Brain Research, 538 (1991) 181-186 Elsevier BRES 16219
181
Research Reports
Chronic selective blockade of/ opioid receptors produces analgesia and augmentation of the effects of a agonist M.J. Katharine Walker 1'2, A.D. L61, Constantine X.
P o u l o s 1'3
and
Howard
CappeU 1'2'3
1Addiction Research Foundation, Toronto, Ont. (Canada) and Departments of 2pharmacology and 3psychology, University of Toronto, Toronto, Ont. (Canada) (Accepted 7 August 1990) Key words: Analgesia; fl-Funaltrexamine; Naloxone; U50-488H; r Opioid analgesia
We have previously demonstrated that, when administered chronically, naloxone and naitrexone have the paradoxical effect of producing analgesia in rats. In this study, rats treated chronically with intracerebroventricular (i.c.v.) microinjections, and mice treated chronically with subcutaneous (s.c.) injections of naloxone or fl-funaltrexamine (fl-FNA) developed analgesia on daily hot plate tests. There was no drug effect on the first day of hot plate testing, but significant increases in paw lick latency developed over subsequent acquisition sessions for animals treated with fl-FNA or naloxone. An augmented analgesic response to a 5 mg/kg s.c. injection of the r opioid agonist, U50-488H, was observed in mice previously treated with naloxone or fl-FNA. The primary findings of the present study were: (1) chronic blockade of/~ opioid receptors is sufficient to produce analgesia on repeated hot plate tests in both rats and mice; (2) chronic blockade of/~ receptors in the presence of stressful stimuli results in augmentation of r agonist-induced analgesia; and (3) the phenomenon of opioid blockade-induced analgesia (OBA) occurs in mice as well as rats. INTRODUCTION We have previously demonstrated that when administered chronically naloxone and naltrexone have the paradoxical effect of producing analgesia in rats 1-5'13'14. Greeley et al. 5 demonstrated that rats implanted with naloxone or naltrexone pellets or receiving 5 mg/kg naloxone injections showed an absolute increase in paw lick latency (PLL). The magnitude of this increase in PLL was such that by the fifth hot plate test the latency of rats injected with naloxone was three times that of controls. Although the mechanism of opioid blockade-induced analgesia (OBA) is not known, important features of this phenomenon have recently been described. Poulos et al. 13 clearly showed that naloxone-induced analgesia develops only when rats receive hot plate testing under the influence of naloxone. In the absence of hot plate testing, mere repeated administration of naloxone failed to produce analgesia. Rats displaying naloxone-induced analgesia also showed an augmented analgesic response to morphine on hot plate and tail-flick tests ~3. Whether O B A is mediated by aspects of the opioid system or by a non-opioid mechanism is still unknown. Opioid analgesia has been associated with p, 6 and opioid receptors 6-8'15. Naloxone binds readily to/~, r and 6 opioid receptors, but its affinity for ~ receptors is
generally more than tenfold higher than for other opioid receptor types 6. Naloxone and naltrexone are also known to produce changes in the numbers of/~ receptors when administered chronically at lower doses than those needed to produce changes in 1¢ or 6 receptors 9'17'23. It may be possible that when one opioid receptor subtype is preferentially blocked under conditions that normally stimulate endogenous opioid analgesia, the analgesic capacity of another opioid receptor subtype is revealed resulting in the development of analgesia. Both the morphine selectivity of the/~ receptor and its distribution in brain regions which have a putative role in pain regulation strongly implicate it as a major factor in supraspinal mediation of opioid analgesia 7'is. Therefore, blockade of p receptors under conditions in which they are normally active, may be sufficient to stimulate the analgesic capacity of other opioid receptor types. Thus far, there has been no demonstration of the contribution of selective opioid receptor blockade to the development of OBA. However, in order to further understand the underlying mechanism of O B A we must first know the limiting conditions under which it will develop. Accordingly, the primary goal of the present study was to determine whether selective blockade of ~ receptors would produce analgesia similar in nature to that produced by naloxone, fl-Funaltrexamine (fl-FNA) is a site
Correspondence: M.J.K. Walker, 6th Floor, Tower, Addiction Research Foundation, 33 Russell St., Toronto, Ont., Canada M5S 2S1. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
182 d i r e c t e d aikylating a g e n t with reversible agonist and irreversible, selective p r e c e p t o r antagonistic activities 12. T h e agonist actions of f l - F N A are o b s e r v e d less t h a n 1 h after a d m i n i s t r a t i o n , and are a n t a g o n i z e d by n a i o x o n e 16'
2o.21 In contrast, the antagonist actions of f l - F N A are d e m o n s t r a t e d within an h o u r of a d m i n i s t r a t i o n and last for at least 4 days. T h e s e antagonist actions o f f l - F N A are m e d i a t e d t h r o u g h p r e c e p t o r s L6'2°'21. B e c a u s e it is an irreversible
and
selective /~ r e c e p t o r
blocker,
using
fl-FNA has a particular a d v a n t a g e in that it does not c o m p e t e for binding with the e n d o g e n o u s p o o l of p r e c e p t o r ligands. T h e r e f o r e , the use of f l - F N A m a k e s it possible to selectively r e m o v e the p r e c e p t o r c o m p o n e n t of the e n d o g e n o u s
o p i o i d system.
