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Identification of a novel receptor mediating substance P-induced behavior in the mouse
European Journal of Pharmacology, 217 (1992) 197-201
197
~" 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52526
Identification of a novel receptor mediating substance P-induced behavior in the mouse D a r r e l l D. M o u s s e a u , X i a o f e n g Sun a n d Alice A. L a r s o n Department of Veterinary PathoBiology, University of Minnesota, 295 Animal Science/Veterinao' Medicine Building, 1988 Fitch At'enue, Saint Paul, MN 55108, USA Received 23 January 1992, accepted 14 April 1992
To determine whether opioid receptors or the more recently characterized naloxone-sensitive substance P (SP) N-terminal binding sites play a role in desensitization to the behavioral effects of SP, we assessed the effects of selective antagonists at /z(naloxonazine and /3-funaltrexamine), 8- (naltrindole) and •- (nor-binaltorphimine) opioid receptors, as well as the effect of [D-ProZ,D-LeuT]SP-(1-7) D-SP-(1-7) (D-SP (1-7)), an inhibitor of [3H]SP-(1-7) binding, on behaviors induced by intrathecally administered SP in mice. Whereas naloxone, a non-selective opioid antagonist, inhibited the development of behavioral desensitization to SP, the response to repeated SP administration remained unaffected by pretreatment with selective opioid antagonists. Like naloxone, however, the SP-(1-7) antagonist inhibited SP-induced desensitization. The protection against desensitization to SP by D-SP-(1-7), but not by selective antagonists of/z, 6 or ~ receptors, suggests that desensitization to the behavioral effects of SP does not appear to be mediated by an action at an opioid receptor but by an action at the SP-(1-7) binding site. Substance P (N-terminal fragments); Substance P-(1-7); Opioid receptor antagonists; Behavioural desensitization
1. Introduction
The undecapeptide substance P (SP) is a neurot r a n s m i t t e r / n e u r o m o d u l a t o r with a wide distribution throughout the central nervous system. Brain subcortical areas (Pernow, 1983) and the dorsal horn of the spinal cord (Yaksh et at., 1980) exhibit high concentrations of SP, with the primary afferent C-fibre system of the latter region being the proposed site for nociception mediated by this peptide (Kellstein et at., 1990; Besson and Chouch, 1987; Lembeck, 1953). That SPcontaining nerves are involved in nociceptive transmission has been demonstrated in the dorsal horn of the spinal cord where SP release has been observed following activation of nociceptive primary afferents (Lembeck et al., 1981; Yaksh et al., 1980). Both depolarization of spinal tissue (Jessell and Iversen, 1977) and electrical stimulation of primary afferent C-fibres (Jessell et al., 1979) result in increased concentrations of SP in the cerebrospinal fluid. This release is inhib-
Correspondence to: A.A. Larson, Department of Veterinary PathoBiology, University of Minnesota, 295 Animal Science/Veterinary Medicine Building, 1988 Fitch Avenue, Saint Paul, MN 55108, USA. Tel, 1.o12.624 3650, fax 1.612.625 0204.
ited by opioids (Yaksh et al., 1980). Furthermore, an increased innervation of SP-containing neurons and an increased amount of immunoreactive SP in the dorsal horn of the spinal cord have been observed in rat models of chronic nociception (Levine et al., 1984; McCarson and Goldstein, 1990). Intrathecal (i.t.) injection of SP elicits a caudally directed biting and scratching (CBS) behavioral syndrome in mice (Hylden and Wilcox, 1981; Piercey et al., 1981) which, following repeated administration of SP, decreases in intensity with each subsequent injection (Larson, 1988). Desensitization has also been reported to develop to SP-induced hyperalgesia (Moochhala and Sawynok, 1984). Selective p~- and 6-opioid agonists have been shown to inhibit CBS behaviour elicited by a single i.t. injection of SP (Moochhala and Sawynok, 1984; Hylden and Wilcox, 1983a) and the development of behavioural desensitization to repeated i.t. injections of SP is blocked by the non-specific opioid antagonist naloxone (Larson, 1988). This last observation suggests opioid receptor activity in SP-induced behavioral desensitization as naloxone is a nonspecific opioid antagonist. It has also been postulated that behavioral desensitization observed following repeated SP injection may be due to its modulation by its N-terminal metabolites, the heptapeptide SP-(1-7) be-
198 ing the predominant fragment (Nyberg et al., 1984; Sakurada et al., 1985). In fact, exogenous SP-(1-7) is capable of inhibiting SP-induced behavior in a naloxone-reversible manner (Igwe et al., 1990a), while the accumulation of SP-(1-7) following repeated SP injection correlates well with behavioral desensitization (Igwe et al., 1990b). Stewart et al. (1982) reported an antinociceptive effect of SP-(1-7) which was naloxonereversible. Subsequently, it was shown that the C- and N-terminal fragments of SP exert opposite effects in terms of nociception and motor activity (Hall and Stewart, 1983). It is therefore possible that the Nterminal metabolites may play a role in both the modulation of pain as well as SP-induced behavioral desensitization. We characterized the interaction between SP and opioid receptors by comparing the effect of selective opioid antagonists on SP-induced behavioral desensitization. In addition, the SP-(1-7) antagonist [DPro2,D-PheT]-sP-(1-7) (D-SP-(1-7)) was investigated in hopes of elucidating whether behavioral desensitization observed following repeated SP administration might be due to activity at the [3H]SP-(1-7) binding site described by Igwe et al. (1990c).
