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Differential effects of specific δ and κ opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain
Brain Research, 623 (1993) 201-207 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
201
BRES 19236
Differential effects of specific and K opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain I. Cattaneo,
V. Kayser
and G. Guilbaud
Unit~ de Recherches de Physiopharmacologie du Syst~meNerveux, INSERM, Paris (France)
(Accepted 27 April 1993)
Key words: Polyarthritic rat; Vocalization threshold; Paw pressure; Endogenous opioid system; Naloxone; Naltrindole; MR-2266
In an attempt to determine the opioid receptor class(es) which underly the two opposing effects of naloxone in models of persistent pain, we tested the action of the selective t5 antagonist naltrindole, and that of the K antagonist MR-2266 on the bidirectional effect of systemic naloxone in arthritic rats. As a nociceptive test, we used the measure of the vocalization thresholds to paw pressure. The antagonists were administered at a dose (1 mg/kg i.v. naltrindole, 0.2 mg/kg i.v. MR-2266), without action per se, but which prevents the analgesic effect of the ~ agonist DTLET (3 mg/kg, i.v.) or the K agonist U-69,593 (1.5 mg/kg, i.v.) respectively, and does not influence the effect of morphine (1 mg/kg i.v.) or the/~ agonist DAMGO (2 mg/kg, i.v.) in these animals. In arthritic rats injected with the 8 antagonist, the paradoxical antinociceptive effect produced by 3/zg/kg i.v. naloxone was not significantly modified (maximal vocalization thresholds (% of control) were 146 _+9% versus 161 + 7% in the control group). By contrast, the hyperalgesic effect produced by 1 mg/kg i.v. naloxone was significantly reduced (maximal vocalization thresholds were 87+4% versus 69+5% in the control group). In rats injected with the K antagonist, the antinociceptive effect of the low dose of naloxone was almost abolished (mean vocalization thresholds were 115 _+3% versus 169 5: 7%) whereas the hyperalgesic effect of naloxone 1 mg/kg i.v. was not significantly modified (mean vocalization thresholds = 70 + 3% and 65 + 3%, respectively). Based on these results, the possible role of each receptor subtype in the putative control exerted by endogenous opioid substances on nociceptive messages in pathological conditions is discussed.
INTRODUCTION T h e F r e u n d ' s a d j u v a n t - i n d u c e d a r t h r i t i c rat is now g e n e r a l l y c o n s i d e r e d as a useful m o d e l for t h e study o f p a i n m e c h a n i s m s 6't°']6. O p i o i d s a d m i n i s t e r e d systemically a r e especially active in t h e s e a n i m a l s with persist e n t pain, as g a u g e d by s e v e r a l n o c i c e p t i v e tests, b u t p a r t i c u l a r l y with t h e v o c a l i z a t i o n t h r e s h o l d to p a w p r e s s u r e 19'29'35'42. This e n h a n c e d effectiveness d o e s n o t s e e m directly r e l a t e d to c h a n g e s in ~ - , 3- a n d K-opioid b i n d i n g sites at c e n t r a l levels 5J2'tS. It c o u l d b e p a r t l y d u e to c h a n g e s in e n d o g e n o u s o p i o i d p e p t i d e s . I n d e e d , b i o c h e m i c a l studies have shown t h a t t h e c o n t e n t a n d b i o s y n t h e s i s o f e n d o g e n o u s o p i o i d s in t h e d o r s a l h o r n o f t h e spinal c o r d a r e i n c r e a s e d 6'12'13'21'25'26'39'41'45 while t h e level o f n e u t r a l e n d o p e p t i d a s e is n o t m o d i f i e d 18 in t h e s e animals. Since 1981, we have t e s t e d extensively t h e activity o f e n d o g e n o u s o p i o i d systems with t h e
o p i o i d a n t a g o n i s t n a l o x o n e in this m o d e l of p e r s i s t e n t pain, using t h e v o c a l i z a t i o n t h r e s h o l d to p a w p r e s s u r e as a n o c i c e p t i v e test. W e have d e m o n s t r a t e d r e p e t e a d l y 28'3t t h a t n a l o x o n e i n d u c e s c l e a r b i d i r e c t i o n a l effects in t h e s e animals: high d o s e s (1 to 3 m g / k g , i.v.) of t h e a n t a g o n i s t i n d u c e d f u r t h e r i n c r e a s e s in t h e hyp e r a l g e s i a , suggesting t h a t e n d o g e n o u s o p i o i d p e p t i d e s e x e r t a p a r t i c u l a r l y p o t e n t tonic inhibitory c o n t r o l on n o c i c e p t i v e m e s s a g e s in t h e s e animals. By c o n t r a s t , e x t r e m e l y low d o s e s (3 to 300 t x g / k g , i.v.) elicit p a r a doxical a n a l g e s i a , p e a k i n g at a d o s e of 3 / ~ g / k g . W e h y p o t h e t i z e d 3°'31, s u p p o r t e d by b i o c h e m i c a l s t u d ies 8'22'5°'51, t h a t t h e p a r a d o x i c a l effect m a y result f r o m t h e i n t e r a c t i o n o f n a l o x o n e with highly sensitive r e c e p tors, r e s p o n s i b l e for t h e o n g o i n g s u p p r e s s i o n o f end o g e n o u s o p i o i d r e l e a s e in c o n d i t i o n s o f p e r s i s t e n t pain. I n d e e d , t h e s e b i d i r e c t i o n a l effects o f naloxone, n o t o b s e r v e d in n o r m a l rats, w e r e also f o u n d in o t h e r
Correspondence: V. Kayser, Unit6 de Recherches de Physiopharmacologie du Syst~me Nerveux, INSERM U 161, 2 rue d'Al6sia, 75014 Paris, France.
202 models of persistent inflammatory or neuropathic pain, using the same test 1-2'3'3°'33. The involvement of different opioid receptors in this dose-dependent effect was also suggested by the modifications observed in arthritic rats rendered tolerant to morphine. In these animals the paradoxical antinociceptive effect of the low dose was dramatically decreased, while the high dose of naloxone still induced hyperalgesia31'32'34. To determine the different opioid receptor subtype(s) involved we studied the effects of specific opiold receptor antagonists on the bidirectional effect of naloxone in polyarthritic rats. We report here the data obtained with the ~ opioid antagonist naltrindole and the x antagonist MR2266. Preliminary data have been reported ~~. MATERIALS
AND METHODS
Animals Male Sprague-Dawley arthritic rats, 9 - 1 0 weeks old, weighing 190-260 g, were obtained from Charles River, France. The rats were allowed to habituate to the colony room for at least 7 days before the experiments. Arthritic rats were used 21 days after the induction of arthritis by inoculation at the breeding center (Charles River, France) of Freund's adjuvant at the base of the tail. At this time, the arthritic lesions and the concomitant humoral changes are at a stable maximum 6,t°A6'24. Since these conditions induce a certain amount of suffering, the guidelines proposed by the committee for Research and Ethical Issue of the IASP (1980) 17 have been followed. The food was directly available ad libitum in the sawdust of the cages to minimise the need for the animals to make potentially painful movements to obtain food. The duration of the experiments was as short as possible and the n u m b e r of animals involved kept to a minimum. Since a relatively high number of animals (n = 116) were necessary in several preliminary experiments to determine the suitable doses of antagonist and to control their specificity, the n u m b e r of rats used in the specific study was minimized by excluding vehicle controls, since it was previously verified that acute injection of saline or Fixanal (a buffer solution with a p H equivalent to that of the naloxone solution) does not alter nociceptive thresholds in this assay 31. Behavioural test Testing sessions, beginning at 10 a.m. were conducted in a quiet room remote from the colony room, by a single experimenter. Prior to the test phase, the rats had no experience with the testing apparatus. The vocalization threshold to paw pressure was measured using the Basile Analgesymeter (Apelex) (tip diameter of the probe was 1 mm). Increasing pressure was applied to the hindpaw, until a squeak was elicited. For each rat, a preliminary threshold or control threshold (mean of two consecutive stable thresholds, expressed in grams) was determined before any injection. Nociceptive pressure thresholds were determined every 5 or 10 min after drug administration, until they returned to baseline. Experimental series In a first step, several groups of animals (n = 116 rats) were used in preliminary experiments to determine antagonist doses that we wish to test, and to control their specificity (Table I). Then, to test the effect of each antagonist on the bidirectional effect of naloxone, 4 groups of rats were used (n = 11 or 9 in each group used for experiments related to 6 and K antagonist respectively). Thus, 80 rats were used for this specific study according to the following protocol:
- one control both naloxone - one control both naloxone
group received naloxone 1 m g / k g alone, one group I m g / k g + the antagonist group received naloxone 3 ~ g / k g alone, one group 3 p~g/kg + the antagonist.