Since analgesia is
m e d i a t e d by b o t h p and'~¢ o p i o i d r e c e p t o r s , it is possible that by b l o c k i n g only ~ r e c e p t o r s u n d e r the influence of stress the analgesic capacity of ~c r e c e p t o r s m a y b e c o m e supersensitive.
To test this hypothesis,
mice t r e a t e d
chronically with f l - F N A or n a l o x o n e in c o n j u n c t i o n with hot plate
testing w e r e
subsequently
tested for their
r e s p o n s e to the analgesic effects of a selective 7c agonist, U 5 0 - 4 8 8 H 19. Finally, the e x p e r i m e n t s w e r e d e s i g n e d to demonstrate
OBA
in two different
species,
rat
and
mouse. MATERIALS AND METHODS
Animals Sixty-four male Sprague-Dawley rats, and 120 male CD1 mice (Charles River Canada, Inc., St. Constant, Que.) weighing 280-300 g and 25-30 g, respectively, were used. Rats and mice were housed and tested in different rooms. They were singly housed in stainless-steel cages with free access to food (Purina Lab Chow) and water. The rat diet consisted of 6 pellets (35 g) per day. This diet promotes healthy weight gain without the development of obesity. The colony room was maintained at 24 °C with a 12-h light-dark cycle (light on during experimental procedures). Surgery Rats were chronically implanted, using standard stereotaxic technique and pentobarbital (50 mg/kg) anaesthesia, with a 23gauge stainless-steel guide cannula at coordinates 0.8 mm posterior to bregma and 1.4 mm lateral to the sagittal suture. The cannula terminated 3.7 mm below the surface of the skull in the lateral ventricle. Five to 7 days was allowed for recovery before drug injections began. At the end of the experiment the rats were killed by pentobarbital overdose and an injection of dye was used to confirm the accuracy of the placement. Apparatus Analgesia was measured using a hot plate apparatus consisting of a plastic storage tub with an aluminum plate on the surface (Grant Instrument Water Bath Model AB02). The temperature of the surface was monitored continually with a thermistor (Yellow Springs Instrument Model 343 TC) and probes (Yellow Springs Banjo Surface Model 408) clamped securely to the surface of the aluminum plate. Rats were confined to the surface of the hot plate within a clear Plexiglas box (27 cm x 16.5 cm × 40 cm high) and mice within a similar Plexiglas box (27 cm × 16.5 cm × 9 cm high) with lids. Procedure Rats were randomly assigned to 4 groups and were given i.c.v.
injections of fl-FNA 10 or 3 ltg, naloxone HCL (Sigma, St. Louis) 20 pg or drug vehicle, in a 3 ~1 volume. Mice were randomly assigned to 7 groups and were given s.c. injections of fl-FNA 10, 5, 2.5 or 1.25 mg/kg, naloxone 10 or 5 mg/kg or drug vehicle, in a 10 ml/kg volume. Naloxone was dissolved in 0.9 % saline, and fl-FNA was dissolved in distilled water. The effects of fl-FNA diminish slowly over time 21 due, probably, to receptor repopulation. Therefore, to insure that p blockade was chronically maintained rats and mice in the fl-FNA groups received drug once every 48 h. Nevertheless, all animals received one injection every 24 h, and rats and mice in the fl-FNA groups received drug alternately with drug vehicle. Prior to hot plate testing all animals were given 4 acclimation trials on an ambient temperature (23 °C) plate in which they were placed individually on the plate for a duration of 60 s and returned to home cages. Animals in the fl-FNA groups received acclimation trials not less than 2 h after a vehicle injection, while animals in the naloxone groups received trials 10-15 min after a vehicle injection. Half of the controls in each experiment received trials 2 h after an injection of distilled water, and the other half received trials 10-15 min after an injection of saline. Hot plate tests were performed using the same procedure as for the acclimation trials with the exceptions that animals received drug injections according to their group prior to testing, and that the temperature of the hot plate was 52 + 0.5 °C for rats and 51 + 0.5 °C for mice. The latency to lick either a front or back paw following placement on the hot plate surface was recorded.