2. Materials and methods
2.1. Animals Male Swiss-Webster mice (Sasco Inc., Omaha, NE) weighing 20-25 g, were housed four per cage in a room maintained at 21-22°C and on a 12 h: 12 h light/dark cycle (lights on at 6:00 a.m.). Animals had free access to food and water. Animals were used strictly in accordance with the Guidelines of the University of Minnesota Animal Care and Use Committee and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHEW publication (N.I.H.) 78-23, revised 1978). 2.2. Drug administration All drugs were administered via i.t. injection unless otherwise indicated. All injections, whether single or multiple (repeated), consisted of a 5 /xl volume (per injection) of the appropriate vehicle either alone (control) or containing the treatment drug. Animals were randomly assigned to treatment groups. Single injections were made according to the method of Hylden and Wilcox (1980). Briefly, a 30-gauge 0.5 inch disposable needle was attached to a 50/zl Luer-tip Microliter syringe (Hamilton, Reno, NV). Unanesthetized mice were held by the iliac crest and the needle was inserted at the L5-L6 intervertebral space. In the case of repeated injections, a slight modification to the needle
was made wherein a 25 cm length of PE-10 tubing, equipped with the barrel of a 30-gauge 0.5 inch needle, was mated to the disposable needle tip attached to the Hamilton syringe. This 'cannula' allowed for i.t. drug administration without the direct handling of, and any potential associated stress to, the animal. Administration of any drug was delayed for a 1 min period following placement of the cannula to allow the animal to acclimate to the presence of the needle. Between animals, the PE-10 tubing was flushed with the drug solution so as to avoid potential dilution of the drug dosage due to adsorption of the drug to the tubing wall, or to temperature-dependent changes in the structure of SP. All drugs were dissolved in normal saline except SP, SP-(1-7) and D-SP-(1-7)which were dissolved in acidified saline (0.85% saline containing 0.01 N acetic acid) to minimize adsorption to synthetic surfaces (Hall and Stewart, 1983). The dose of SP (10 ng (7.5 pmol); 4 × at 2 min intervals) chosen for the present experiments is known to elicit CBS behaviour (Hylden and Wilcox, 1981; Piercey et al., 1981) of an intensity which could be attenuated or potentiated without inducing seizure activity. SP-(1-7) (20 ng) was coadministered with SP, whereas D-SP-(1-7) was administered as a single injection pretreatment (1 ~g, 5 rain). Opioid antagonists employed in this series of experiments were administered as a single dose pretreatment. Doses of selective antagonists were obtained from the literature based on their ability to selectively antagonize opioid receptor subtypes: naloxone, a non-selective opioid antagonist (1 tzg, 15 min); naloxonazine, selective for high-affinity (/z 1)/z receptors (Paul et al., 1989; 35 mg/kg i.p., 24 h); /3-funaltrexamine, selective for low-affinity (/-*2) /~ receptors (Hylden and Wilcox, 1983b; 0.5 t~g, 24 h); naltrindole, selective for the 3 receptor subtype (Drower et al., 1989; 0.1 ~g, 24 h); nor-binaltorphimine, selective for the K receptor subtype (Takemori et al., 1988, 4 ~g, 15 min). Naloxonazine, /3-funaltrexamine and naltrindole were purchased from Research Biochemicals Incorporated (Natick, MA). Nor-binaltorphimine was kindly provided by Dr. A.E. Takemori, Department of Pharmacology, University of Minnesota (Minneapolis, MN). Naloxone hydrochloride was a gift from Endo Laboratory (Garden City, NY). Substance P and SP-(1-7) were purchased from Peninsula Laboratories (Belmont, CA). D-SP-(1-7) was synthesized by the University of Minnesota Microchemical Facilities (Minneapolis, MN).
2.3. Behavioral testing Immediately following the insertion of the cannula, the test animal (n = 5 - 6 per treatment group) was placed in a large (2 1) glass cylinder. After the appropriate acclimation period (see above), SP was injected
199
and the intensity of CBS behaviours were recorded. Each incident (i.e. a bite or a scratch) was counted as a single CBS behaviour. In the event that the duration of CBS activity extended beyond that of a 'single' bite or scratch, then the duration, in seconds (one count per 1 s), was used as an index of CBS activity.
._= ==
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2.4. Statistical analysis Animals were randomly assigned to treatment groups. Behavioural desensitization was defined as the decrease in the average number of CBS behaviours induced by the fourth injection of SP compared to that following the first injection for each individual animal. Behavioral data are expressed as means + S.E.M. in CBS behaviors for each group and were analyzed by the two-tailed unpaired Student's t-test or by A N O V A (followed by the Dunnett's test for significance) using the Macintosh StatView TM program. Statistical significance was determined as P < 0.05.