Drugs and tests dosages The following drugs and dosages were used, unless specified otherwise: naloxone hydrochloride, 3 /zg and 1 m g / k g (Narcan, Dupont de Nemours, Paris, France), naltrindole hydrochloride, 1 m g / k g ( 17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy6,7-2',3'-indolomorphinan, a fi antagonist, gift from Pr. Roques, Paris, France), and MR-2266, 0.2 m g / k g , (( - )-5,9-diethyl-2-hydroxy2-(3-furylmethyl)-6,7-benzomorphan, K antagonist, gift from Dr. Neil, Uppsala, Finland). Naloxone (3 ~ g and 1 m g / k g ) was given at doses that induce potent analgesic and hyperalgesic effects in arthritic animals 31. The doses of 1 m g / k g of naltrindole and 0.2 m g / k g of MR-2266 were determined by preliminary experiments to be without action per se, but to prevent specifically the analgesic effect of 3 m g / k g of the ~ agonist D T L E T (o-Thr2, Leu5-enkephalyI-Thr, Bachem Feinkemikalien A G Bubendorf, Switzerland) or 1.5 m g / k g of the x agonist U-69,593 (5-,7-,8B-(-)-N-methyl-N(7-(1-pyrrolidinyl)-l-oxaspiro(4,5)-dec-7-8-yl)benzene-acetamide), gift from Dr. Duffet, Upjohn, Paris, France), respectively. We also controlled that these doses of fi and K antagonists did not influence significantly the effects of 1 m g / k g morphine or 2 m g / k g of t h e / , agonist D A M G O (D-Ala2,MePhe4,Gly-ol-5-enkephalin, Bachem Feinkemikalien A G Bubendorf, Switzerland) in these animals (Table I). The doses of agonists used for these controls were choosen according to earlier studies showing that their peak effect was roughly comparable to that of 1 m g / k g i.v. morphine in arthritic rats 29,4z. Drugs were freshly prepared in sterile physiological saline (0.9% NaC1) and all injections were given intravenously into the lateral tail vein. Statistical procedures Post-drugs thresholds were calculated for each animal as percentages of m e a n control thresholds. Statistical calculations were done with a one- or multiple-way analysis of variance ( A N O V A procedure), Tukey test (with vocalization thresholds expressed in grams) or Student's t-test (with vocalization thresholds expressed as percentage of the control values) for comparison of the area under the curves. Differences were considered significant for P-values < 0.05. TABLE 1 Control of the specificity of the antagonists naltrindole and MR-2266 towards tz, 6 and K agonists
morphine DAMGO DTLET U-69,593
Ant. = 0
naltrindole
MR-226
191 _+6 n=t3 171 + 10 n=10 1425:6 n=8 215 + 27 n=8
103+4 n=7 191 _+9 n=ll 170 +_39 n=7 1025:4 * * n=7 207 5:55 n=6
114+4 n=10 184 5:7 n=8 163 _+5 n=7 151 _+ 11 n=5 111 + 6 * * n=9
Mean peak effects of: the 6-antagonist, naltrindole (1 m g / k g i.v.) and the K-antagonist, MR-2266 (0.2 m g / k g , i.v.) administered alone, the agonists, morphine (1 m g / k g , i.v.), D A M G O (2 m g / k g , i.v.), D T L E T (3 m g / k g , i.v.) and U-69,593 (1.5 m g / k g , i.v.) injected alone (ant = 0) or co-injected with naltrindole or MR-2266. The vocalization threshold to paw pressure was determined every 5 or 10 rain after drug administration, until they returned to baseline. Each value is expressed as a percentage of the baseline threshold_+S.E.M., n = n u m b e r of rats per group. * * Denotes difference from the corresponding control group receiving the agonist only at the P < 0.01 level, Tukey test. Time of maximum effect of the drugs: naltrindole and M R - 2 2 6 6 : 1 5 min; morphine: 30 min; D A M G O : 20 min; DTLET: 20 min; U-69,593:40 min.
203 RESULTS
260
Before any drug injection, the vocalization threshold to paw pressure was similar for the various animals. The m e a n control threshold determined for all the arthritic rats used in the experiments to test the effect of the antagonists on the bidirectional effect of naloxone was 148 + 12 g (n = 80), roughly comparable to previous studies 28'31.
t, >
220
u
180
[ ] control [ ] NTI-treated [ ] MR2266-treatecl rats
140 "0
100
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60
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20
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Effects of the antagonists on the hyperalgesic effect of naloxone
5_
Naloxone 1 mg/kg NALOXONE 3 ug/kg
-60
-I00
Effect of the 6 antagonist naltrindole, 1 m g / kg Lv. In the control group of rats receiving 1 m g / k g i.v. naloxone alone, an overall significant hyperalgesic effect (F7,79---10.37; A N O V A , P < 0.001, n = 11) was elicited, as described in previous studies (Fig. 1A). The effect was significant at 5 min, and maximal at 15 min
A o 100
§ 8O
"="*~9/kg **
60
iv MR-2266
T
•
+ NTI
**
•
+
B o
100
u
x
-140
Fig. 2. Mean areas under the curves ( + S . E . M . ) for naloxone 3 # g / k g i.v. and naloxone 1 m g / k g i.v. injected alone (open bars) or co-injected with naltrindole (NTI) 1 m g / k g i.v. (hatched bars) or MR-2266 0.2 m g / k g i.v. (filled bars), as illustrated in Fig. 1 and Fig. 3. Total response area (percentage increase from baseline x min) was calculated for the whole duration of the response in each animal. ** * Denotes difference from the corresponding control rat group at the P < 0.001 level, Student's t-test.
after the injection. At this time, the m e a n vocalization threshold expressed as percentage of preinjection value was 69 + 5% ( P < 0.01, Tukey test, n = 11). Recovery was observed by 35-40 min. In rats receiving both 1 m g / k g i.v. naloxone and naltrindole, the hyperalgesic effect was markedly reduced (F7,79 = 4.44; A N O V A , P < 0.001, n = 11), although still significant at 10 min (Fig. 1A). At this time, the mean value was 87 + 4% of the control ( P < 0.05, Tukey test, n = 11). The total recovery was reached at 25 min after the injection (difference from the controls for the overall effect of naloxone: Fl,173 = 26.1, A N O V A , P < 0.001, n = 22). The difference between the effects of naloxone in the two groups of rats is also well illustrated by comparison between the mean areas under the curves calculated for naloxone 1 m g / k g i.v. and naloxone 1 m g / k g i.v. + naltrindole (Fig. 2).
80
Effect of the K-antagonist MR-2266, 0.2 mg / kg i.v. 60
** I
I
I
I
10
20
30
40 min
Fig. 1. M e a n curves illustrating the effects on the vocalization threshold to paw pressure of naloxone (Nx) 1 m g / k g i.v. injected alone or co-injected with: (A) naltrindole (NTI) 1 m g / k g i.v., (B) MR-2266, 0.2 m g / k g i.v. Each value is expressed as a percentage _+S.E.M. of the control threshold value m e a s u r e d just before injection. Tukey test was used for statistical analysis (with vocalization thresholds expressed in grams): * P < 0.05, * * P < 0.01. T h e overall effect of each dose was calculated with a multiple-way analysis of variance ( A N O V A procedure), fully described in the results paragraph, n = 11 rats in each group in A; n = 9 rats in each group in B.