Test of K agonist-induced analgesia On the day after the 4th hot plate test, mice from all groups were tested for analgesia produced by the selective r receptor agonist, U50-488H. Half of the mice in each group were given a 5 mg/kg s.c. injection of U50-488H in an injection volume of 1 ml/kg. The other half of the mice in each group were given a l ml/kg s.c. injection of 0.9% saline. Thirty rain after the injections each mouse was tested on the hot plate as described above. RESULTS A total of 11 rats (3 f r o m c o n t r o l , 3 f r o m 3 p g f l - F N A , 4 f r o m 10/~g f l - F N A , and 1 f r o m n a l o x o n e g r o u p ) w e r e r e m o v e d f r o m the e x p e r i m e n t d u e to d a m a g e d or m i s p l a c e d cannulas. D a t a f r o m t h e s e animals w e r e not i n c l u d e d in analyses. Fig. l a depicts t h e m e a n (_+ S . E . M . ) P L L for the rat groups t r e a t e d with f l - F N A o v e r t h e 4 test days. A n a l y s i s of v a r i a n c e r e v e a l e d significant effects o f f l - F N A treatm e n t (F2,33 -= 4.08, P < 0.02) a n d test day (F3.99 = 15.80, P < 0.001). Fig. l b depicts the m e a n ( + S . E . M . ) P L L for the rats t r e a t e d with n a l o x o n e o v e r t h e 4 test days. A n a l y s i s of v a r i a n c e r e v e a l e d significant effects of n a l o x o n e treatm e n t (F1,21 = 4.83, P < 0.03) and test day (F3.63 = 6.90, P < 0.001). Since s u b s e q u e n t analysis r e v e a l e d no significant diff e r e n c e b e t w e e n the f l - F N A and n a l o x o n e v e h i c l e - t r e a t e d control g r o u p s t h e y w e r e c o m p i l e d in o r d e r to p e r f o r m an overall analysis of v a r i a n c e on all of the rat g r o u p s in this e x p e r i m e n t . T h e o v e r a l l analysis r e v e a l e d significant effects of drug t r e a t m e n t
(F3.49 = 4.51, P < 0.007), test
d a y (F3,147 = 33.53, P < 0.001) and d r u g x day i n t e r a c t i o n (F9,147 = 2.45, P < 0.01). Post h o c T u k e y tests
a)
b)
PJ,PPJ,,'TOF CHRONIC/I-FNA TREAllME)ff ON THE DEVELOPMENT OF ANALGESIA IN THE RAT
40.
40.
[]
.i ~ /].
30
~
,~.~
~
I
/L
30PLL
PLL
(=)
ETREEr Of" CHRONIC N/COXONE TRF.AIMENT ON 11.1E DEVELOPMENT OF A N A L ~ IN 114E RAT
20
•/./1 ----o-------~ i
(')
20
"&
i
/
l
v
10
10-
I 1
I 2
I 3
I
I
0
I 1
4.
I 2
I 3
I 4
I
TEST DAY TEST DAY Fig. 1. a: mean (+ S.E.M.) PLL of rats pre-treated with i.c.v, microinjections of 0 (O), 3 (A) or 10 (F1)/~g fl-FNA, b: mean (+ S.E.M.) PLL of rats pre-treated with i.c.v, microinjections of 0 (0) or 20 (A) #g naloxone. Measurements of PLL for each animal were made during daily hot plate tests over 4 consecutive days.
believed to be a form of stress-induced analgesia 3. Fig. 2a depicts the mean (_+ S.E.M.) P L L for the mouse groups treated with fl-FNA. Analysis of variance revealed significant effects of fl-FNA treatment (F4,7o = 5.18, P < 0.001), test day (F3,21o = 3.41, P < 0.01) and fl-FNA treatment x day interaction (Fi2,21o = 5.53, P 0.001). Fig. 2b depicts the mean (_+ S.E.M.) P L L for the mouse groups treated with naloxone. Analysis of variance revealed significant effects of naloxone treatment (Fz,a2 = 7.52, P < 0.002), test day (F3,126 = 11.44, P < 0.001) and naloxone treatment x day interaction (F6jz6 = 7.47, P < 0.001).