3. Results
The effect of a single i.t. injection of SP on CBS activity is depicted in fig. 1. SP-induced CBS behavior was significantly inhibited when coadministered with the SP-(1-7) metabolite. This effect of SP-(1-7) was blocked by pretreatment with the SP-(1-7) antagonist D-SP-(1-7). D-SP-(1-7) did not elicit an effect on SP-induced CBS activity on its own. Repeated injections with SP resulted in a successive decrease in CBS
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80 m
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60-
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B
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0-2
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2-4
i
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6-8
Time (min) Fig, 2. Reduction in the behavioral response to repeated (4 x 10 ng) i.t. injections of SP (open squares). Shaded squares depict the effect of repeated injections with the acid saline vehicle. The extent of desensitization in each treatment group described in fig. 3 is determined as the mean difference between the first and fourth injection, or A and B, as shown above.
behaviour (i.e. behavioral desensitization) in every animal tested in this manner (fig. 2). The acid saline vehicle did not elicit any significant CBS activity. Pretreatment with 1/xg of D-SP-(1-7) resulted in a reduction of the extent of desensitization when compared to saline-pretreated control mice, whereas pretreatment with 1 /xg of naloxone completely blocked the SP-induced desensitization (fig. 3). The effects of single injection pretreatments with the opioid antagonists chosen for this study, at individual doses previously shown to be selective for subtypes of opioid receptors, are displayed next to their respective vehicle controls for ease of interpretation (fig. 3). Neither the selective /z-antagonists naloxonazine (i.e. /x~) and /3-funaltrexamine (i.e. /x2), nor the 3- and K receptor antagonists naltrindole and nor-binaltorphimine, respectively, were effective at inhibiting behavioral desensitization.
@ 4. D i s c u s s i o n SP
SP
SP
SP
D-SP(1-7)
SP(1-T)
SP(1.7) D-SP(I-7)
Treatment
Fig. 1. Effect of i.t. administration of the SP N-terminal metabolite SP-(1-7) (20 ng, 0 min) and the SP-(1-7) antagonist D-SP-(1-7) (1 /zg, 5 min) on SP (10 ng)-induced CBS behaviors. SP-induced CBS activity is attenuated by SP-(1-7) (t p < 0.05 using A N O V A followed by Dunnett's test). This attenuation is inhibited by pretreatment with the SP-(1-7) antagonist D-SP-(1-7).
The CBS model, useful as a reflection of the activities of compounds in vivo, was chosen to study the interaction of SP with receptors believed to be involved in the mediation of nociceptive (both tonic and chronic) information. The behaviors may reflect pain perception, but more importantly, it is a simple and reproducible system that reflects quantifiable activity produced by stimulation of receptors that are postulated
200 40
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20,
NAL
D-SP(1-7)
NLX
II-FNA
nor-BNI
NTL
Pretreatment
Fig. 3. Effect of the SP-(1-7) antagonist D-SP-(1-7) (1 /,g, 5 rain) and non-selective (NAL: naloxone, 1 p-g, 15 rain) and selective (NLX: naloxonazine (P-l), 35 mg/kg i.p., 24 h; /3-FNA: /3funaltrexamine (P-2), 0.5 p-g, 24 h; nor-BNI: nor-binaltorphimine (K), 4 p.g, 15 rain; NTL: naltrindole (6), 0.1 p-g, 24 h) opioid antagonists on the extent of SP-induced behavioral desensitization. The extent of behavioral desensitizationin each treatment group is defined as the mean difference between the 1st and 4th injection (i.e. A and B shown in fig. 1) in a series of four injections. SP-induced behavioral desensitization was attenuated by both naloxone and D-SP-(1-7) (* P < 0.05 using Student's t-test) but remained unaffected by selective opioid antagonists.
to be important in pain transmission at the level of the spinal cord. Using this model, we have previously shown that there is an attenuation of CBS behaviors in response to repeated intrathecal injections of SP (Larson, 1988) and N-methyl-D-aspartate (Sun and Larson, 1991) while a similar injection protocol using kainic acid or quisqualic acid results in sensitization of the CBS effects (Sun and Larson, 1991). We refer to this diminution of CBS behaviors as a result of repeated SP and N-methyl-D-aspartate administration as behavioral 'desensitization'. Pretreatment with naloxone inhibited the development of behavioral desensitization to SP, thus confirming our previous results (Larson, 1988). Subsequent use of more selective opioid antagonists, and the analysis of their effects on SP-induced behavioral desensitization, allowed us to determine which opioid receptors might be involved in this phenomenon. It is apparent from the data depicted in fig. 3 that, of the opioid receptor antagonists chosen for the present study, all failed to prevent SP-induced desensitization. It would therefore a p p e a r that SP-induced behavioral desensitization is mediated by an action at a naloxone-sensitive receptor which is not one of the classical opioid receptors.