In the control group of rats receiving 1 m g / k g i.v. naloxone alone, a significant hyperalgesic effect (F7, 63 = 13.5; A N O V A , P < 0.001, n = 9), comparable to that described above, was elicited (Fig. 1B). The effect was clear 5 min after injection, and maximal at 15 min. At this time, the mean vocalization threshold was 65 _+ 3% of the pre-injection control value ( P < 0.01, Tukey test, n = 9). In rats receiving both 1 m g / k g i.v. naloxone and MR-2266, the hyperalgesic effect was still observed (F7,63 = 15.2; A N O V A , P < 0.001, n = 9), comparable to that in the control group of rats. The mean peak
204 value was 70 + 3% of the control ( P < 0.01, Tukey test, n = 9), 15 min after the injection. Thus, in arthritic rats, the hyperalgesic effect of naloxone 1 m g / k g i.v. was greatly reduced by naltrindole and not influenced by MR-2266.
Effects of the antagonists on the paradoxical antinociceptive effect of naloxone Effect of the ~-antagonist naltrindole In the control group of rats receiving 3 tzg/kg i.v. naloxone alone, an overall significant antinociceptive effect (F7.55 = 22.1, ANOVA, P < 0.001, n = 9) was observed, as described in previous studies (Fig. 3A). Starting 5 min after the injection it was maximum at 15 rain (mean vocalization threshold was = 161 + 7% of the control value, P < 0.01, Tukey test, n = 9). The recovery was reached progressively at 40 min after the injection. In naltrindole-injected rats, there was still a potent antinociceptive effect of the low dose of naloxone
"K" "X"
A
-6 160 "X" "X"
(F7,55 = 20.6, ANOVA, P < 0.001, n = 9). The mean peak value was 146 _+ 9% of the control value ( P < 0.01, Tukey test, n = 9) and the whole time course was not markedly affected (Figs. 2 and 3A).
Effect of the K-antagonist MR-2266 In the control group receiving 3 / z g / k g i.v. naloxone, significant antinociceptive effect (F7.55= 21.8, ANOVA, P < 0.001, n = 8) was observed (Fig. 3B). Starting 5 min after the injection it was 169 + 7% of the control value at 15 min ( P < 0.01, Tukey test, n -- 8). The recovery to controls was reached at 40 min. In MR-2266-injected rats, the antinociceptive effect of 3 /zg/kg i.v. naloxone (F7.55 = 3.7, ANOVA, P < 0.05, n = 8) was profoundly decreased (difference between the two groups of rats F1,126 = 41.59, ANOVA, P < 0.001, n = 16 rats) (Fig. 3B). The maximal increase in vocalization threshold was 115 + 3% of the control ( P < 0.05, Tukey test, n = 8) at 20 min, reduced by about 60%. These results are clearly seen by comparison of the mean areas under the curves, calculated for the two doses of naloxone in each group of arthritic rats (Fig. 2).
"X" "K"
o u
DISCUSSION 140
")('"IF
o
120
100
h.>.i 1 t'?l I
f
f
t
I
160
I
•
o
+
MR-2266
o
~ 140
120
100 I i
i
i
i
10
20
30
40 rnin
Fig. 3. Mean curves illustrating the effects of naloxone (Nx) 3/~g/kg i.v. injected alone or co-injected with: (A) naltrindole (NTI) 1 mg/kg i.v., (B) MR-2266, 0.2 mg/kg i.v.. Each value is expressed as a percentage +S.E.M. of the control threshold value measured just before injection, n = 9 rats in each group. * P < 0.05, ** P < 0.01, Tukey test (with vocalization thresholds expressed in g).
The present data clearly confirm that the bidirectional dose-dependent effects of naloxone in arthritic rats results from the interaction of the drug with opioid receptors of different characteristics, as suggested by extensive behavioural studies performed in this model and in models of localized inflammatory or neuropathic pain ~'2'3°'31. Simultaneously, they provide some indications on the putative role of receptor subtypes with 6- and K-site characteristics in the control of nociceptive messages exerted by endogenous opioid peptides under pathological conditions. (1) We observed that the hyperalgesic effect produced by the high dose of naloxone (1 mg/kg) in arthritic rats, was significantly reduced by the selective ~ antagonist naltrindole (by about 3 - 4 fold when gauged by the mean areas under the curves). By contrast, the same close of the 3 antagonist did not influence the paradoxical antinociceptive effect produced by the low dose of naloxone in these animals. It should be noted that the same effects were obtained with naltrindole co-injected with naloxone, and with another 6-antagonist ICI-174,864 (10 mg/kg, i.v.) or when naltrindole was administered 15 min before naloxone (not shown). The antagonists were used at doses without action per se, without influence on the antinociceptive effect of K- and /z-preferential ago-
205 nists, but which prevent selectively the antinociceptive effect of the 6-agonists DTLET (3 mg/kg) or BUBU (3 mg/kg) in arthritic rats 19. The functional relationship between endogenous opioid peptides and the types of opioid receptors remains unclear 23'46, with the results obtained with enkephalinase inhibitors suggesting the activation of either tJ,14 o r 3 9,20 opioid receptors by the protected endogenous opioids to produce antinociception. The results obtained here, with both naltrindole and ICI174,864, seem to indicate that ~ opioid receptors are, to some extent, involved in the actions of endogenous opioids, if we assume, as proposed initially, that the further hyperalgesia elicited by the high dose of naloxone in arthritic rats or in other models of pain 1'2'31'33, is due to the lifting of tonic inhibitory opioid control(s) on nociceptive messages. It should be stressed that level and biosynthesis of endogenous opioids are increased in various models of persistent pain compared to normal rats 12'21'25'26'39'45. In direct contrast, the paradoxical antinociceptive effect produced by the low doses of naloxone in arthritic rats was not modified in rats injected with the same dose of naltrindole. Comparable data (not shown) have been obtained with ICI-174,864 (10 mg/kg, i.v.). This suggests that 6 opioid receptors are not importantly involved in the negative feedback control of the endogenous opioid release proposed as an explanation of the paradoxical naloxone effect 31. These data appear in contradiction with other studies, using ICI-174,864 in normal rats 48 or naltrindole in diabetic mice 27, which indicate that the paradoxical analgesia produced by naloxone is mediated by interaction at a site with characteristics of the 6 opioid receptor. It should be noted that in the particular case of diabetic mice 27, very high doses of naloxone (5 mg/kg s.c.) were used to produce analgesia. Although we have not a definitive explanation for the discrepancy between the study of Taiwo et al. 48 and ours, differences in the experimental conditions (tests, training of the rats before the experiments, intrathecal administration of the antagonists) must be noted. It has been suggested that chronic intrathecal cannulation may affect spinal and brain endogenous opioid concentrations 37. In addition, as mentioned by the authors, only very weak antinociceptive effects of naloxone were seen in the 'normal' rats. Finally, it may be noted that the action of the antagonists on the hyperalgesic effect of naloxone was not tested in these experiments. (2) The second conclusions refer to the effect of the preferential K antagonist on the bidirectional effect of naloxone. The antinociceptive effect of the low dose of naloxone was almost abolished in arthritic rats receiv-
