgesia depending upon the species and pain model tested (for review see [8]). * Corresponding author. Tel.: 11-403-220-4507; fax: 11-403270-0617. E-mail address: [email protected] (J.J. Mc Dougall).
In addition to a central role for nociceptin, the neuropeptide has also been found to occur in the periphery. Immunohistochemical data clearly demonstrate the presence of nociceptin throughout the alimentary tract as well as in the kidney, pancreas and bronchi [9,18] where it acts to alter smooth muscle tone and regulate water/sodium excretion [2,4,14]. Despite its principal nociceptive function, in vivo physiological assessment of nociceptin on peripheral pain control mechanisms has not been fully investigated. The aim of the present study was to examine the effect of nociceptin on knee joint afferent mechanosensitivity and to determine whether these responses are altered by acute in¯ammation. To this end, single unit recordings were made from normal and in¯amed knee joint primary afferents in response to normal (within the normal working range of the joint) and extreme rotation of the knee. Eighteen male Wistar rats (320±410 g) were deeply anaesthetized by intraperitoneal injection of sodium thiopental (100±120 mg/kg) and placed in dorsal recumbency on a thermostatically controlled heating pad which maintained core body temperature at 378C. Nine of the rats underwent unilateral intraarticular injection of 2% kaolin/ carrageenan about 4 h prior to afferent recording (acute in¯ammation group) with the remaining nine animals constituting the normal control group. Following abolition of the ¯exor withdrawal re¯ex, the trachea was cannulated
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 21 1- 8
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J.J. McDougall et al. / Neuroscience Letters 288 (2000) 123±126
and connected to a respiratory pump to permit arti®cial ventilation of the rat with 100% oxygen with a respiratory frequency of about 52 breaths/min. End tidal CO2 was continuously monitored and maintained within the physiological range. The left femoral artery was isolated and cannulated with a heparinized saline-®lled cannula before attachment to a pressure transducer to allow blood pressure monitoring. The left femoral vein was also cannulated so that additional doses of anaesthetic could be administered to the animal as required. A ®nal catheter was inserted into the right saphenous artery at a level distal to the knee joint before being advanced retrogradely to a point just distal to the medial articular artery branch. This cannula served as a portal to administer test substances to the knee by close intraarterial infusion. Finally, a vice-like grip was attached to the right femur and subsequently ®xed to a stereotactic frame thereby immobilizing the proximal aspect of the hindlimb. An oil pool was created using hindlimb skin ¯aps and ®lled with warmed paraf®n oil. The saphenous nerve was isolated distal to the knee joint and transected to prevent the conduction of afferent impulses from the foot. The saphenous nerve was also exposed in the inguineal region and transected as central as possible. Fine neuro®laments were dissected free from the nerve and subsequently placed over a platinum electrode to enable single unit extracellular recordings. Afferent nerve ®bres originating from the knee were identi®ed by recording neural discharges generated in response to probing of the joint and consequently their receptive ®eld with a blunt glass rod. The conduction velocities of the ®bres were ascertained by electrically stimulating the receptive ®eld with a pair of silver wire bipolar electrodes (0.2 Hz, 100 ms pulse width, 8±20 V). Afferent activity was then recorded in response to a speci®ed regiment of torque being applied to the knee consisting of normal outward, extreme outward, normal inward, and extreme inward joint rotations. Normal joint movements are described here as occurring within the normal working range of the knee while extreme movements refer to hyper-rotation of the joint against tissue resistance without imparting soft tissue injury. Each movement lasted 10 s with each movement cycle being repeated every 3 min. The amount of torque applied to the joint was measured by a force transducer attached to the foot and standardized for each of the individual movements (approx. 15±20 mN for normal and 40±50 mN for extreme rotations). Knee joint afferent mechanosensitivity was tested before (three movement cycles) and after (®ve movement cycles) close intraarterial infusion of 0.2 ml nociceptin (0.01, 1 and 100 mM). The number of action potentials/movement was determined using analysis software (MRATE, Cambridge Electronic Design Limited) and the percentage change in afferent activity before and after administration of nociceptin was calculated. All inward and outward movement data were pooled for normal and hyper-rotations, respectively, and the mean ^ SEM calculated for each dose of the drug. A positive effect of nociceptin on knee joint mechanosensi-