The p h e n o m e n o n of desensitization is consistent with SP's role as a nociceptive transmitter that has the capacity to initiate the rapid development of an endogenously mediated, naloxone-reversible analgesia. The mechanism by which naloxone-reversible desensitization to SP occurs is not fully understood. We have gathered evidence, however, to support the hypothesis that SP is metabolized in the spinal cord to N-terminal peptide fragments (Igwe et al., 1990b). That these fragments are important in the development of desensitization to SP is reflected by their ability to attenuate CBS activity in response to SP (Igwe et al., 1990a; present study) and by the ability of the SP-(1-7) antagonist D-SP-(1-7) to inhibit SP-induced desensitization (present study). This desensitization may occur by a direct interaction of the metabolites with a /3funaltrexamine-insensitive subset of high-affinity, /~selective, opioid receptors, as SP-(1-7) has been found to interact with this select subset of [D-AIa 2, M e P h e 4,Gly-(ol)5]enkephalin ( D A M G O ) - [ a b e l l e d binding sites (Krumins et al., 1989a, b). Tritiated Nterminal fragments of SP have also been found to label a novel population of [3H]SP-(1-7) binding sites that are displaced by D A M G O but relatively insensitive to /3-funaltrexamine, sufentanyl, naloxone and morphine (Igwe et al., 1990c). It appears that in addition to an interaction with ~ opioid receptors D A M G O also interacts with the same population of [3H]SP-(1-7) binding sites. Binding studies appear to support this since we have shown that /3-funaltrexamine failed to decrease either SP N-terminal displaceable high-affinity [ 3 H ] D A M G O binding (Krumins et al., 1989a) or [3H]SP-(1-7) binding (Igwe et al., 1990c). The existence of a unique set of SP N-terminal binding sites sensitive to only relatively high concentrations of naloxone is also supported by the present data which indicates that selective opioid antagonists, including /3funaltrexamine, failed to mimic the ability of a high dose of naloxone to protect completely against SP-induced desensitization. The present work confirms our previous study of the development of desensitization to SP and extends these data to indicate that binding sites which are naloxone-, as well as D-SP-(1-7)-, sensitive may be involved in the regulation of tonic SP activity in the spinal cord. Recent microdialysis studies (Brodin et al., 1987) indicate very low basal concentrations of SP in the extracellular fluid of the cat dorsal spinal cord. This, together with our results, suggest that even in the absence of a SP trigger, there may be tonic SP-(1-7) binding site activity that affects the magnitude of SP-induced CBS. The present data are consistent with our hypothesis that an accumulation of SP N-terminal peptide fragments interacts with novel, high-affinity SP N-terminal binding sites to inhibit subsequent SP activity.
201
Acknowledgements This work was supported by United States Public Health Service Grants NIDA04090, DA04190 and DA00124.
References Besson, J. and A. Chouch, 1987, Peripheral and spinal mechanisms of nociception, Physiol. Rev. 67, 67. Brodin, E., B. Linderoth, B. Gazelius and U. Ungerstedt, 1987, In vivo release of substance P in cat dorsal horn studied with microdialysis, Neurosci. Lett. 76, 357. Drower, E.J,, A. Staplefeld, M.F. Rafferty, D,L. Hammond, K.C. Rice and B. DeCosta, 1989, Specific antagonism of the antinociceptive effects of intrathecal DPDPE by naltrindole in rats, Soc. Neurosci. (Abstr.) 303, 756. Hall, M.E. and J.M. Stewart, 1983, Substance P and behavior: opposite effects of N-terminal and C-terminal fragments, Peptides 4, 763. Hylden, J.L.K. and G.L. Wilcox, 1980, Intrathecal morphine in mice: a new technique, Eur. J. Pharmacol. 67, 313. ttylden, J.L.K. and G.L. Wilcox, 1981, Intrathecal substance P elicits a caudally-directed biting and scratching behavior, Brain Res 217, 212. Hylden, J.L.K. and G.L. Wilcox, 1983a, Pharmacological characterization of substance P-induced nociception in mice: modulation by opioid and noradrenergic agonists at the spinal level, J. Pharmacol. Exp. Ther. 226, 398. Hylden, J.L.K. and G.L. Wilcox, 1983b, Intrathecal opioids block a spinal action of substance P in mice: functional importance of both p.- and &receptors, Eur. J. Pharmacol. 86, 95. Igwe, O.J., X. Sun and A.A. Larson, 1990a, Role of substance P amino terminal metabolites in substance P-induced desensitization in mice, Neuroscience 36, 535. Igwe, O.J., X. Sun and A.A. Larson, 199()b, Correlation of substance P-induced desensitization with substance P amino terminal metabolites in the mouse spinal cord, Peptides 11, 817. lgwe, O.J., D.C. Kim, V.S. Seybold and A.A. Larson, 1990c, Specific binding of substance P aminoterminal heptapeptides [SP(1-7)] to mouse brain and spinal cord membranes, J. Neurosci. 10, 3653. Jessell, T.M. and L.L. Iversen, 1977, Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (London) 268, 549. Jessell, T.M., A.W. Mudge, S.E. Leeman and T.L. Yaksh, 1979, Release of substance P and somatostatin, in vivo, from primary afferent terminals in mammalian spinal cord, Soc. Neurosci. (Abstr.) 5, 611. Kellstein, D.E., D.D. Price, R.L. Hayes and D.J. Meyer, 1990, Evidence that substance P selectively modulates C-fiber-evoked discharges of dorsal horn nociceptive neurons, Brain Res. 526, 291.