(P < 0.05) indicated that the 10 # g fl-FNA and naloxone groups displayed significantly longer P L L than the controls on days 2 - 4 while the 3 /.tg fl-FNA group displayed significantly longer P L L than the controls on days 3 and 4. In this experiment a P L L increase was also observed for vehicle-treated rats. This increase began on the second trial and reached a peak on the third trial. As described above, the analgesic response of control rats was of significantly lower magnitude than that of the fl-FNA- or naloxone-treated rats. A hot plate temperature-dependent P L L increase in saline-treated rats has also been observed by Greeley and Westbrook 3 and is
o) 30.
pil
,',-P¢.C1"OF CHRONICp-F'NA TREATMENT ON ~ DL~ME~FI" OF ANALGESIA IN MICE
0 - - 0 conf.'~l ~
1.,2am
O
b) 30. l l ~ I
kg
PJ-i-l:.GTOF' CHRONIC NN.OXONETREATME~'T' ON THE DEVELOPMENT OF N~N.GESIA IN MICE control 5 rno/
f
T
/ll'~'--A~
20.
PU.
20-
e/
(.)
C=) 10-
0
10-
I 1
I 2
I 3
TEST DAY
I 4
I
0
I 1
I 2
I 3
I 4.
TEST DAY
Fig. 2. a: mean (_+ S.E.M.) PLL of mice pre-treated with s.c. injections of 0 (O), 1.25 (A), 2.5 (n), 5 (~7) or 10 (~) mg/kg fl-FNA, b: mean (+ S.E.M.) PLL of mice pre-treated with s.c. injections of 0 (O), 5 (1) or 10 (A) mg/kg naloxone. Measurements of PLL for each animal were made during daily hot plate tests over 4 consecutive days.
184 a)
60-
ANALGESICrJ-I-P.CTSOF U50-4.88H IN MICE RECEIVING CHRONIC PRE-TREATMENTWITH/Y-F'NA
b)
60-
n~a U S O ~ - t r e o t e d i--I vohl~e-b'etfl:e(l
50-
ANALGESICEFFECI~ OF USO-488H IN MICE RECEIVING CHRONIC PRE-TREATME1'~TWITH NALOXONE USO-4.88H-treated r'--I vehlcle-b'eatod
lib
50-
PLL 40
PLL 40
(,)
(s)
30
30
20
20
10
10
O
0 O
1.25 2.5 5 10 ~-FNA PRE-TREATMENTDOSE (mg/kg)
0
5
10
NALOXONE PRE-TREATMENT DOSE (mg/kg)
Fig. 3. a: mean (+ S.E.M.) PLL of mice treated chronically with 0, 1.25, 2.5, 5 or 10 mg/kg fl-FNA, b: mean (+ S.E.M.) PLL of mice treated chronically with 0, 5 or 10 mg/kg naloxone. Half of the animals from each fl-FNA and naloxone dose group received a s.c. injection of U50-488H (5 mg/kg) and the other half received a s.c. injection of vehicle 30 min prior to hot plate testing.