tivity was established by either a two-tailed one sample Student's t-test (normally distributed data) or the Wilcoxon signed rank test (non-normally distributed data). Changes were only considered signi®cantly different from zero when P , 0:05. The conduction velocities for the primary afferents ranged from 0.97 to 8.5 m/s indicating that recordings were made from group III and IV knee joint sensory nerve ®bres. Close intraarterial infusion of nociceptin had no direct stimulatory effect on any of the knee joint afferents as evidenced by a lack of evoked activity following administration of the peptide prior to joint movements. In normal knees, nociceptin signi®cantly (P , 0:025, n 70±83) sensitized articular afferents in response to normal mechan-
Fig. 1. Effect of close intraarterial injection of nociceptin on articular primary afferent activity in response to normal and hyper-rotation of the rat knee. In normal joints (A), nociceptin sensitized afferent nerve ®bres to normal joint movements but had no effect on hyper-rotations. In acutely in¯amed knees (B), the top dose of nociceptin enhanced afferent discharge rate with normal movements, but produced a desensitizing effect during extreme mechanical manipulation. (Data are shown as mean ^ SEM. *P , 0:05; **P , 0:01; ***P , 0:001 two-tailed one sample t-test and Wilcoxon signed rank test; n 70±83 for normal joints and n 71±73 for in¯amed knees).
J.J. McDougall et al. / Neuroscience Letters 288 (2000) 123±126
ical stimulation of the joint (Fig. 1A). The maximum effect of the peptide occurred with the 1 mM dose which caused afferent activity to increase by 35.12 ^ 8.9%. In contrast, nociceptin had no signi®cant effect on knee joint mechanosensitivity in response to hyper-rotation of the knee (P 0:2509, P 0:07, and P 0:5586 for the 0.01, 1, and 100 mM doses, respectively). With normal movements of acutely in¯amed knees, nociceptin appeared to have a sensitizing effect on joint afferents (Fig. 1B), however, this was only found to be signi®cant with the 100 mM dose (P 0:001, n 71±73) which caused afferent activity to increase by 34.74 ^ 10.1%. Interestingly, the level of afferent discharge produced by hyperrotation of the joint was reduced by prior administration of nociceptin, however, once again this effect was only significant with the top dose of the drug (P , 0:05). Specimen records of the differential responses from normal and in¯amed joints are shown in Fig. 2. Although ORL-1 receptors have been clearly demonstrated in several peripheral tissues [9,24], their functional characterization has been restricted to non-nociceptive processes such as smooth muscle activation and renal function [2,4,14]. The present study shows for the ®rst time the modulatory effects of nociceptin on peripheral nociceptive mechanisms and provides the ®rst physiological evidence of the presence of ORL-1 receptors in rat knee joints. Despite the lack of an evoked effect on knee joint afferents, nociceptin was able to alter the sensitivity of articular ®bres in
125
response to mechanical manipulation of the joint. Close intraarterial infusion of nociceptin resulted in a sensitization of articular afferents during normal rotation of normal and acutely in¯amed knees suggesting a pronociceptive function of the peptide during low load joint movement. In contrast, afferent discharges in response to hyper-rotation of the knee were unaltered by nociceptin treatment in normal joints but were diminished in the in¯amed group of animals. It appears, therefore, that in situations of extreme sensory activity, the sensitizing effect of nociceptin is reversed so that the neuropeptide now exhibits antinociceptive properties. One can only speculate as to how this plasticity change is achieved, but radical alteration of the ORL-1 receptor by the in¯ammatory process may be one explanation. The mechanism by which nociceptin modulates nociception in the periphery will require further investigation, but may be related to the secondary release of other excitatory agents. Recent reports indicate that nociceptin may stimulate capsaicin-sensitive nerve terminals to cause the release of tachykinins such as substance P (SP) [13,15]. Since SP sensitizes rat knee joint primary afferents [12], then the pronociceptive effects of nociceptin described here may be due to an indirect excitatory action via SP release with subsequent activation of prejunctional neurokinin-1 receptors. Interestingly, Heppelmann and Pawlak [12] found that in in¯amed joints, the sensitizing effect of SP was diminished during hyper-rotation such that the majority of afferents were unaffected or even showed a reduction in afferent
Fig. 2. Specimen records from single experiments illustrating the change in primary afferent ®ring rate exerted by 100 mM nociceptin on normal (A) and in¯amed (B) rat knee joint mechanosensitivity during normal and hyper-rotations of the joint. Conduction velocities were 8.5 and 1.9 m/s for the normal and in¯amed joint afferents, respectively.