ing the K antagonist, while the hyperalgesic effect produced by the high dose was not significantly modified. Again, the K antagonist was used at a dose (0.2 mg/kg MR-2266) without action per se, without influence on the antinociceptive effect of/x or 6 agonists, but selectively attenuating the antinociceptive effect of the K agonist U-69,593 (1.5 mg/kg) in these animals. These results strongly support the involvement of K opioid receptors in the paradoxical effect of low doses of naloxone in arthritic rats, and thus in the putative control of endogenous opioid peptide release. They are in agreement with previous studies 5°'51 showing that the K antagonist MR-2266 enhanced the evoked release of Met-enkephalin in the rat brainstem to a greater extent than /z or ~ antagonists5°. Recent biochemical studies in the rat demonstrated that K receptors modulate the /z-(but not the 3-)mediated inhibitory control of spinal Met-enkephalin release in vivo ~5. In fact, results of biochemical investigations in this model of polyarthritis as well as in models of unilateral inflammation or peripheral mononeuropathy, are consistent with a role of K receptors in the modulation of nociception under persistent painful conditions 37'38'45. Although there are very few K sites in the rat spinal cord as compared to the total population of opioid binding sites 4'47, the anatomical organization of both K-receptors and dynorphin supports their physiological role in the control of nociception, since they are concentrated in strategic sites of the spinal cord in pain processing 4'39'4°. Although it is unknown whether the increased endogenous opioid content of the dorsal horn reflects an increase in available opioid able to bind K receptors or only a cellular accumulation in vesicles, recent studies have demonstrated a pronounced increase in the spinal cord dynorphin pool both in polyarthritic rats or in rats with a localized inflammation of a single hindlimb 25'26'41'44. The parallel elevation in levels of mRNA encoding dynorphin indicates that the functional activity of dynorphinergic neurons is enhanced in these animals 25'26. Interestingly, the activity of dynorphin-A converting enzyme is decreased in the cerebrospinal fluid during the acute phase of arthritis in collagen-arthritic rats 43 and the decrease in the activity of this enzyme occurs in parallel with the increase in both the synthesis and the tissue content of dynorphin, in the dorsal horn of arthritic rats. However, the functional significance of these data remains unclear. It has been reported that dynorphin may induced hypercitability and expansion of the receptive fields of some dorsal horn neurons in animal models of persistent pain 45. In agreement with these experiments, we observed (data not shown) that
206 0.3 mg/kg i.v. MR-2266 produced a significant antinociceptive effect in arthritic rats (maximal mean vocalization threshold: 130 +_8% of the control value, P < 0.01, Tukey test, n = 10) (the reason why we only used the dose of 0.2 mg/kg i.v. MR-2266 in this study). On the basis of all these data it appears that K receptor sites could participate in a complex modulation of nociceptive messages, in particular in models of inflammatory pain. However, data obtained with the K receptor antagonist address several questions. The apparant high sensitivity of some opioid sites with K characteristics to extremely low doses of naloxone is somewhat puzzling, especially as the participation of/z sites in the paradoxical analgesic effect of naloxone has been suggested by several earlier experiments: (a) cross-tolerance phenomena have been observed between low doses of morphine and naloxone, in particular the paradoxical antinociceptive effects induced by low doses of naloxone were significantly attenuated in morphine-pretreated arthritic rats, whereas by contrast, the hyperalgesia induced by higher doses of naloxone persisted3°'3t'34; (b) extremely low doses of morphine (6 /zg/kg, i.v.) elicit hyperalgesic effect in these animals 31, suggesting the involvement o f / z receptors in the control of endogenous opioid peptide release. Thus, it is possible that there is some coupling between /z- and K-sites36'49, in these pathological conditions at least. In fact, in arthritic rats, an unexpected link between /z and K binding sites was indicated by the potent reduction of the antinociceptive action of the K opioid receptor agonist, U-50,488H, in morphine tolerant animals 42. In light of the high degree of complexity of the functional role of each opioid receptor subtype and of endogenous opioid substances 7, it is obvious that further studies combining blockade of/z and K a n d / o r receptors with a wide range of agonist and antagonist doses, or the irreversible block of /z-sites with /3funaltrexamine are required for a better understanding. Similar results as in the present study have been obtained recently, using i.v. naltrindole and the K antagonist nor-binaltorphamine in a model of persistent 'pain' due to peripheral nerve injury 3. Acknowledgements. The authors wish to thank Dr. A.H. Dickenson for the English revision and Mr. G. Corvalan for the photography. This research was supported partly by a grant from the Fondation pour la Recherche Medicale to I.C.
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207 common site of action in spinal antinociception, Eur. J. Pharmacol., 148 (1988) 37-45. 21 Draisci, G., Kajander, K.C., Dubner, R., Bennett, G.J. and Iadarola, M.J., Up-regulation of opioid gene expression in spinal cord evoked by experimental nerve injuries and inflammation, Brain Res., 560 (1991) 186-192. 22 Giros, B., De La Baume, S., Gros, C. and Schwartz, J.C., Inhibition de la lib6ration 6voqu6e de ( M e t S) enk6phaline de coupes de globus pallidus en pr6sence de thiorphan et de bestatine, C.R. Acad. Sci. (Paris), 297 (1983) 609-612. 23 Goldstein, A. and Naidu, A., Multiple opioid receptors: ligand selectivity profiles and binding site signatures, Mol. Pharmacol., 36 (1989) 265-272. 24 Gouret, C., Mocquet, G. and Raynaud, G., Use of Freund's adjuvant arthritis test in anti-inflammatory drug screening in the rat: value of animal selection and preparation at the breeding center, Lab. Anita. Sci., 26 (1976) 281-287. 25 Hollt, V., Haarman, I., Millan, M.J. and Herz, A., Prodynorphin gene expression is enhanced in the spinal cord of chronic arthritic rats, Neurosci. Lett., 73 (1987) 90-94. 26 Iadorola, M.J., Douglass, J., Civelli, O. and Naranjo, J.R., Differential activation of spinal cord dynorphin and enkephalin neurones during hyperalgesia: evidence using cDNA hybridization, Brain Res., 455 (1988) 205-212. 27 Kamei, J , Kawashima, N. and Kasuya, Y., Naloxone-induced analgesia in diabetic mice, Eur. J. Pharmacol., 210 (1992) 339-341. 28 Kayser, V. and Guilbaud, G., Dose-dependent analgesic and hyperalgesic effects of systemic naloxone in arthritic rats, Brain Res., 267 (1981) 131-138. 29 Kayser, V. and Guilbaud, G., The analgesic effects of morphine, but not those of the enkephalinase inhibitor thiorphan, are enhanced in arthritic rats, Brain Res., 267 (1983) 131-138. 30 Kayser, V., Besson, J.M. and Guilbaud, G., Paradoxical effects of low doses of naloxone in experimental models of inflammatory pain, Prog. Brain Res., 77 (1988) 301-312. 31 Kayser, V., The reactivity of arthritic rat to acute and chronic administration of various opioid substances. In: J.M. Besson and G. Guilbaud (Eds), The Arthritic Rat as a Model of Clinical Pain? 1988, Elsevier, pp. 111-138. 32 Kayser, V. and Guilbaud, G., Cross tolerance between analgesic low doses of morphine and naloxone in arthritic rats, Brain Res., 405 (1987) 123-129. 33 Kayser, V. and Guilbaud, G., Physiological relevance and timecourse of a tonic endogenous opioid modulation of nociceptive messages, based on the effects of naloxone in a rat model of localized hyperalgesic inflammation, Brain Res., 567 (1991) 197203. 34 Kayser, V., Besson, J.M. and Guilbaud, G., Analgesia produced by low doses of the opiate-antagonist naloxone in arthritic rats is reduced in morphine-tolerant animals, Brain Res., 371 (1986) 37-41. 35 Kayser, V., Fourni6-Zaluski, M.C., Guilbaud, G. and Roques, B.P., Potent antinociceptive effects of kelatorphan (a highly efficient inhibitor of enkephalin degrading enzymes) systemically administered in normal and arthritic rats, Brain Res., 497 (1989) 94-101. 36 Miakowski, C., Sutters, K.A., Talwo, Y. and Levine, J.D., Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists, Pain, 49 (1992) 137144.
