Naltrexone, but not atropine or yohimbine, antagonizes suppression of formalin-induced spinal sensitization by intrathecal nociceptin

p11 8o&%%3205(98)00359-2

ELSEVIER

Life Sciences, Vol. 63, No. 11, pp. PL 167-173,1998 Coppight o 1998 Ekvier Science Inc. Printed in the USA. All rights resewed 0024-3%s/98 $19.00 + .I0

PhTARMACOLOGY LETTERS Accelemted Communication

NALTREXONE, SUPPRESSION

BUT

NOT

ATROPINE

OR YOHIMBINE,

OF FORMALIN-INDUCED INTRATHECAL

SPINAL

ANTAGONIZES

SENSITIZATION

BY

NOCICEPTIN

Shuanglin Hao and Hidemichi

Ogawa

Department of Anesthesiology and Critical Care Medicine Asahikawa Medical College, 078-85 10, Japan (Submitted February 24, 1998; accepted June 13, 1998; received in final form June 30, 1998)

Abstract We investigated the effects of spinal nociceptin on formalin-induced spinal sensitization and examined the role of the opioidergic, alpha 2-adrenergic and muscarinic cholinergicreceptors in the nociceptin-produced suppression ofspinal sensitization. The results demonstrated that spinal nociceptin suppressed the formalin-induced spinal sensitization in a dose-dependent manner ( 1, 5 and 10 nmol). The inhibitory effect of 10 nmol of nociceptin on spinal sensitization, was readily antagonized by naltrexone, but not by atropine or yohimbine. Each ofthe antagonists, naltrexone, atropine or yohimbine, alone had no effect on the formalin-induced spinal sensitization. Our results show that spinal nociceptin elicits dose-dependent, naltrexone-reversible suppression of spinal sensitization evoked by injection of formalin. 0 1998 Elsevier Science Inc.

Key Words: nociceptin, formalin test, naltrexone, atropine, yohimbine

Introduction The Opioid Receptor-like I (ORLi) receptor has been identified as a G protein-coupled receptor whose amino acid sequenceis closest to those ofopioid receptors (1). Nociceptin, a newly identified heptadecapeptide, is an endogenous ligand for the ORLr receptor (2,3). This heptadecapeptide shares considerable structural homology with other endogenous opioid peptides and binds with high affinity and selectivity to its receptor (2,3). The wide distribution of ORLt n-RNA and Corresponding author: Shuanglin Hao, MD, Department of Anesthesiology, Asahikawa College, 078-85 10, Japan, Fax: 81-166-659443, E-mail: haoaasahikawa-med.ac jp

Medical

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nociceptin precursor in the central nervous system(CNS), particularly in the limbic system regions and in several areas known to be involved in pam perception, suggests that nociceptin is potentially endowed with various central functions (4). Although transcripts ofthe ORLI receptor are expressed in several brain regions including the dorsal horn of the spinal cord (5-7). the physiological significance of the receptor is poorly understood Nociceptin performs a complex series of pharmacological actions. Lnitial studies reported that intracerebroventricularly administered nociceptin produced thermal hyperalgesia in the mouse, an action opposite to that typically seen with the opioid peptides (2,3). But among a numberof other actions that have been examined, the most intri,gtring has been the observation that nociceptin can elicit spinal analgesia in different nociceptive tests (8 11). Electrophysiological evidence showed that mtrathecally administered nociceptin dose-relatedly inhibited the C-fiber evoked wind-up and post-discharge of dorsal horn neurons ( 12). The antinociceptive effect of spinal nociceptin was not reversed by antagonists of opioidergic, alpha 2-adrenergrc or pammaaminobutyric acid (GABA)receptors in a flexor reflex model ( 10) The formalin test is different from most nociceptive tests wrth mechanical or thermal stimuli in that tt allows the assessment of animal responses to continuous pam generated by tissue injury The present study sought to define the effects of nociceptin on formalin-induced sensitization in the spinal cord and to determine the role of opioid, noradrenergic and choline& receptors in mediating the action of nociceptin.