As in the rat experiment, subsequent analysis revealed no significant difference between the fl-FNA and naloxone vehicle-treated controls and, therefore, they were compiled in order to perform an overall analysis of variance on all the data. The overall analysis of variance revealed significant effects of drug t r e a t m e n t (F6,113 = 6.53, P < 0.001), test day (F3,339 = 15.95, P < 0.001) and drug × day interaction (F18,339 ~-- 6.95, P < 0.001). Post hoc Tukey tests (P < 0.05) revealed the following differences between groups on specific days. The two naloxone groups did not differ from each other over the four test days, however they displayed significantly longer PLL than the control group on days 2-4. The 10 mg/kg fl-FNA group showed PLL similar to those of the control group on the first two tests, but performed similarly to the naioxone groups on the last two trials. The three lower fl-FNA groups displayed a less pronounced effect. The 5 and 2.5 mg/kg groups did not differ from each other over the 4 test days and showed significant differences from the control group only on day 3. The 1.25 mg/kg group differed from the control only on day 4. Fig. 3a depicts mean (_+ S.E.M.) PLL for mice pre-treated with fl-FNA on the test of K agonist-induced analgesia. Recall that half of the animals in each group received injections of U50-488H and the other half received injections of the drug vehicle. Analysis of variance revealed significant effects of fl-FNA pretreatment (F4,65 = 5.44, P < 0.001) and U50-488H treatment (F~,65 = 30.64, P < 0.001). Post hoc Tukey tests (P < 0.05) indicated that the U50-488H-treated animals displayed significantly longer PLL than vehicle-treated animals at all pre-treatment dose levels except 1.25 mg/kg.
Fig. 3b depicts the mean (+ S.E.M.) PLL for mice receiving naloxone pre-treatment on the test of r agonistinduced analgesia. Analysis of variance revealed significant effects of naloxone pre-treatment (F2.39 = 7.12, P < 0.002) and U50-488H treatment (Fi,39 = 22.07, P < 0.001). Post hoc Tukey tests (P < 0.05) indicated that the U50488H-treated mice displayed significantly longer PLL than vehicle-treated mice and all pre-treatment doses. DISCUSSION
The results of the present experiments clearly showed that chronic blockade of /~ receptors is sufficient to produce analgesia on repeated hot plate tests, and an augmented analgesic response to a r agonist. O B A can be produced when naloxone or fl-FNA are delivered via i.c.v, or s.c. routes. Finally, O B A was shown to develop in both rats and mice. As previously indicated, chronic treatment with nonselective opioid antagonists has the paradoxical effect of producing analgesia in rats 1-SA3A4. The present study demonstrates that chronic selective blockade of/~ receptors is sufficient to induce analgesia in both rats and mice. Rats treated with i.c.v, microinjections of the selective antagonist, fl-FNA, or naloxone developed similar levels of analgesia at a comparable rate. In a second experiment it was shown that treatment with fl-FNA or naloxone induced an analgesic response in mice that was similar to that observed with rats. Mice treated with the largest dose (10 mg/kg) of fl-FNA displayed an analgesic response comparable to that of the rat. In contrast, mice treated with fl-FNA doses in the range of 1.25-5 mg/kg developed an analgesic response of similar magnitude
185 which was not as great as that displayed by the highest dose group. Therefore, although this experiment did not contain enough fl-FNA dose groups to construct a proper dose response curve, there is evidence to suggest that an increase in fl-FNA treatment dose is accompanied by an increased O B A response. There is one issue that is important to discuss with regard to the analgesia produced by fl-FNA. It is highly improbable that the analgesia seen in fl-FNA-treated animals was due to agonist activity for two reasons. First, the analgesia was measured 24 h after drug administration when the agonist effects of fl-FNA are no longer present. As mentioned earlier, the agonist effects of fl-FNA occur within the first hour after administration, whereas the antagonist effects of fl-FNA are observed within an hour after administration and last for at least 4 days 2~. Second, no acute agonist effects of fl-FNA were visible in mice or rats on the first hot plate test. Accordingly, these observations constitute the first demonstration of analgesia induced by chronic selective blockade of/~ opioid receptors, and support our previous demonstrations of OBA. This study has also provided the important finding that O B A develops in the mouse as well as in the rat. Although, there were some differences with respect to the onset of OBA, the basic analgesic response to naloxone and fl-FNA treatment was very similar in rats and mice. In both species treatment with fl-FNA or naloxone resulted in the development of a similar analgesic response. Another similarity between mice and rats was observed in the test of r agonist-induced analgesia. In this experiment, mice from the naloxone pre-treatment groups that were injected with saline displayed a conditioned analgesia. Previous experiments 4'14 have clearly established that naloxone-induced analgesia reflects a conditioned analgesia in rats. Thus, the nature and effects of O B A are very similar in both species. Preceding studies 4'13"14 have demonstrated an augmented analgesic response to the analgesic effects of the /~ receptor agonist, morphine, in rats displaying OBA. In this study, an augmented analgesic response was observed to the analgesic effects of a highly selective r receptor agonist, U50-488H. This effect occurred in mice that had been previously treated with fl-FNA or naloxone. REFERENCES 1 Cappell, H., Knoke, D.M., Ld, A.D. and Poulos, C.X., Naloxone-induced analgesia: effects of the benzodiazepine antagonist Ro 15-1788, Pharmacol. Biochem. Behav., 34 (1989) 197-200, 2 Cappell, H., Poulos, C.X. and L6, A.D., Enhancement of naloxone-induced analgesia by pretreatment with morphine, Pharmacol. Biochem. Behav., 34 (1989) 425-427.