37 Millan, M.J. and Colpaert, F.C., Opioid systems in the response to inflammatory pain: sustained blockade suggests role of K- but not ~-opioid receptors in the modulation of nociception, behaviour and pathology, Neuroscience, 42 (1991) 541-553. 38 Millan, M.J., Czlonkowski, A., Lipowski, A. and Herz, A., Kappa-opioid receptor-mediated antinociception in the rat. II. Supraspinal in addition to spinal sites of action, J. PharmacoL Exp. Ther., 251 (1989) 342-350. 39 Millan, M,J., Czlonkowski, A., Morris, B., Stein, C., Arendt, R., Huber, A., Hollt, V. and Herz, A., Inflammation of the hind limb as a model of unilateral, localized pain: influence on multiple opioid systems in the spinal cord of the rat, Pain, 35 (1987) 299-312. 40 Morris, B.J. and Herz, A., Distinct distribution of opioid receptor types in rat lumbar spinal cord. Naunym-Schiedeberg's Arch. Pharmacol., 336 (1987) 240-243. 41 Nahin, R.L., Hylden, J.K.L., Iadarola, M.J. and Dubner, R., Peripheral inflammation is associated with increased dynorphin immunoreactivity in both projection and local circuit neurons in the superficial dorsal horn of the rat lumbar spinal cord, Neurosci. Lett., 96 (1989) 247-250. 42 Neil, A., Kayser, V., Gacel, G., Besson, J.M. and Guilbaud, G., Opioid receptor types and antinociceptive activity in chronic inflammation: both r- and p.-opiate agonistic effects are enhanced in arthritic rats, Eur. J. Pharmacol., 130 (1986) 203-208. 43 Persson, S., Post, C. and Nyberg, F., Decrease of neuropeptideconverting enzyme activities in cerebrospinal fluid during acute but not chronic phases of collagen-induced arthritis in rats, Brain Res., 581 (1992) 273-279. 44 Przewlocka, B., Lason, W. and Przewlocki, R., Time-dependent changes in the activity of opioid systems in the spinal cord of monoarthritic rats-a release and in situ hybridization study, Neuroscience, 46 (1992) 209-216. 45 Ruda, M.A. and Dubner, R., Molecular and biochemical events mediate neuronal plasticity following inflammation and hyperalgesia. In: Willis W.D. (Ed.), Hyperalgesia and Allodynia, Raven, NY, 1992, pp. 311-325. 46 Simon, E.J., Subunit structure and purification of opioid receptors, J. Receptor Res., 7 (1987) 105-132. 47 Stevens, C.W., Lacey, C.B., Miller, K.E., Elde, R.P. and Seybold, V.S., Biochemical characterization and regional quantification of /z-, ~- and K-opioid binding sites in rat spinal cord, Brain Res., 550 (1991) 77-80. 48 Taiwo, Y.O., Basbaum, A.I., Perry, F. and Levine, J.D., Paradoxical analgesia produced by low doses of the opiate-antagonist naloxone is mediated by interaction at a site with characteristics of the delta-opioid receptor, J. Pharmacol. Exp. Ther, 249-1 (1989) 97-100. 49 Takahashi, M., Senda, T. and Kaneto, H., Role of spinal x opioid receptors in the blockade of the development of antinociceptive tolerance to morphine, Eur. J. Pharmacol., 200 (1991) 293-297. 50 Ueda, H., Fukushima, N., Ge, M., Takagi, H. and Satoh, M., Presynaptic opioid K- receptor and regulation of the release of Met-enkephalin in the rat brainstem, Neurosci. Lett., 81 (1987) 309-313. 51 Ueda, H., Fukushima, N., Kitao, T., Ge, M. and Takagi, H., Low doses of naloxone produce analgesia in the mouse brain by blocking presynaptic autoinhibition of enkephalin release, Neurosci. Lett., 65 (1986) 247-252.
201
BRES 19236
Differential effects of specific and K opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain I. Cattaneo,
V. Kayser
and G. Guilbaud
Unit~ de Recherches de Physiopharmacologie du Syst~meNerveux, INSERM, Paris (France)
(Accepted 27 April 1993)
Key words: Polyarthritic rat; Vocalization threshold; Paw pressure; Endogenous opioid system; Naloxone; Naltrindole; MR-2266
In an attempt to determine the opioid receptor class(es) which underly the two opposing effects of naloxone in models of persistent pain, we tested the action of the selective t5 antagonist naltrindole, and that of the K antagonist MR-2266 on the bidirectional effect of systemic naloxone in arthritic rats. As a nociceptive test, we used the measure of the vocalization thresholds to paw pressure. The antagonists were administered at a dose (1 mg/kg i.v. naltrindole, 0.2 mg/kg i.v. MR-2266), without action per se, but which prevents the analgesic effect of the ~ agonist DTLET (3 mg/kg, i.v.) or the K agonist U-69,593 (1.5 mg/kg, i.v.) respectively, and does not influence the effect of morphine (1 mg/kg i.v.) or the/~ agonist DAMGO (2 mg/kg, i.v.) in these animals. In arthritic rats injected with the 8 antagonist, the paradoxical antinociceptive effect produced by 3/zg/kg i.v. naloxone was not significantly modified (maximal vocalization thresholds (% of control) were 146 _+9% versus 161 + 7% in the control group). By contrast, the hyperalgesic effect produced by 1 mg/kg i.v. naloxone was significantly reduced (maximal vocalization thresholds were 87+4% versus 69+5% in the control group). In rats injected with the K antagonist, the antinociceptive effect of the low dose of naloxone was almost abolished (mean vocalization thresholds were 115 _+3% versus 169 5: 7%) whereas the hyperalgesic effect of naloxone 1 mg/kg i.v. was not significantly modified (mean vocalization thresholds = 70 + 3% and 65 + 3%, respectively). Based on these results, the possible role of each receptor subtype in the putative control exerted by endogenous opioid substances on nociceptive messages in pathological conditions is discussed.
INTRODUCTION T h e F r e u n d ' s a d j u v a n t - i n d u c e d a r t h r i t i c rat is now g e n e r a l l y c o n s i d e r e d as a useful m o d e l for t h e study o f p a i n m e c h a n i s m s 6't°']6. O p i o i d s a d m i n i s t e r e d systemically a r e especially active in t h e s e a n i m a l s with persist e n t pain, as g a u g e d by s e v e r a l n o c i c e p t i v e tests, b u t p a r t i c u l a r l y with t h e v o c a l i z a t i o n t h r e s h o l d to p a w p r e s s u r e 19'29'35'42. This e n h a n c e d effectiveness d o e s n o t s e e m directly r e l a t e d to c h a n g e s in ~ - , 3- a n d K-opioid b i n d i n g sites at c e n t r a l levels 5J2'tS. It c o u l d b e p a r t l y d u e to c h a n g e s in e n d o g e n o u s o p i o i d p e p t i d e s . I n d e e d , b i o c h e m i c a l studies have shown t h a t t h e c o n t e n t a n d b i o s y n t h e s i s o f e n d o g e n o u s o p i o i d s in t h e d o r s a l h o r n o f t h e spinal c o r d a r e i n c r e a s e d 6'12'13'21'25'26'39'41'45 while t h e level o f n e u t r a l e n d o p e p t i d a s e is n o t m o d i f i e d 18 in t h e s e animals. Since 1981, we have t e s t e d extensively t h e activity o f e n d o g e n o u s o p i o i d systems with t h e
o p i o i d a n t a g o n i s t n a l o x o n e in this m o d e l of p e r s i s t e n t pain, using t h e v o c a l i z a t i o n t h r e s h o l d to p a w p r e s s u r e as a n o c i c e p t i v e test. W e have d e m o n s t r a t e d r e p e t e a d l y 28'3t t h a t n a l o x o n e i n d u c e s c l e a r b i d i r e c t i o n a l effects in t h e s e animals: high d o s e s (1 to 3 m g / k g , i.v.) of t h e a n t a g o n i s t i n d u c e d f u r t h e r i n c r e a s e s in t h e hyp e r a l g e s i a , suggesting t h a t e n d o g e n o u s o p i o i d p e p t i d e s e x e r t a p a r t i c u l a r l y p o t e n t tonic inhibitory c o n t r o l on n o c i c e p t i v e m e s s a g e s in t h e s e animals. By c o n t r a s t , e x t r e m e l y low d o s e s (3 to 300 t x g / k g , i.v.) elicit p a r a doxical a n a l g e s i a , p e a k i n g at a d o s e of 3 / ~ g / k g . W e h y p o t h e t i z e d 3°'31, s u p p o r t e d by b i o c h e m i c a l s t u d ies 8'22'5°'51, t h a t t h e p a r a d o x i c a l effect m a y result f r o m t h e i n t e r a c t i o n o f n a l o x o n e with highly sensitive r e c e p tors, r e s p o n s i b l e for t h e o n g o i n g s u p p r e s s i o n o f end o g e n o u s o p i o i d r e l e a s e in c o n d i t i o n s o f p e r s i s t e n t pain. I n d e e d , t h e s e b i d i r e c t i o n a l effects o f naloxone, n o t o b s e r v e d in n o r m a l rats, w e r e also f o u n d in o t h e r
Correspondence: V. Kayser, Unit6 de Recherches de Physiopharmacologie du Syst~me Nerveux, INSERM U 161, 2 rue d'Al6sia, 75014 Paris, France.