Krumins, S.A., D.C. Kim and A.A. Larson, 1989a, Substance P modulation of DAMGO binding in the brain of CXBK and Swiss-Webster mice, Peptides 11,281. Krumins, S.A., D.C. Kim, V.S. Seybold and A.A. Larson, 1989b, Modulation of [3H]DAGO binding by substance P (SP) and SP fragments in the mouse brain and spinal cord via mu~ interactions, Neuropeptides 13, 225. Larson, A.A., 1988, Desensitization to intrathecal substance P in mice: possible involvement of opioids, Pain 32, 367. Lembeck, F., 1953, Zur Frage der zentralen lSbertragung afferenter Impulse. IlI. Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Ruckenmarks, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 219, 197. Lembeck, F., J. Donnerer and F.C. Colpaert, 1981, Increase of substance P in primary afferent nerves during chronic pain, Neuropeptides 1, 175. Levine, J.D., R. Clark, M. Devor, C. Helms, M.A. Mokowitz and A.I. Basbaum, 1984, Intraneuronal substance P contributes to the severity of experimental arthritis, Science 226, 547. McCarson, K.E. and B.D. Goldstein, 1990. Time course of the alteration in dorsal horn substance P levels following formalin: blockade by naloxone, Pain 41, 95. Moochhala, S.M. and J. Sawynok, 1984, Hyperalgesia produced by intrathecal substance P and related peptides: desensitization and cross desensitization, Br. J. Pharmacol. 82, 381. Nyberg F., P. Legreves, S. Sunqvist and L. Terenius, 1984, Characterization of substance P(1-7) and (1-8) generating enzyme in human cerebralspinal fluid, Biochem. Biophys. Res. Commun. 125,244. Paul, D., RJ. Bodnar, M.A. Gistrak and G.W. Pasternak, 1989, Different ~ receptor subtypes mediate spinal and supraspinal analgesia in mice, Eur. J. Pharmacol. 168. 307. Pernow, B., 1983, Substance P, Pharmacol. Rev. 35, 86. Piercey, M.F., P.J.K. Dobry, L.A, Schroeder and F.J. Einspahr, 1981, Behavioral evidence that substance P may be a spinal cord neurotransmitter, Brain Res. 210, 407. Sakurada, T., P. Le Greves, J. Stewart and L. Terenius, 1985, Measurement of substance P metabolites in rat CNS, J. Neurochem. 44, 718. Stewart, J.M., M.E. Hall, J. Harkins, R.C.A. Frederickson, L. Terenius, T. H6kfelt and W.A. Krivoy, 1982, A fragment of substance P with specific central activity: SP(1-7), Peptides 3, 851. Sun, X. and A.A. Larson, 1991, Behavioral sensitization to kainic acid and quisqualic acid in mice: comparison to NMDA and substance P responses, J. Neurosci. 11, 311l. Takemori, A.E., B.Y. Ho, J.S. Naeseth and P.S. Portoghese, 1988, Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 246, 255. Yaksh, T.L., T.M. Jessell, R. Gamse, A.W. Mudge and S.E. Leeman, 1980, Intrathecal morphine inhibits substance P release, in vivo, from mammalian spinal cord, Nature (London) 286, 155.
197
~" 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52526
Identification of a novel receptor mediating substance P-induced behavior in the mouse D a r r e l l D. M o u s s e a u , X i a o f e n g Sun a n d Alice A. L a r s o n Department of Veterinary PathoBiology, University of Minnesota, 295 Animal Science/Veterinao' Medicine Building, 1988 Fitch At'enue, Saint Paul, MN 55108, USA Received 23 January 1992, accepted 14 April 1992
To determine whether opioid receptors or the more recently characterized naloxone-sensitive substance P (SP) N-terminal binding sites play a role in desensitization to the behavioral effects of SP, we assessed the effects of selective antagonists at /z(naloxonazine and /3-funaltrexamine), 8- (naltrindole) and •- (nor-binaltorphimine) opioid receptors, as well as the effect of [D-ProZ,D-LeuT]SP-(1-7) D-SP-(1-7) (D-SP (1-7)), an inhibitor of [3H]SP-(1-7) binding, on behaviors induced by intrathecally administered SP in mice. Whereas naloxone, a non-selective opioid antagonist, inhibited the development of behavioral desensitization to SP, the response to repeated SP administration remained unaffected by pretreatment with selective opioid antagonists. Like naloxone, however, the SP-(1-7) antagonist inhibited SP-induced desensitization. The protection against desensitization to SP by D-SP-(1-7), but not by selective antagonists of/z, 6 or ~ receptors, suggests that desensitization to the behavioral effects of SP does not appear to be mediated by an action at an opioid receptor but by an action at the SP-(1-7) binding site. Substance P (N-terminal fragments); Substance P-(1-7); Opioid receptor antagonists; Behavioural desensitization
1. Introduction
The undecapeptide substance P (SP) is a neurot r a n s m i t t e r / n e u r o m o d u l a t o r with a wide distribution throughout the central nervous system. Brain subcortical areas (Pernow, 1983) and the dorsal horn of the spinal cord (Yaksh et at., 1980) exhibit high concentrations of SP, with the primary afferent C-fibre system of the latter region being the proposed site for nociception mediated by this peptide (Kellstein et at., 1990; Besson and Chouch, 1987; Lembeck, 1953). That SPcontaining nerves are involved in nociceptive transmission has been demonstrated in the dorsal horn of the spinal cord where SP release has been observed following activation of nociceptive primary afferents (Lembeck et al., 1981; Yaksh et al., 1980). Both depolarization of spinal tissue (Jessell and Iversen, 1977) and electrical stimulation of primary afferent C-fibres (Jessell et al., 1979) result in increased concentrations of SP in the cerebrospinal fluid. This release is inhib-
Correspondence to: A.A. Larson, Department of Veterinary PathoBiology, University of Minnesota, 295 Animal Science/Veterinary Medicine Building, 1988 Fitch Avenue, Saint Paul, MN 55108, USA. Tel, 1.o12.624 3650, fax 1.612.625 0204.