There are two possible explanations for this effect. First, it is possible that supersensitivity to opioid agonists is related to up-regulation of opioid receptors after chronic treatment with antagonists. Functional supersensitivity to opiates after chronic treatment with opioid antagonists has been reliably demonstrated by various investigators 9'~8'22'23. In addition, an increase in opioid receptor density has been observed in many studies following chronic treatment with naloxone or naltrexone 1°'22'23. There are, however, some important differences with respect to these studies and the present findings. Specifically, previous demonstrations of opiate supersensitivity and receptor up-regulation have followed treatment with doses of naloxone or naltrexone which are considerably higher than those used in this study 8-~1'~7. Furthermore, these studies have always involved chronic, continuous administration of drug 9A°'22'23. In this study the fl-FNA groups would constitute a continuous treatment condition, however, it is clear that this is not the case with naloxone. It should be noted, that unlike the present study, previous studies were performed in the absence of repeated stressful or painful stimulation. Therefore, the question remains as to whether chronic treatment with lower doses of naloxone in the presence of such stimuli results in opiate supersensitivity. A second possible explanation of this finding involves the observation that mice from the naloxone pretreatment groups displayed a conditioned analgesia after an injection of saline on the test of r agonist-induced analgesia. It has been clearly established that naloxoneinduced analgesia reflects a conditioned analgesia 4"14, accordingly, an augmented response to ~ and r receptor agonists may reflect summation between their analgesic effects and this conditioned analgesia. On the basis of the present data, however, it is impossible to choose which of these explanations truly underlies the increased opioid agonist-induced analgesia observed in animals displaying OBA. Acknowledgements. This study was funded by National Science and Engineering Council of Canada Grant 3-640-118-80 to H.C. and C.X.P.M.J.K.W. was supported by an Addiction Research Foundation Scholarship. We are grateful to the Upjohn Company for the generous donation of U50-488H. We would also like to thank Ms. Annette Goebel and Ms. Chris Cotie for their excellent technical assistance.
3 Greeley, J.D. and Westbrook, R.E, Some effects of exposure to a heat stressor upon the rat's subsequent reactions to that stressor, Quart. J. Exp. Psychol. B, 42 (1990) 1-40. 4 Greeley, J.D., Conditioned Inhibition in a Homeostatic Response System: Evidence from Pharmacological Conditioning with Naloxone and Morphine, A thesis submitted in conformity with the
requirements of the degree of Doctor of Philosophy, Department of Psychology, University of Toronto, Toronto, 1987. 5 Greeley, J.D., L~, A.D., Poulos, C.X. and CappeU, H.,
186
6
7
8 9
10
11
12
13
"Paradoxical' analgesia induced by naloxone and naltrexone, Psychopharmacology, 96 (1988) 36-39. Jaffe, J.H. and Martin, W.R., Opioid analgesics and antagonists. In A.G. Gilman, L.S. Goodman, T.W. Rail and E Murad (Eds.), Goodman and Gilman's, The Pharmacological Basis of Therapeutics, MacMillan, New York, 1985, pp. 491-531. Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J., Anatomy of CNS opioid receptors, Trends. Neurosci., 11 (1988) 308-316. Martin, W.R., Pharmacology of opioids, Pharmacol. Rev., 35 (1984) 283-323. Millan, M.J., Morris, B.J. and Herz, A., Antagonist-induced opioid receptor up-regulation. I. Characterization of supersensitivity to selective mu and kappa agonists, J. Pharmacol. Exp. Ther., 247 (1988) 721-728. Morris, B.J., Millan, M.J. and Herz, A., Antagonist-induced opioid receptor up-regulation. II. Regionally specific modulation of mu, delta and kappa binding sites in rat brain revealed by quantitative autoradiography, J. Pharmacol. Exp. Ther., 247 (1988) 729-736. Pfeiffer, A., Pfeiffer, D.G., Feuerstein, G., Faden, A.I. and Kopin, I.J., An increase in opiate receptor-sites is associated with enhanced cardiovascular depressant, but not respiratory depressant action of morphine, Brain Research, 296 (1984) 305-311. Portoghese, P.S., Larson, D.L., Sayer, L.M., Fries, D.S. and Takemori, A.E., A novel opioid receptor site directed alkylating agent with irreversible narcotic antagonistic and reversible agonistic activities, J. Med. Chem. 23 (1980) 233-234. Pouios, C.X., Knoke, D.M., L6, A.D. and Cappell, H., Naloxone-induced analgesia and morphine supersensitivity effects are contingent upon prior exposure to analgesic testing,