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J.J. McDougall et al. / Neuroscience Letters 288 (2000) 123±126
activity in response to the peptide. This ®nding is consistent with the present study providing further evidence of possible tachykinergic involvement in nociceptin-mediated pain modulation in the periphery. In summary, the present study has clearly shown that nociceptin alters knee joint mechanosensitivity implying a role for the neuropeptide in peripheral nociceptive control mechanisms. The behaviour of the peptide appears to be dichotomous depending upon the in¯ammatory status of the tissue and this ®nding may help explain the divergent results obtained from behavioural studies. The authors would like to thank Mrs M. Oppmann and T. Hoffmann for their technical expertise. This study was supported by the Deutsche Forschungsgemeinschaft SFB 353/B10. JJMCD received ®nancial support from the Medical Research Council of Canada, and the Ernst and Young Joint Injury and Arthritis Research Fellowship. [1] Anton, B., Fein, J., To, T., Li, X., Silberstein, L. and Evans, C.J., Immunohistochemical localization of ORL-1 in the central nervous system of the rat, J. Comp. Neurol., 368 (1996) 229±251. [2] Bigoni, R., Giuliani, S., Calo, G., Rizzi, A., Guerrini, R., Salvadori, S., Regoli, D. and Maggi, C.A., Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies, Naunyn-Schmiedeberg's Arch. Pharmacol., 359 (1999) 160±167. [3] Bunzow, J.R., Saez, C., Mortrud, M., Bouvier, C., Williams, J.T., Low, M. and Grandy, D.K., 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., 347 (1994) 284±288. [4] Champion, H.C., Pierce, R.L. and Kadowitz, P.J., Nociceptin, a novel endogenous ligand for the ORL-1 receptor, dilates isolated resistance arteries from the rat, Regul. Pept., 78 (1998) 69±74. [5] Chen, Y., Fan, Y., Liu, J., Mestek, A., Tian, M., Kozak, C.A. and Yu, L., Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family, FEBS Lett., 347 (1994) 279±283. [6] Civelli, O., Nothacker, H.P. and Reinscheid, R., Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin, CRC Crit. Rev. Neurobiol, 12 (1998) 163±176. [7] Darland, T. and Grandy, D.K., The orphanin FQ system: an emerging target for the management of pain? Br. J. Anaesth., 81 (1998) 29±37. [8] Darland, T., Heinricher, M.M. and Grandy, D.K., Orphanin FQ/nociceptin: a role in pain and analgesia, but so much more, Trends Neurosci., 21 (1998) 215±221. [9] Fischer, A., Forssmann, W.G. and Undem, B.J., Nociceptininduced inhibition of tachykinergic neurotransmission in guinea pig bronchus, J. Pharmacol. Exp. Ther., 285 (1998) 902±907. [10] Fukuda, K., Kato, S., Mori, K., Nishi, M., Takeshima, H., Iwabe, N., Miyata, T., Houtani, T. and Sugimoto, T., cDNA
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cloning and regional distribution of a novel member of the opioid receptor family, FEBS Lett., 343 (1994) 42±46. Goda, Y. and Mutneja, M., Memory mechanisms: the nociceptin connection, Curr. Biol., 8 (1998) R889±R891. Heppelmann, B. and Pawlak, M., Sensitization of articular afferents in normal and in¯amed knee joints by substance P in the rat, Neurosci. Lett., 223 (1997) 97±100. Inoue, M., Kobayashi, M., Kozaki, S., Zimmer, A. and Ueda, H., Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice, Proc. Natl. Acad. Sci. USA, 95 (1998) 10949±10953. Kapusta, D.R., Sezen, S.F., Chang, J.K., Lippton, H. and Kenigs, V.A., Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ), Life Sci., 60 (1997) L15±L21. Lecci, A., Giuliani, S., Tramontana, M., Meini, S., Santicioli, P. and Maggi, C.A., Tachykinin-mediated effect of nociceptin in the rat urinary bladder in vivo, Eur. J. Pharmacol., 389 (2000) 99±102. Manabe, T., Noda, Y., Mamiya, T., Katagiri, H., Houtani, T., Nishi, M., Noda, T., Takahashi, T., Sugimoto, T., Nabeshima, T. and Takeshima, H., Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors, Nature, 394 (1998) 577±581. Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.L., Guillemot, J.C., Ferrara, P., Monsarrat, B., Mazarquil, H., Vassart, G., Parmentier, M. and Costentin, J., Isolation and structure of the endogenous agonist of opioid receptor-like ORL-1 receptor, Nature, 377 (1995) 532±535. Mitsuma, T., Rhue, H., Kayama, M., Adachi, K., Mori, Y., Nogimori, T., Sakai, J., Ping, J. and Hirooka, Y., Distribution of orphanin FQ in the rat - an immunohistochemical study, Med. Sci. Res., 26 (1998) 403±504. Mollereau, C., Parmentier, M., Mailleux, P., Butour, J.L., Moisand, C., Chalon, P., Caput, D., Vassart, G. and Meunier, J.C., ORL-1, a novel member of the opioid receptor family. Cloning, functional expression and localization, FEBS Lett., 341 (1994) 33±38. Neal Jr., C.R., Mansour, A., Reinscheid, R., Nothacker, H.P., Civelli, O. and Watson Jr., S.J., Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat, J. Comp. Neurol., 406 (1999) 503±547. Riedl, M., Shuster, S., Vulchanova, L., Wang, J., Loh, H.H. and Elde, R., Orphanin FQ/nociceptin-immunoreactive nerve ®bres parallel those containing endogenous opioids in rat spinal cord, NeuroReport, 7 (1996) 1369±1372. Reinscheid, R.K., Nothacker, H.P., Bourson, A., Ardati, A., Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H., Monsma Jr., F.J. and Civelli, O., Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor, Science, 270 (1995) 792±794. Schuligoi, R., Amann, R., Angelberger, P. and Peskar, B.A., Determination of nociceptin-like immunoreactivity in the rat dorsal spinal cord, Neurosci. Lett., 224 (1997) 136±138. Wang, J.B., Johnson, P.S., Imai, Y., Persico, A.M., Ozenberger, B.A., Eppler, C.M. and Uhl, G.R., cDNA cloning of an orphan opiate receptor gene family member and its splice variant, FEBS Lett., 348 (1994) 75±79.
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Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ Jason J. McDougall a,*, Matthias Pawlak b, Ulrike Hanesch b, Robert F. Schmidt b a
McCaig Centre for Joint Injury and Arthritis Research, University of Calgary, 3330 Hospital Drive NW, Calgary AB., T2N 4N1 Canada b Physiologisches Institut der UniversitaÈt WuÈrzburg, RoÈntgenring 9, D-97070 WuÈrzburg, Germany Received 4 May 2000; received in revised form 22 May 2000; accepted 22 May 2000
Abstract The peripheral effects of nociceptin were examined in normal and acutely in¯amed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 mM) caused a sensitization of normal and in¯amed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no signi®cant effect on afferent discharge rate in normal knees, however, in in¯amed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These ®ndings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the in¯ammatory status of the tissue. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Arthritis; In¯ammation; Knee joint; Neuropeptides; Pain
Following the genomic sequencing of the d-, m- and kopioid receptors, a novel receptor was unearthed which possessed 65% homology to the conventional opioid receptors but which did not bind any of the known endorphins [3,5,10,19,24]. Consigned to orphan status, the receptor was termed the opioid receptor-like receptor (ORL-1) and which has been found to be widely distributed throughout the central and peripheral nervous systems [1,7,19]. The endogenous ligand for ORL-1 was later found to be a heptadecapeptide called orphanin FQ [22], also known and hereafter referred to as nociceptin [17]. With neuropeptidergic characteristics, nociceptin has been localized in various regions of the central nervous system [20,21,23] where it is involved in processes such as pain modulation, long term potentiation, and the regulation of locomotor activity [6,8,11,16]. The experimental evidence pertaining to the role of nociceptin in central nociceptive control mechanisms is contentious. Behavioural studies have indicated that intracerebroventricular or intrathecal administration of the peptide can produce either hyperalgesia, have no effect or even cause analgesia depending upon the species and pain model tested (for review see [8]). * Corresponding author. Tel.: 11-403-220-4507; fax: 11-403270-0617. E-mail address: [email protected] (J.J. Mc Dougall).