Methods Anrmals and lntrathecal catheters The study was carried out under a protocol approved by the Institutional Animal Care Committee ofAsahikawa Medical College. All rats (Male Sprague-Dawley, 250-3 50g) were housed in individual cages with free access to food and water and maintained on a 12 hour light-dark cycle at an ambient temperature of 20-30 “C. Chronic intrathecal catheters were implanted under the isoflurane anesthesia as previously described (13). Briefly, through an incision in the atlanto-occipital membrane, a polyethylene (PE- 10) catheter, filled wtth 0 9% saline, was advanced 8 cm caudally to position its tip at the level of the lumbar enlargement. The rostra1 tip of the catheter was passed subcutaneously, externalized on top of the skull, and sealed with a stainless steel plug. Animals showing neurological deficits after implantation were excluded. Rats were not used sooner than 4 days nor later than 10 days after implantation Formalin Test For formalin injection, 50 K 1 of 5% formalin was injected subcutaneously into the dorsal surface of the right hind paw using 27-G needle. Animals were then placed in a clear Plexiglas cylinder (20 X30 cm) for observation. A mirror was placed below the floor (Plexiglas) at a 45 ’ angle for unencumbered observation during the test. The number of flinches per minute was counted 1 and 5 min after formalin injection (phase 1) and at 5 min intervals thereafter for 60 min (phase 2). Criteria for exclusion from the study included more than 20% weight loss, incomplete formalin injection, or excessive bleeding from injection site

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Experimental paradigms Saline was injected intrathecally for control data (group 1). For the dose-response study, 1,5 and 10 nmol of nociceptin were administered intrathecally 20 min before the formalin injection for groups 2-4, respectively. In separate groups, we studied whether the effects of nociceptin on the formalin test were mediated by spinal opioid receptors, muscarinic receptoror alpha 2-adrenergic receptor activity. 10 nmol naltrexone (group 5), 35 nmol atropine (group 6) or 28 nmol yohimbine (group 7) was coadministered intrathecally with 10 nmol nociceptin, respectively, 20 min before the formalin injection. To study whether antagonists have any effects on the formalin test, we administered intrathecally 10 nmol naltrexone (group S), 35 nmol atropine (group 9) or 28 nmol yohimbine (group IO), 20 min before formalin injection. Drug and Data analysis Drugs used in the study included nociceptin (Peptide Institute,Osaka, Japan), naltrexone hydrochloride, yohimbine hydrochloride and atropine sulfate (Sigma, St Louis, MO,USA). The intrathecally administered agents were delivered with a microsyringe in a total volume of 10 K 1 followed immediately by a 10 iu.1saline to flush the catheter. All agents were dissolved in saline such that the final dose was delivered in 10 M1. The total number of flinches was recorded for phase 1 (O-10 min) and phase 2 (lo-60 min) for each animal, and data were compared by one-way analysis of variance (StatView J 4.2). Post hoc comparisons were done using Scheffe’ s F test.

Results Subcutaneous injection of formalin resulted in a reliable biphasic display of flinching of the injected paw in intrathecally saline-administered rats (group 1) (Table 1). Intrathecally administered nociceptin decreased the number of flinches in both phase 1 and phase 2 in a dose-dependent manner. Although 1 nmol nociceptin (group 2) did not suppress either phase 1 or phase 2 activities, 5 nmol nociceptin (group 3) showed a significant suppression of both phase 1 and phase 2 activities (pO.O5) (group 8-10).

Discussion The results of this study demonstrate that intrathecally administered nociceptin decreased both the phase 1 and phase 2 activities in a dose dependent manner. The antinociceptive effects of nociceptin on the formalin test were antagonized by naltrexone, but not by atropine or yohimbine.

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Spinal Nociceptin

TABLE

Total Number

Vol. 63, No. 11, 1998

and Formalin Test

1

of Flinches for Phase 1 and Phase 2 of the Formalin Test

n

phase

1. 10 yl saline

6

2227

705 179

2. 1 nmol

nociceptin

6

18i4

540&53

3. 5 nmol

nociceptin

5

14-t5*

480?118#

5

8k6*

5. 10 nmol nociceptin

5

2Ok4 ::

64Ok81 P

+ 10 nmol naltrexone 6. 10 nmol nociceptin

5

13*3*

301+71

+35 nmol atropine 7. 10 nmol nociceptin

5

lot-5*

8. 10 nmol naltrexone

5

2Ok4

687k50

9. 35 nmol atropine

5

2Ok5

730-t 129

10. 28 nmol yohimbine

5

25-t2

895t- 10

drug

group

4.

10 nmol

nociceptin

1

*

*

phase 2

310293

# ##

# # #

3922 137# # #

+28 nmol yohimbine

Values are mean& SEM.