ited by opioids (Yaksh et al., 1980). Furthermore, an increased innervation of SP-containing neurons and an increased amount of immunoreactive SP in the dorsal horn of the spinal cord have been observed in rat models of chronic nociception (Levine et al., 1984; McCarson and Goldstein, 1990). Intrathecal (i.t.) injection of SP elicits a caudally directed biting and scratching (CBS) behavioral syndrome in mice (Hylden and Wilcox, 1981; Piercey et al., 1981) which, following repeated administration of SP, decreases in intensity with each subsequent injection (Larson, 1988). Desensitization has also been reported to develop to SP-induced hyperalgesia (Moochhala and Sawynok, 1984). Selective p~- and 6-opioid agonists have been shown to inhibit CBS behaviour elicited by a single i.t. injection of SP (Moochhala and Sawynok, 1984; Hylden and Wilcox, 1983a) and the development of behavioural desensitization to repeated i.t. injections of SP is blocked by the non-specific opioid antagonist naloxone (Larson, 1988). This last observation suggests opioid receptor activity in SP-induced behavioral desensitization as naloxone is a nonspecific opioid antagonist. It has also been postulated that behavioral desensitization observed following repeated SP injection may be due to its modulation by its N-terminal metabolites, the heptapeptide SP-(1-7) be-
198 ing the predominant fragment (Nyberg et al., 1984; Sakurada et al., 1985). In fact, exogenous SP-(1-7) is capable of inhibiting SP-induced behavior in a naloxone-reversible manner (Igwe et al., 1990a), while the accumulation of SP-(1-7) following repeated SP injection correlates well with behavioral desensitization (Igwe et al., 1990b). Stewart et al. (1982) reported an antinociceptive effect of SP-(1-7) which was naloxonereversible. Subsequently, it was shown that the C- and N-terminal fragments of SP exert opposite effects in terms of nociception and motor activity (Hall and Stewart, 1983). It is therefore possible that the Nterminal metabolites may play a role in both the modulation of pain as well as SP-induced behavioral desensitization. We characterized the interaction between SP and opioid receptors by comparing the effect of selective opioid antagonists on SP-induced behavioral desensitization. In addition, the SP-(1-7) antagonist [DPro2,D-PheT]-sP-(1-7) (D-SP-(1-7)) was investigated in hopes of elucidating whether behavioral desensitization observed following repeated SP administration might be due to activity at the [3H]SP-(1-7) binding site described by Igwe et al. (1990c).
2. Materials and methods
2.1. Animals Male Swiss-Webster mice (Sasco Inc., Omaha, NE) weighing 20-25 g, were housed four per cage in a room maintained at 21-22°C and on a 12 h: 12 h light/dark cycle (lights on at 6:00 a.m.). Animals had free access to food and water. Animals were used strictly in accordance with the Guidelines of the University of Minnesota Animal Care and Use Committee and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHEW publication (N.I.H.) 78-23, revised 1978). 2.2. Drug administration All drugs were administered via i.t. injection unless otherwise indicated. All injections, whether single or multiple (repeated), consisted of a 5 /xl volume (per injection) of the appropriate vehicle either alone (control) or containing the treatment drug. Animals were randomly assigned to treatment groups. Single injections were made according to the method of Hylden and Wilcox (1980). Briefly, a 30-gauge 0.5 inch disposable needle was attached to a 50/zl Luer-tip Microliter syringe (Hamilton, Reno, NV). Unanesthetized mice were held by the iliac crest and the needle was inserted at the L5-L6 intervertebral space. In the case of repeated injections, a slight modification to the needle
was made wherein a 25 cm length of PE-10 tubing, equipped with the barrel of a 30-gauge 0.5 inch needle, was mated to the disposable needle tip attached to the Hamilton syringe. This 'cannula' allowed for i.t. drug administration without the direct handling of, and any potential associated stress to, the animal. Administration of any drug was delayed for a 1 min period following placement of the cannula to allow the animal to acclimate to the presence of the needle. Between animals, the PE-10 tubing was flushed with the drug solution so as to avoid potential dilution of the drug dosage due to adsorption of the drug to the tubing wall, or to temperature-dependent changes in the structure of SP. All drugs were dissolved in normal saline except SP, SP-(1-7) and D-SP-(1-7)which were dissolved in acidified saline (0.85% saline containing 0.01 N acetic acid) to minimize adsorption to synthetic surfaces (Hall and Stewart, 1983). The dose of SP (10 ng (7.5 pmol); 4 × at 2 min intervals) chosen for the present experiments is known to elicit CBS behaviour (Hylden and Wilcox, 1981; Piercey et al., 1981) of an intensity which could