In addition to a central role for nociceptin, the neuropeptide has also been found to occur in the periphery. Immunohistochemical data clearly demonstrate the presence of nociceptin throughout the alimentary tract as well as in the kidney, pancreas and bronchi [9,18] where it acts to alter smooth muscle tone and regulate water/sodium excretion [2,4,14]. Despite its principal nociceptive function, in vivo physiological assessment of nociceptin on peripheral pain control mechanisms has not been fully investigated. The aim of the present study was to examine the effect of nociceptin on knee joint afferent mechanosensitivity and to determine whether these responses are altered by acute in¯ammation. To this end, single unit recordings were made from normal and in¯amed knee joint primary afferents in response to normal (within the normal working range of the joint) and extreme rotation of the knee. Eighteen male Wistar rats (320±410 g) were deeply anaesthetized by intraperitoneal injection of sodium thiopental (100±120 mg/kg) and placed in dorsal recumbency on a thermostatically controlled heating pad which maintained core body temperature at 378C. Nine of the rats underwent unilateral intraarticular injection of 2% kaolin/ carrageenan about 4 h prior to afferent recording (acute in¯ammation group) with the remaining nine animals constituting the normal control group. Following abolition of the ¯exor withdrawal re¯ex, the trachea was cannulated
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 21 1- 8
124
J.J. McDougall et al. / Neuroscience Letters 288 (2000) 123±126
and connected to a respiratory pump to permit arti®cial ventilation of the rat with 100% oxygen with a respiratory frequency of about 52 breaths/min. End tidal CO2 was continuously monitored and maintained within the physiological range. The left femoral artery was isolated and cannulated with a heparinized saline-®lled cannula before attachment to a pressure transducer to allow blood pressure monitoring. The left femoral vein was also cannulated so that additional doses of anaesthetic could be administered to the animal as required. A ®nal catheter was inserted into the right saphenous artery at a level distal to the knee joint before being advanced retrogradely to a point just distal to the medial articular artery branch. This cannula served as a portal to administer test substances to the knee by close intraarterial infusion. Finally, a vice-like grip was attached to the right femur and subsequently ®xed to a stereotactic frame thereby immobilizing the proximal aspect of the hindlimb. An oil pool was created using hindlimb skin ¯aps and ®lled with warmed paraf®n oil. The saphenous nerve was isolated distal to the knee joint and transected to prevent the conduction of afferent impulses from the foot. The saphenous nerve was also exposed in the inguineal region and transected as central as possible. Fine neuro®laments were dissected free from the nerve and subsequently placed over a platinum electrode to enable single unit extracellular recordings. Afferent nerve ®bres originating from the knee were identi®ed by recording neural discharges generated in response to probing of the joint and consequently their receptive ®eld with a blunt glass rod. The conduction velocities of the ®bres were ascertained by electrically stimulating the receptive ®eld with a pair of silver wire bipolar electrodes (0.2 Hz, 100 ms pulse width, 8±20 V). Afferent activity was then recorded in response to a speci®ed regiment of torque being applied to the knee consisting of normal outward, extreme outward, normal inward, and extreme inward joint rotations. Normal joint movements are described here as occurring within the normal working range of the knee while extreme movements refer to hyper-rotation of the joint against tissue resistance without imparting soft tissue injury. Each movement lasted 10 s with each movement cycle being repeated every 3 min. The amount of torque applied to the joint was measured by a force transducer attached to the foot and standardized for each of the individual movements (approx. 15±20 mN for normal and 40±50 mN for extreme rotations). Knee joint afferent mechanosensitivity was tested before (three movement cycles) and after (®ve movement cycles) close intraarterial infusion of 0.2 ml nociceptin (0.01, 1 and 100 mM). The number of action potentials/movement was determined using analysis software (MRATE, Cambridge Electronic Design Limited) and the percentage change in afferent activity before and after administration of nociceptin was calculated. All inward and outward movement data were pooled for normal and hyper-rotations, respectively, and the mean ^ SEM calculated for each dose of the drug. A positive effect of nociceptin on knee joint mechanosensi-