* Significantly different from group 1 of phase 1 (p
different different

from group 1 of phase 1 (p
,

from group 4 of phase l(p
# Significantly different from group 1 of phase 2 (p
Significantly

f Significantly

different

different

from group

1 of phase 2 (p
from group 4 of phase 2 (p
The mechanism of spinal nociceptive transmission of the formalin test may be different from that of hot plate test or electrical stimulation. Phase1 of the formalin test is representative of a short-lasting burst of small afferent activity Phase 2 is believed to reflect a state of spinal sensitization of the dorsal horn WDR neurons driven by the moderate ongoing peripheral input from C-fibers (14). Repetitive afferent input evokes release of excitatory amino acids that lead to the initiation of a state of hyperexcitability (wind-up phenomenon), mediated in part by activation of N-methyl-D-aspartate (NMDA) receptors. Wind-up phenomenon is reported to be inhibited by NMDA receptor antagonist (15)

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Electrophysiological evidence demonstrated that nociceptin applied microiontophoretically or intracerebroventricularly produced a predominantly inhibitory modulation of NMDA-evoked responses of nociceptive neurons (16). IntrathecaJly administered nociceptin dose-relatedly inhibited the C-fiber evoked wind-up and post-discharge of dorsal horn neurons, but not the baseline C-fiber evoked response (12). Nociceptin was reported to inhibit glutamate@ transmission and appears to be acting as an inhibitory peptide at the spinal level through a naloxone-insensitive opioid receptor (17). These electrophysiological data show that nociceptin can suppress the glutamatergic transmission and reduce the C-fiber activity. This pharmacological effect of nociceptin on the wind-up phenomena is comparable with that of nociceptin on the formalin test. Thus, we think that one of the antinociceptive mechanisms of nociceptin is through inhibiting transmission of excitatory amino acids anddepressing the C-fiber activity in the formalin test. The site and mechanism of analgesia produced by nociceptin remains unclear. Supraspinally,two initial studies demonstrated that nociceptin produces hyperalgesia (2,3), while nociceptin was also reported to produce a delayed analgesia which was readily reversed by opioid antagonists (18,19). Spinally, on the other hand, nociceptin produced antinociception in the rat formalin test, but the antinociceptive effect was not reversed by naloxone (20, 21) However, naltrexone was reported to antagonize the analgesic effect of spinal nociceptin in the tail flick assay (8). Our results showed that nociceptin produced naltrexone-reversible suppression of formalin-induced spinal sensitization in a dose-dependent manner. An interesting result was that supraspinal nociceptin has no effect on basal tail-flick latency by itself, but antagonizes systemic morphine analgesia, and that spinal nociceptin displays an analgesic effect and potentiates morphine analgesia, demonstrating that nociceptin has bidirectional modulatory effects (22). Thus, the different results from the different sites of administration of nociceptin indicate that nociceptin performs a complex series of pharmacological characteristics. Also, there is no cross-tolerance between nociceptin and morphine in eliciting spinal antinociception, supporting the notion that nociception produced spinal antinociception through a site which is different from opioid receptors (11). Opioid receptors are localized in the same area of the spinal cord as muscarinic receptors (23). Evidence that systemic or spinal morphine analgesia was potentiated by physostigmine and inhibited by a&opine (24-26), demonstrated that cholinergic mechanisms were relevant to opiate effectiveness (27). Our results showed that atropine, a muscarinic choline@ antagonist, did not reverse the inhibitory effect of nociceptin on the formalin-induced sensitization, suggesting that spinal action of nociceptin was not mediated by spinal muscarinic cholinergic system. Electrophysiological results showed that the reflex depression produced by nociceptin was not reversed by alpha 2- adrenergic antagonists in flexor reflex model (IO). Our study also indicated that yohimbine did not block the action of nociceptin in the formalin test. The present results showed that intrathecal nociceptin suppressed spinal sensitization induced by injection of formalin in a dose dependent manner, which is reversed by naltrexone, but not by atropine and yohimbine. From the references above and our data, nociceptin performs acomplex series ofpharmacological actions. Although, up to now, we have no selective antagonists for the OIW receptor to show that the effect ofnociceptin on the formalin-induced

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spinal sensitization is mediated through the activition of ORLt receptor, the effect of nociceptin is presumably mediated by the ORLr receptor rather than by classical opioid receptors

Acknowledgement

We wish to thank Dr T. Yamamoto, Department of Anesthesioloa, and Dr. M. Kubota,

assistant

professor

of Our Department

Simon Bayley and Dr. Noel Whelan for checking

Chiba University,

for their help

Japan,

We also thank Dr.

English of the manuscript.

References

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