be attenuated or potentiated without inducing seizure activity. SP-(1-7) (20 ng) was coadministered with SP, whereas D-SP-(1-7) was administered as a single injection pretreatment (1 ~g, 5 rain). Opioid antagonists employed in this series of experiments were administered as a single dose pretreatment. Doses of selective antagonists were obtained from the literature based on their ability to selectively antagonize opioid receptor subtypes: naloxone, a non-selective opioid antagonist (1 tzg, 15 min); naloxonazine, selective for high-affinity (/z 1)/z receptors (Paul et al., 1989; 35 mg/kg i.p., 24 h); /3-funaltrexamine, selective for low-affinity (/-*2) /~ receptors (Hylden and Wilcox, 1983b; 0.5 t~g, 24 h); naltrindole, selective for the 3 receptor subtype (Drower et al., 1989; 0.1 ~g, 24 h); nor-binaltorphimine, selective for the K receptor subtype (Takemori et al., 1988, 4 ~g, 15 min). Naloxonazine, /3-funaltrexamine and naltrindole were purchased from Research Biochemicals Incorporated (Natick, MA). Nor-binaltorphimine was kindly provided by Dr. A.E. Takemori, Department of Pharmacology, University of Minnesota (Minneapolis, MN). Naloxone hydrochloride was a gift from Endo Laboratory (Garden City, NY). Substance P and SP-(1-7) were purchased from Peninsula Laboratories (Belmont, CA). D-SP-(1-7) was synthesized by the University of Minnesota Microchemical Facilities (Minneapolis, MN).
2.3. Behavioral testing Immediately following the insertion of the cannula, the test animal (n = 5 - 6 per treatment group) was placed in a large (2 1) glass cylinder. After the appropriate acclimation period (see above), SP was injected
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and the intensity of CBS behaviours were recorded. Each incident (i.e. a bite or a scratch) was counted as a single CBS behaviour. In the event that the duration of CBS activity extended beyond that of a 'single' bite or scratch, then the duration, in seconds (one count per 1 s), was used as an index of CBS activity.
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2.4. Statistical analysis Animals were randomly assigned to treatment groups. Behavioural desensitization was defined as the decrease in the average number of CBS behaviours induced by the fourth injection of SP compared to that following the first injection for each individual animal. Behavioral data are expressed as means + S.E.M. in CBS behaviors for each group and were analyzed by the two-tailed unpaired Student's t-test or by A N O V A (followed by the Dunnett's test for significance) using the Macintosh StatView TM program. Statistical significance was determined as P < 0.05.
3. Results
The effect of a single i.t. injection of SP on CBS activity is depicted in fig. 1. SP-induced CBS behavior was significantly inhibited when coadministered with the SP-(1-7) metabolite. This effect of SP-(1-7) was blocked by pretreatment with the SP-(1-7) antagonist D-SP-(1-7). D-SP-(1-7) did not elicit an effect on SP-induced CBS activity on its own. Repeated injections with SP resulted in a successive decrease in CBS
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Time (min) Fig, 2. Reduction in the behavioral response to repeated (4 x 10 ng) i.t. injections of SP (open squares). Shaded squares depict the effect of repeated injections with the acid saline vehicle. The extent of desensitization in each treatment group described in fig. 3 is determined as the mean difference between the first and fourth injection, or A and B, as shown above.
behaviour (i.e. behavioral desensitization) in every animal tested in this manner (fig. 2). The acid saline vehicle did not elicit any significant CBS activity. Pretreatment with 1/xg of D-SP-(1-7) resulted in a reduction of the extent of desensitization when compared to saline-pretreated control mice, whereas pretreatment with 1 /xg of naloxone completely blocked the SP-induced desensitization (fig. 3). The effects of single injection pretreatments with the opioid antagonists chosen for this study, at individual doses previously shown to be selective for subtypes of opioid receptors, are displayed next to their respective vehicle controls for ease of interpretation (fig. 3). Neither the selective /z-antagonists naloxonazine (i.e. /x~) and /3-funaltrexamine (i.e. /x2), nor the 3- and K receptor antagonists naltrindole and nor-binaltorphimine, respectively, were effective at inhibiting behavioral desensitization.
@ 4. D i s c u s s i o n SP
SP
SP
SP
D-SP(1-7)
SP(1-T)
SP(1.7) D-SP(I-7)
Treatment
Fig. 1. Effect of i.t. administration of the SP N-terminal metabolite SP-(1-7) (20 ng, 0 min) and the SP-(1-7) antagonist D-SP-(1-7) (1 /zg, 5 min) on SP (10 ng)-induced CBS behaviors. SP-induced CBS activity is attenuated by SP-(1-7) (t p < 0.05 using A N O V A followed by Dunnett's test). This attenuation is inhibited by pretreatment with the SP-(1-7) antagonist D-SP-(1-7).