tivity was established by either a two-tailed one sample Student's t-test (normally distributed data) or the Wilcoxon signed rank test (non-normally distributed data). Changes were only considered signi®cantly different from zero when P , 0:05. The conduction velocities for the primary afferents ranged from 0.97 to 8.5 m/s indicating that recordings were made from group III and IV knee joint sensory nerve ®bres. Close intraarterial infusion of nociceptin had no direct stimulatory effect on any of the knee joint afferents as evidenced by a lack of evoked activity following administration of the peptide prior to joint movements. In normal knees, nociceptin signi®cantly (P , 0:025, n 70±83) sensitized articular afferents in response to normal mechan-
Fig. 1. Effect of close intraarterial injection of nociceptin on articular primary afferent activity in response to normal and hyper-rotation of the rat knee. In normal joints (A), nociceptin sensitized afferent nerve ®bres to normal joint movements but had no effect on hyper-rotations. In acutely in¯amed knees (B), the top dose of nociceptin enhanced afferent discharge rate with normal movements, but produced a desensitizing effect during extreme mechanical manipulation. (Data are shown as mean ^ SEM. *P , 0:05; **P , 0:01; ***P , 0:001 two-tailed one sample t-test and Wilcoxon signed rank test; n 70±83 for normal joints and n 71±73 for in¯amed knees).
J.J. McDougall et al. / Neuroscience Letters 288 (2000) 123±126
ical stimulation of the joint (Fig. 1A). The maximum effect of the peptide occurred with the 1 mM dose which caused afferent activity to increase by 35.12 ^ 8.9%. In contrast, nociceptin had no signi®cant effect on knee joint mechanosensitivity in response to hyper-rotation of the knee (P 0:2509, P 0:07, and P 0:5586 for the 0.01, 1, and 100 mM doses, respectively). With normal movements of acutely in¯amed knees, nociceptin appeared to have a sensitizing effect on joint afferents (Fig. 1B), however, this was only found to be signi®cant with the 100 mM dose (P 0:001, n 71±73) which caused afferent activity to increase by 34.74 ^ 10.1%. Interestingly, the level of afferent discharge produced by hyperrotation of the joint was reduced by prior administration of nociceptin, however, once again this effect was only significant with the top dose of the drug (P , 0:05). Specimen records of the differential responses from normal and in¯amed joints are shown in Fig. 2. Although ORL-1 receptors have been clearly demonstrated in several peripheral tissues [9,24], their functional characterization has been restricted to non-nociceptive processes such as smooth muscle activation and renal function [2,4,14]. The present study shows for the ®rst time the modulatory effects of nociceptin on peripheral nociceptive mechanisms and provides the ®rst physiological evidence of the presence of ORL-1 receptors in rat knee joints. Despite the lack of an evoked effect on knee joint afferents, nociceptin was able to alter the sensitivity of articular ®bres in
125