Psychopharmacology, 100 (1990) 87-103. 14 Rochford, J. and Stewart, J., Activation and expression of endogenous pain control mechanisms in rats given repeated nociceptive tests under the influence of naloxone, Behav. Neurosci., 101 (1987) 87-103. 15 Snyder, S.H., Drug and neurotransmitter receptors in the brain, Science, 224 (1984) 22-31. 16 Takemori, A.E., Larson, D.L. and Portoghese, P.S., The irreversible narcotic antagonistic and reversible agonistic properties of the fumaramate methyl ester derivative of naltrexone, Eur. J. Pharmacol., 70 (1981) 445-451. 17 Tempel, A., Gardner, E.L. and Zukin, R.S., Neurochemical and functional correlates of naltrexone-induced opiate receptor up-regulation, J. Pharmacol. Exp. Ther., 232 (1985) 439-444. 18 Tempel, A., Zukin, R.S. and Gardner, E.L., Supersensitivity of brain opiate receptor subtypes after chronic naltrexone treatment, Life Sci., 31 (1982) 1401-1404. 19 von Voigtlander, P.E, Lahti, R.A. and Ludens, J.H., U-50,488: A selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther., 224 (1983) 7-12. 20 Ward, S.J., Portoghese, P.S. and Takemori, A.E., Pharmacological profiles of fl-funaltrexamine (fl-FNA) and fl-chlornaltrexamine (fl-CNA) on the mouse vas deferens preparation, Eur. J. Pharmacol., 80 (1982) 377-384. 21 Ward, S.J., Portoghese, P.S. and Takemori, A.E., Pharmacological characterization in vivo of the novel opiate, fl-funaltrexamine, J. Pharmacol. Exp. Ther., 220 (1982b) 494-498. 22 Yoburn, B.C., Sierra, V. and Lutfy, K., Chronic opioid antagonist treatment: assessment of receptor up-regulation, Eur. J. Pharmacol., 170 (1989) 193-200. 23 Zukin, R.S. and Tempel, A., Neurochemical correlates of opiate receptor regulation, Biochem. Pharmacol., 35 (1986) 1623-1627.
181
Research Reports
Chronic selective blockade of/ opioid receptors produces analgesia and augmentation of the effects of a agonist M.J. Katharine Walker 1'2, A.D. L61, Constantine X.
P o u l o s 1'3
and
Howard
CappeU 1'2'3
1Addiction Research Foundation, Toronto, Ont. (Canada) and Departments of 2pharmacology and 3psychology, University of Toronto, Toronto, Ont. (Canada) (Accepted 7 August 1990) Key words: Analgesia; fl-Funaltrexamine; Naloxone; U50-488H; r Opioid analgesia
We have previously demonstrated that, when administered chronically, naloxone and naitrexone have the paradoxical effect of producing analgesia in rats. In this study, rats treated chronically with intracerebroventricular (i.c.v.) microinjections, and mice treated chronically with subcutaneous (s.c.) injections of naloxone or fl-funaltrexamine (fl-FNA) developed analgesia on daily hot plate tests. There was no drug effect on the first day of hot plate testing, but significant increases in paw lick latency developed over subsequent acquisition sessions for animals treated with fl-FNA or naloxone. An augmented analgesic response to a 5 mg/kg s.c. injection of the r opioid agonist, U50-488H, was observed in mice previously treated with naloxone or fl-FNA. The primary findings of the present study were: (1) chronic blockade of/~ opioid receptors is sufficient to produce analgesia on repeated hot plate tests in both rats and mice; (2) chronic blockade of/~ receptors in the presence of stressful stimuli results in augmentation of r agonist-induced analgesia; and (3) the phenomenon of opioid blockade-induced analgesia (OBA) occurs in mice as well as rats. INTRODUCTION We have previously demonstrated that when administered chronically naloxone and naltrexone have the paradoxical effect of producing analgesia in rats 1-5'13'14. Greeley et al. 5 demonstrated that rats implanted with naloxone or naltrexone pellets or receiving 5 mg/kg naloxone injections showed