202 models of persistent inflammatory or neuropathic pain, using the same test 1-2'3'3°'33. The involvement of different opioid receptors in this dose-dependent effect was also suggested by the modifications observed in arthritic rats rendered tolerant to morphine. In these animals the paradoxical antinociceptive effect of the low dose was dramatically decreased, while the high dose of naloxone still induced hyperalgesia31'32'34. To determine the different opioid receptor subtype(s) involved we studied the effects of specific opiold receptor antagonists on the bidirectional effect of naloxone in polyarthritic rats. We report here the data obtained with the ~ opioid antagonist naltrindole and the x antagonist MR2266. Preliminary data have been reported ~~. MATERIALS
AND METHODS
Animals Male Sprague-Dawley arthritic rats, 9 - 1 0 weeks old, weighing 190-260 g, were obtained from Charles River, France. The rats were allowed to habituate to the colony room for at least 7 days before the experiments. Arthritic rats were used 21 days after the induction of arthritis by inoculation at the breeding center (Charles River, France) of Freund's adjuvant at the base of the tail. At this time, the arthritic lesions and the concomitant humoral changes are at a stable maximum 6,t°A6'24. Since these conditions induce a certain amount of suffering, the guidelines proposed by the committee for Research and Ethical Issue of the IASP (1980) 17 have been followed. The food was directly available ad libitum in the sawdust of the cages to minimise the need for the animals to make potentially painful movements to obtain food. The duration of the experiments was as short as possible and the n u m b e r of animals involved kept to a minimum. Since a relatively high number of animals (n = 116) were necessary in several preliminary experiments to determine the suitable doses of antagonist and to control their specificity, the n u m b e r of rats used in the specific study was minimized by excluding vehicle controls, since it was previously verified that acute injection of saline or Fixanal (a buffer solution with a p H equivalent to that of the naloxone solution) does not alter nociceptive thresholds in this assay 31. Behavioural test Testing sessions, beginning at 10 a.m. were conducted in a quiet room remote from the colony room, by a single experimenter. Prior to the test phase, the rats had no experience with the testing apparatus. The vocalization threshold to paw pressure was measured using the Basile Analgesymeter (Apelex) (tip diameter of the probe was 1 mm). Increasing pressure was applied to the hindpaw, until a squeak was elicited. For each rat, a preliminary threshold or control threshold (mean of two consecutive stable thresholds, expressed in grams) was determined before any injection. Nociceptive pressure thresholds were determined every 5 or 10 min after drug administration, until they returned to baseline. Experimental series In a first step, several groups of animals (n = 116 rats) were used in preliminary experiments to determine antagonist doses that we wish to test, and to control their specificity (Table I). Then, to test the effect of each antagonist on the bidirectional effect of naloxone, 4 groups of rats were used (n = 11 or 9 in each group used for experiments related to 6 and K antagonist respectively). Thus, 80 rats were used for this specific study according to the following protocol:
- one control both naloxone - one control both naloxone
group received naloxone 1 m g / k g alone, one group I m g / k g + the antagonist group received naloxone 3 ~ g / k g alone, one group 3 p~g/kg + the antagonist.
Drugs and tests dosages The following drugs and dosages were used, unless specified otherwise: naloxone hydrochloride, 3 /zg and 1 m g / k g (Narcan, Dupont de Nemours, Paris, France), naltrindole hydrochloride, 1 m g / k g ( 17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy6,7-2',3'-indolomorphinan, a fi antagonist, gift from Pr. Roques, Paris, France), and MR-2266, 0.2 m g / k g , (( - )-5,9-diethyl-2-hydroxy2-(3-furylmethyl)-6,7-benzomorphan, K antagonist, gift from Dr. Neil, Uppsala, Finland). Naloxone (3 ~ g and 1 m g / k g ) was given at doses that induce potent analgesic and hyperalgesic effects in arthritic animals 31. The doses of 1 m g / k g of naltrindole and 0.2 m g / k g of MR-2266 were determined by preliminary experiments to be without action per se, but to prevent specifically the analgesic effect of 3 m g / k g of the ~ agonist D T L E T (o-Thr2, Leu5-enkephalyI-Thr, Bachem Feinkemikalien A G Bubendorf, Switzerland) or 1.5 m g / k g of the x agonist U-69,593 (5-,7-,8B-(-)-N-methyl-N(7-(1-pyrrolidinyl)-l-oxaspiro(4,5)-dec-7-8-yl)benzene-acetamide), gift from Dr. Duffet, Upjohn, Paris, France), respectively. We also controlled that these doses of fi and K antagonists did not influence significantly the effects of 1 m g / k g morphine or 2 m g / k g of t h e / , agonist D A M G O (D-Ala2,MePhe4,Gly-ol-5-enkephalin, Bachem Feinkemikalien A G Bubendorf, Switzerland) in these animals (Table I). The doses of agonists used for these controls were choosen according to earlier studies showing that their peak effect was roughly comparable to that of 1 m g / k g i.v. morphine in arthritic rats 29,4z. Drugs were freshly prepared in sterile physiological saline (0.9% NaC1) and all injections were given intravenously into the lateral tail vein. Statistical procedures Post-drugs thresholds were calculated for each animal as percentages of m e a n control thresholds. Statistical calculations were done with a one- or multiple-way analysis of variance ( A N O V A procedure), Tukey test (with vocalization thresholds expressed in grams) or Student's t-test (with vocalization thresholds expressed as percentage of the control values) for comparison of the area under the curves. Differences were considered significant for P-values < 0.05. TABLE 1 Control of the specificity of the antagonists naltrindole and MR-2266 towards tz, 6 and K agonists
morphine DAMGO DTLET U-69,593
Ant. = 0
naltrindole
MR-226
191 _+6 n=t3 171 + 10 n=10 1425:6 n=8 215 + 27 n=8
103+4 n=7 191 _+9 n=ll 170 +_39 n=7 1025:4 * * n=7 207 5:55 n=6
114+4 n=10 184 5:7 n=8 163 _+5 n=7 151 _+ 11 n=5 111 + 6 * * n=9
Mean peak effects of: the 6-antagonist, naltrindole (1 m g / k g i.v.) and the K-antagonist, MR-2266 (0.2 m g / k g , i.v.) administered alone, the agonists, morphine (1 m g / k g , i.v.), D A M G O (2 m g / k g , i.v.), D T L E T (3 m g / k g , i.v.) and U-69,593 (1.5 m g / k g , i.v.) injected alone (ant = 0) or co-injected with naltrindole or MR-2266. The vocalization threshold to paw pressure was determined every 5 or 10 rain after drug administration, until they returned to baseline. Each value is expressed as a percentage of the baseline threshold_+S.E.M., n = n u m b e r of rats per group. * * Denotes difference from the corresponding control group receiving the agonist only at the P < 0.01 level, Tukey test. Time of maximum effect of the drugs: naltrindole and M R - 2 2 6 6 : 1 5 min; morphine: 30 min; D A M G O : 20 min; DTLET: 20 min; U-69,593:40 min.