The CBS model, useful as a reflection of the activities of compounds in vivo, was chosen to study the interaction of SP with receptors believed to be involved in the mediation of nociceptive (both tonic and chronic) information. The behaviors may reflect pain perception, but more importantly, it is a simple and reproducible system that reflects quantifiable activity produced by stimulation of receptors that are postulated
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Fig. 3. Effect of the SP-(1-7) antagonist D-SP-(1-7) (1 /,g, 5 rain) and non-selective (NAL: naloxone, 1 p-g, 15 rain) and selective (NLX: naloxonazine (P-l), 35 mg/kg i.p., 24 h; /3-FNA: /3funaltrexamine (P-2), 0.5 p-g, 24 h; nor-BNI: nor-binaltorphimine (K), 4 p.g, 15 rain; NTL: naltrindole (6), 0.1 p-g, 24 h) opioid antagonists on the extent of SP-induced behavioral desensitization. The extent of behavioral desensitizationin each treatment group is defined as the mean difference between the 1st and 4th injection (i.e. A and B shown in fig. 1) in a series of four injections. SP-induced behavioral desensitization was attenuated by both naloxone and D-SP-(1-7) (* P < 0.05 using Student's t-test) but remained unaffected by selective opioid antagonists.
to be important in pain transmission at the level of the spinal cord. Using this model, we have previously shown that there is an attenuation of CBS behaviors in response to repeated intrathecal injections of SP (Larson, 1988) and N-methyl-D-aspartate (Sun and Larson, 1991) while a similar injection protocol using kainic acid or quisqualic acid results in sensitization of the CBS effects (Sun and Larson, 1991). We refer to this diminution of CBS behaviors as a result of repeated SP and N-methyl-D-aspartate administration as behavioral 'desensitization'. Pretreatment with naloxone inhibited the development of behavioral desensitization to SP, thus confirming our previous results (Larson, 1988). Subsequent use of more selective opioid antagonists, and the analysis of their effects on SP-induced behavioral desensitization, allowed us to determine which opioid receptors might be involved in this phenomenon. It is apparent from the data depicted in fig. 3 that, of the opioid receptor antagonists chosen for the present study, all failed to prevent SP-induced desensitization. It would therefore a p p e a r that SP-induced behavioral desensitization is mediated by an action at a naloxone-sensitive receptor which is not one of the classical opioid receptors.
The p h e n o m e n o n of desensitization is consistent with SP's role as a nociceptive transmitter that has the capacity to initiate the rapid development of an endogenously mediated, naloxone-reversible analgesia. The mechanism by which naloxone-reversible desensitization to SP occurs is not fully understood. We have gathered evidence, however, to support the hypothesis that SP is metabolized in the spinal cord to N-terminal peptide fragments (Igwe et al., 1990b). That these fragments are important in the development of desensitization to SP is reflected by their ability to attenuate CBS activity in response to SP (Igwe et al., 1990a; present study) and by the ability of the SP-(1-7) antagonist D-SP-(1-7) to inhibit SP-induced desensitization (present study). This desensitization may occur by a direct interaction of the metabolites with a /3funaltrexamine-insensitive subset of high-affinity, /~selective, opioid receptors, as SP-(1-7) has been found to interact with this select subset of [D-AIa 2, M e P h e 4,Gly-(ol)5]enkephalin ( D A M G O ) - [ a b e l l e d binding sites (Krumins et al., 1989a, b). Tritiated Nterminal fragments of SP have also been found to label a novel population of [3H]SP-(1-7) binding sites that are displaced by D A M G O but relatively insensitive to /3-funaltrexamine, sufentanyl, naloxone and morphine (Igwe et al., 1990c). It appears that in addition to an interaction with ~ opioid receptors D A M G O also interacts with the same population of [3H]SP-(1-7) binding sites. Binding studies appear to support this since we have shown that /3-funaltrexamine failed to decrease either SP N-terminal displaceable high-affinity [ 3 H ] D A M G O binding (Krumins et al., 1989a) or [3H]SP-(1-7) binding (Igwe et al., 1990c). The existence of a unique set of SP N-terminal binding sites sensitive to only relatively high concentrations of naloxone is also supported by the present data which indicates that selective opioid antagonists, including /3funaltrexamine, failed to mimic the ability of a high dose of naloxone to protect completely against SP-induced desensitization. The present work confirms our previous study of the development of desensitization to SP and extends these data to indicate that binding sites which are naloxone-, as well as D-SP-(1-7)-, sensitive may be involved in the regulation of tonic SP activity in the spinal cord. Recent microdialysis studies (Brodin et al., 1987) indicate very low basal concentrations of SP in the extracellular fluid of the cat dorsal spinal cord. This, together with our results, suggest that even in the absence of a SP trigger, there may be tonic SP-(1-7) binding site activity that affects the magnitude of SP-induced CBS. The present data are consistent with our hypothesis that an accumulation of SP N-terminal peptide fragments interacts with novel, high-affinity SP N-terminal binding sites to inhibit subsequent SP activity.
201
Acknowledgements This work was supported by United States Public Health Service Grants NIDA04090, DA04190 and DA00124.
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