response to mechanical manipulation of the joint. Close intraarterial infusion of nociceptin resulted in a sensitization of articular afferents during normal rotation of normal and acutely in¯amed knees suggesting a pronociceptive function of the peptide during low load joint movement. In contrast, afferent discharges in response to hyper-rotation of the knee were unaltered by nociceptin treatment in normal joints but were diminished in the in¯amed group of animals. It appears, therefore, that in situations of extreme sensory activity, the sensitizing effect of nociceptin is reversed so that the neuropeptide now exhibits antinociceptive properties. One can only speculate as to how this plasticity change is achieved, but radical alteration of the ORL-1 receptor by the in¯ammatory process may be one explanation. The mechanism by which nociceptin modulates nociception in the periphery will require further investigation, but may be related to the secondary release of other excitatory agents. Recent reports indicate that nociceptin may stimulate capsaicin-sensitive nerve terminals to cause the release of tachykinins such as substance P (SP) [13,15]. Since SP sensitizes rat knee joint primary afferents [12], then the pronociceptive effects of nociceptin described here may be due to an indirect excitatory action via SP release with subsequent activation of prejunctional neurokinin-1 receptors. Interestingly, Heppelmann and Pawlak [12] found that in in¯amed joints, the sensitizing effect of SP was diminished during hyper-rotation such that the majority of afferents were unaffected or even showed a reduction in afferent
Fig. 2. Specimen records from single experiments illustrating the change in primary afferent ®ring rate exerted by 100 mM nociceptin on normal (A) and in¯amed (B) rat knee joint mechanosensitivity during normal and hyper-rotations of the joint. Conduction velocities were 8.5 and 1.9 m/s for the normal and in¯amed joint afferents, respectively.
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activity in response to the peptide. This ®nding is consistent with the present study providing further evidence of possible tachykinergic involvement in nociceptin-mediated pain modulation in the periphery. In summary, the present study has clearly shown that nociceptin alters knee joint mechanosensitivity implying a role for the neuropeptide in peripheral nociceptive control mechanisms. The behaviour of the peptide appears to be dichotomous depending upon the in¯ammatory status of the tissue and this ®nding may help explain the divergent results obtained from behavioural studies. The authors would like to thank Mrs M. Oppmann and T. Hoffmann for their technical expertise. This study was supported by the Deutsche Forschungsgemeinschaft SFB 353/B10. JJMCD received ®nancial support from the Medical Research Council of Canada, and the Ernst and Young Joint Injury and Arthritis Research Fellowship. [1] Anton, B., Fein, J., To, T., Li, X., Silberstein, L. and Evans, C.J., Immunohistochemical localization of ORL-1 in the central nervous system of the rat, J. Comp. Neurol., 368 (1996) 229±251. [2] Bigoni, R., Giuliani, S., Calo, G., Rizzi, A., Guerrini, R., Salvadori, S., Regoli, D. and Maggi, C.A., Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies, Naunyn-Schmiedeberg's Arch. Pharmacol., 359 (1999) 160±167. [3] Bunzow, J.R., Saez, C., Mortrud, M., Bouvier, C., Williams, J.T., Low, M. and Grandy, D.K., 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., 347 (1994) 284±288. [4] Champion, H.C., Pierce, R.L. and Kadowitz, P.J., Nociceptin, a novel endogenous ligand for the ORL-1 receptor, dilates isolated resistance arteries from the rat, Regul. Pept., 78 (1998) 69±74. [5] Chen, Y., Fan, Y., Liu, J., Mestek, A., Tian, M., Kozak, C.A. and Yu, L., Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family, FEBS Lett., 347 (1994) 279±283. [6] Civelli, O., Nothacker, H.P. and Reinscheid, R., Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin, CRC Crit. Rev. Neurobiol, 12 (1998) 163±176. [7] Darland, T. and Grandy, D.K., The orphanin FQ system: an emerging target for the management of pain? Br. J. Anaesth., 81 (1998) 29±37. [8] Darland, T., Heinricher, M.M. and Grandy, D.K., Orphanin FQ/nociceptin: a role in pain and analgesia, but so much more, Trends Neurosci., 21 (1998) 215±221. [9] Fischer, A., Forssmann, W.G. and Undem, B.J., Nociceptininduced inhibition of tachykinergic neurotransmission in guinea pig bronchus, J. Pharmacol. Exp. Ther., 285 (1998) 902±907. [10] Fukuda, K., Kato, S., Mori, K., Nishi, M., Takeshima, H., Iwabe, N., Miyata, T., Houtani, T. and Sugimoto, T., cDNA
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