203 RESULTS
260
Before any drug injection, the vocalization threshold to paw pressure was similar for the various animals. The m e a n control threshold determined for all the arthritic rats used in the experiments to test the effect of the antagonists on the bidirectional effect of naloxone was 148 + 12 g (n = 80), roughly comparable to previous studies 28'31.
t, >
220
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180
[ ] control [ ] NTI-treated [ ] MR2266-treatecl rats
140 "0
100
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Effects of the antagonists on the hyperalgesic effect of naloxone
5_
Naloxone 1 mg/kg NALOXONE 3 ug/kg
-60
-I00
Effect of the 6 antagonist naltrindole, 1 m g / kg Lv. In the control group of rats receiving 1 m g / k g i.v. naloxone alone, an overall significant hyperalgesic effect (F7,79---10.37; A N O V A , P < 0.001, n = 11) was elicited, as described in previous studies (Fig. 1A). The effect was significant at 5 min, and maximal at 15 min
A o 100
§ 8O
"="*~9/kg **
60
iv MR-2266
T
•
+ NTI
**
•
+
B o
100
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x
-140
Fig. 2. Mean areas under the curves ( + S . E . M . ) for naloxone 3 # g / k g i.v. and naloxone 1 m g / k g i.v. injected alone (open bars) or co-injected with naltrindole (NTI) 1 m g / k g i.v. (hatched bars) or MR-2266 0.2 m g / k g i.v. (filled bars), as illustrated in Fig. 1 and Fig. 3. Total response area (percentage increase from baseline x min) was calculated for the whole duration of the response in each animal. ** * Denotes difference from the corresponding control rat group at the P < 0.001 level, Student's t-test.
after the injection. At this time, the m e a n vocalization threshold expressed as percentage of preinjection value was 69 + 5% ( P < 0.01, Tukey test, n = 11). Recovery was observed by 35-40 min. In rats receiving both 1 m g / k g i.v. naloxone and naltrindole, the hyperalgesic effect was markedly reduced (F7,79 = 4.44; A N O V A , P < 0.001, n = 11), although still significant at 10 min (Fig. 1A). At this time, the mean value was 87 + 4% of the control ( P < 0.05, Tukey test, n = 11). The total recovery was reached at 25 min after the injection (difference from the controls for the overall effect of naloxone: Fl,173 = 26.1, A N O V A , P < 0.001, n = 22). The difference between the effects of naloxone in the two groups of rats is also well illustrated by comparison between the mean areas under the curves calculated for naloxone 1 m g / k g i.v. and naloxone 1 m g / k g i.v. + naltrindole (Fig. 2).
80
Effect of the K-antagonist MR-2266, 0.2 mg / kg i.v. 60
** I
I
I
I
10
20
30
40 min
Fig. 1. M e a n curves illustrating the effects on the vocalization threshold to paw pressure of naloxone (Nx) 1 m g / k g i.v. injected alone or co-injected with: (A) naltrindole (NTI) 1 m g / k g i.v., (B) MR-2266, 0.2 m g / k g i.v. Each value is expressed as a percentage _+S.E.M. of the control threshold value m e a s u r e d just before injection. Tukey test was used for statistical analysis (with vocalization thresholds expressed in grams): * P < 0.05, * * P < 0.01. T h e overall effect of each dose was calculated with a multiple-way analysis of variance ( A N O V A procedure), fully described in the results paragraph, n = 11 rats in each group in A; n = 9 rats in each group in B.
In the control group of rats receiving 1 m g / k g i.v. naloxone alone, a significant hyperalgesic effect (F7, 63 = 13.5; A N O V A , P < 0.001, n = 9), comparable to that described above, was elicited (Fig. 1B). The effect was clear 5 min after injection, and maximal at 15 min. At this time, the mean vocalization threshold was 65 _+ 3% of the pre-injection control value ( P < 0.01, Tukey test, n = 9). In rats receiving both 1 m g / k g i.v. naloxone and MR-2266, the hyperalgesic effect was still observed (F7,63 = 15.2; A N O V A , P < 0.001, n = 9), comparable to that in the control group of rats. The mean peak
204 value was 70 + 3% of the control ( P < 0.01, Tukey test, n = 9), 15 min after the injection. Thus, in arthritic rats, the hyperalgesic effect of naloxone 1 m g / k g i.v. was greatly reduced by naltrindole and not influenced by MR-2266.
Effects of the antagonists on the paradoxical antinociceptive effect of naloxone Effect of the ~-antagonist naltrindole In the control group of rats receiving 3 tzg/kg i.v. naloxone alone, an overall significant antinociceptive effect (F7.55 = 22.1, ANOVA, P < 0.001, n = 9) was observed, as described in previous studies (Fig. 3A). Starting 5 min after the injection it was maximum at 15 rain (mean vocalization threshold was = 161 + 7% of the control value, P < 0.01, Tukey test, n = 9). The recovery was reached progressively at 40 min after the injection. In naltrindole-injected rats, there was still a potent antinociceptive effect of the low dose of naloxone
"K" "X"
A
-6 160 "X" "X"
(F7,55 = 20.6, ANOVA, P < 0.001, n = 9). The mean peak value was 146 _+ 9% of the control value ( P < 0.01, Tukey test, n = 9) and the whole time course was not markedly affected (Figs. 2 and 3A).
Effect of the K-antagonist MR-2266 In the control group receiving 3 / z g / k g i.v. naloxone, significant antinociceptive effect (F7.55= 21.8, ANOVA, P < 0.001, n = 8) was observed (Fig. 3B). Starting 5 min after the injection it was 169 + 7% of the control value at 15 min ( P < 0.01, Tukey test, n -- 8). The recovery to controls was reached at 40 min. In MR-2266-injected rats, the antinociceptive effect of 3 /zg/kg i.v. naloxone (F7.55 = 3.7, ANOVA, P < 0.05, n = 8) was profoundly decreased (difference between the two groups of rats F1,126 = 41.59, ANOVA, P < 0.001, n = 16 rats) (Fig. 3B). The maximal increase in vocalization threshold was 115 + 3% of the control ( P < 0.05, Tukey test, n = 8) at 20 min, reduced by about 60%. These results are clearly seen by comparison of the mean areas under the curves, calculated for the two doses of naloxone in each group of arthritic rats (Fig. 2).
"X" "K"
o u
DISCUSSION 140
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o
120
100
h.>.i 1 t'?l I
f
f
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160
I
•
o
+
MR-2266
o
~ 140
120
100 I i
i
i
i
10
20
30
40 rnin
Fig. 3. Mean curves illustrating the effects of naloxone (Nx) 3/~g/kg i.v. injected alone or co-injected with: (A) naltrindole (NTI) 1 mg/kg i.v., (B) MR-2266, 0.2 mg/kg i.v.. Each value is expressed as a percentage +S.E.M. of the control threshold value measured just before injection, n = 9 rats in each group. * P < 0.05, ** P < 0.01, Tukey test (with vocalization thresholds expressed in g).
The present data clearly confirm that the bidirectional dose-dependent effects of naloxone in arthritic rats results from the interaction of the drug with opioid receptors of different characteristics, as suggested by extensive behavioural studies performed in this model and in models of localized inflammatory or neuropathic pain ~'2'3°'31. Simultaneously, they provide some indications on the putative role of receptor subtypes with 6- and K-site characteristics in the control of nociceptive messages exerted by endogenous opioid peptides under pathological conditions. (1) We observed that the hyperalgesic effect produced by the high dose of naloxone (1 mg/kg) in arthritic rats, was significantly reduced by the selective ~ antagonist naltrindole (by about 3 - 4 fold when gauged by the mean areas under the curves). By contrast, the same close of the 3 antagonist did not influence the paradoxical antinociceptive effect produced by the low dose of naloxone in these animals. It should be noted that the same effects were obtained with naltrindole co-injected with naloxone, and with another 6-antagonist ICI-174,864 (10 mg/kg, i.v.) or when naltrindole was administered 15 min before naloxone (not shown). The antagonists were used at doses without action per se, without influence on the antinociceptive effect of K- and /z-preferential ago-
205 nists, but which prevent selectively the antinociceptive effect of the 6-agonists DTLET (3 mg/kg) or BUBU (3 mg/kg) in arthritic rats 19. The functional relationship between endogenous opioid peptides and the types of opioid receptors remains unclear 23'46, with the results obtained with enkephalinase inhibitors suggesting the activation of either tJ,14 o r 3 9,20 opioid receptors by the protected endogenous opioids to produce antinociception. The results obtained here, with both naltrindole and ICI174,864, seem to indicate that ~ opioid receptors are, to some extent, involved in the actions of endogenous opioids, if we assume, as proposed initially, that the further hyperalgesia elicited by the high dose of naloxone in arthritic rats or in other models of pain 1'2'31'33, is due to the lifting of tonic inhibitory opioid control(s) on nociceptive messages. It should be stressed that level and biosynthesis of endogenous opioids are increased in various models of persistent pain compared to normal rats 12'21'25'26'39'45. In direct contrast, the paradoxical antinociceptive effect produced by the low doses of naloxone in arthritic rats was not modified in rats injected with the same dose of naltrindole. Comparable data (not shown) have been obtained with ICI-174,864 (10 mg/kg, i.v.). This suggests that 6 opioid receptors are not importantly involved in the negative feedback control of the endogenous opioid release proposed as an explanation of the paradoxical naloxone effect 31. These data appear in contradiction with other studies, using ICI-174,864 in normal rats 48 or naltrindole in diabetic mice 27, which indicate that the paradoxical analgesia produced by naloxone is mediated by interaction at a site with characteristics of the 6 opioid receptor. It should be noted that in the particular case of diabetic mice 27, very high doses of naloxone (5 mg/kg s.c.) were used to produce analgesia. Although we have not a definitive explanation for the discrepancy between the study of Taiwo et al. 48 and ours, differences in the experimental conditions (tests, training of the rats before the experiments, intrathecal administration of the antagonists) must be noted. It has been suggested that chronic intrathecal cannulation may affect spinal and brain endogenous opioid concentrations 37. In addition, as mentioned by the authors, only very weak antinociceptive effects of naloxone were seen in the 'normal' rats. Finally, it may be noted that the action of the antagonists on the hyperalgesic effect of naloxone was not tested in these experiments. (2) The second conclusions refer to the effect of the preferential K antagonist on the bidirectional effect of naloxone. The antinociceptive effect of the low dose of naloxone was almost abolished in arthritic rats receiv-
ing the K antagonist, while the hyperalgesic effect produced by the high dose was not significantly modified. Again, the K antagonist was used at a dose (0.2 mg/kg MR-2266) without action per se, without influence on the antinociceptive effect of/x or 6 agonists, but selectively attenuating the antinociceptive effect of the K agonist U-69,593 (1.5 mg/kg) in these animals. These results strongly support the involvement of K opioid receptors in the paradoxical effect of low doses of naloxone in arthritic rats, and thus in the putative control of endogenous opioid peptide release. They are in agreement with previous studies 5°'51 showing that the K antagonist MR-2266 enhanced the evoked release of Met-enkephalin in the rat brainstem to a greater extent than /z or ~ antagonists5°. Recent biochemical studies in the rat demonstrated that K receptors modulate the /z-(but not the 3-)mediated inhibitory control of spinal Met-enkephalin release in vivo ~5. In fact, results of biochemical investigations in this model of polyarthritis as well as in models of unilateral inflammation or peripheral mononeuropathy, are consistent with a role of K receptors in the modulation of nociception under persistent painful conditions 37'38'45. Although there are very few K sites in the rat spinal cord as compared to the total population of opioid binding sites 4'47, the anatomical organization of both K-receptors and dynorphin supports their physiological role in the control of nociception, since they are concentrated in strategic sites of the spinal cord in pain processing 4'39'4°. Although it is unknown whether the increased endogenous opioid content of the dorsal horn reflects an increase in available opioid able to bind K receptors or only a cellular accumulation in vesicles, recent studies have demonstrated a pronounced increase in the spinal cord dynorphin pool both in polyarthritic rats or in rats with a localized inflammation of a single hindlimb 25'26'41'44. The parallel elevation in levels of mRNA encoding dynorphin indicates that the functional activity of dynorphinergic neurons is enhanced in these animals 25'26. Interestingly, the activity of dynorphin-A converting enzyme is decreased in the cerebrospinal fluid during the acute phase of arthritis in collagen-arthritic rats 43 and the decrease in the activity of this enzyme occurs in parallel with the increase in both the synthesis and the tissue content of dynorphin, in the dorsal horn of arthritic rats. However, the functional significance of these data remains unclear. It has been reported that dynorphin may induced hypercitability and expansion of the receptive fields of some dorsal horn neurons in animal models of persistent pain 45. In agreement with these experiments, we observed (data not shown) that
206 0.3 mg/kg i.v. MR-2266 produced a significant antinociceptive effect in arthritic rats (maximal mean vocalization threshold: 130 +_8% of the control value, P < 0.01, Tukey test, n = 10) (the reason why we only used the dose of 0.2 mg/kg i.v. MR-2266 in this study). On the basis of all these data it appears that K receptor sites could participate in a complex modulation of nociceptive messages, in particular in models of inflammatory pain. However, data obtained with the K receptor antagonist address several questions. The apparant high sensitivity of some opioid sites with K characteristics to extremely low doses of naloxone is somewhat puzzling, especially as the participation of/z sites in the paradoxical analgesic effect of naloxone has been suggested by several earlier experiments: (a) cross-tolerance phenomena have been observed between low doses of morphine and naloxone, in particular the paradoxical antinociceptive effects induced by low doses of naloxone were significantly attenuated in morphine-pretreated arthritic rats, whereas by contrast, the hyperalgesia induced by higher doses of naloxone persisted3°'3t'34; (b) extremely low doses of morphine (6 /zg/kg, i.v.) elicit hyperalgesic effect in these animals 31, suggesting the involvement o f / z receptors in the control of endogenous opioid peptide release. Thus, it is possible that there is some coupling between /z- and K-sites36'49, in these pathological conditions at least. In fact, in arthritic rats, an unexpected link between /z and K binding sites was indicated by the potent reduction of the antinociceptive action of the K opioid receptor agonist, U-50,488H, in morphine tolerant animals 42. In light of the high degree of complexity of the functional role of each opioid receptor subtype and of endogenous opioid substances 7, it is obvious that further studies combining blockade of/z and K a n d / o r receptors with a wide range of agonist and antagonist doses, or the irreversible block of /z-sites with /3funaltrexamine are required for a better understanding. Similar results as in the present study have been obtained recently, using i.v. naltrindole and the K antagonist nor-binaltorphamine in a model of persistent 'pain' due to peripheral nerve injury 3. Acknowledgements. The authors wish to thank Dr. A.H. Dickenson for the English revision and Mr. G. Corvalan for the photography. This research was supported partly by a grant from the Fondation pour la Recherche Medicale to I.C.
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