an absolute increase in paw lick latency (PLL). The magnitude of this increase in PLL was such that by the fifth hot plate test the latency of rats injected with naloxone was three times that of controls. Although the mechanism of opioid blockade-induced analgesia (OBA) is not known, important features of this phenomenon have recently been described. Poulos et al. 13 clearly showed that naloxone-induced analgesia develops only when rats receive hot plate testing under the influence of naloxone. In the absence of hot plate testing, mere repeated administration of naloxone failed to produce analgesia. Rats displaying naloxone-induced analgesia also showed an augmented analgesic response to morphine on hot plate and tail-flick tests ~3. Whether O B A is mediated by aspects of the opioid system or by a non-opioid mechanism is still unknown. Opioid analgesia has been associated with p, 6 and opioid receptors 6-8'15. Naloxone binds readily to/~, r and 6 opioid receptors, but its affinity for ~ receptors is
generally more than tenfold higher than for other opioid receptor types 6. Naloxone and naltrexone are also known to produce changes in the numbers of/~ receptors when administered chronically at lower doses than those needed to produce changes in 1¢ or 6 receptors 9'17'23. It may be possible that when one opioid receptor subtype is preferentially blocked under conditions that normally stimulate endogenous opioid analgesia, the analgesic capacity of another opioid receptor subtype is revealed resulting in the development of analgesia. Both the morphine selectivity of the/~ receptor and its distribution in brain regions which have a putative role in pain regulation strongly implicate it as a major factor in supraspinal mediation of opioid analgesia 7'is. Therefore, blockade of p receptors under conditions in which they are normally active, may be sufficient to stimulate the analgesic capacity of other opioid receptor types. Thus far, there has been no demonstration of the contribution of selective opioid receptor blockade to the development of OBA. However, in order to further understand the underlying mechanism of O B A we must first know the limiting conditions under which it will develop. Accordingly, the primary goal of the present study was to determine whether selective blockade of ~ receptors would produce analgesia similar in nature to that produced by naloxone, fl-Funaltrexamine (fl-FNA) is a site
Correspondence: M.J.K. Walker, 6th Floor, Tower, Addiction Research Foundation, 33 Russell St., Toronto, Ont., Canada M5S 2S1. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
182 d i r e c t e d aikylating a g e n t with reversible agonist and irreversible, selective p r e c e p t o r antagonistic activities 12. T h e agonist actions of f l - F N A are o b s e r v e d less t h a n 1 h after a d m i n i s t r a t i o n , and are a n t a g o n i z e d by n a i o x o n e 16'
2o.21 In contrast, the antagonist actions of f l - F N A are d e m o n s t r a t e d within an h o u r of a d m i n i s t r a t i o n and last for at least 4 days. T h e s e antagonist actions o f f l - F N A are m e d i a t e d t h r o u g h p r e c e p t o r s L6'2°'21. B e c a u s e it is an irreversible
and
selective /~ r e c e p t o r
blocker,
using
fl-FNA has a particular a d v a n t a g e in that it does not c o m p e t e for binding with the e n d o g e n o u s p o o l of p r e c e p t o r ligands. T h e r e f o r e , the use of f l - F N A m a k e s it possible to selectively r e m o v e the p r e c e p t o r c o m p o n e n t of the e n d o g e n o u s
o p i o i d system.
Since analgesia is
m e d i a t e d by b o t h p and'~¢ o p i o i d r e c e p t o r s , it is possible that by b l o c k i n g only ~ r e c e p t o r s u n d e r the influence of stress the analgesic capacity of ~c r e c e p t o r s m a y b e c o m e supersensitive.
To test this hypothesis,
mice t r e a t e d
chronically with f l - F N A or n a l o x o n e in c o n j u n c t i o n with hot plate
testing w e r e
subsequently
tested for their
r e s p o n s e to the analgesic effects of a selective 7c agonist, U 5 0 - 4 8 8 H 19. Finally, the e x p e r i m e n t s w e r e d e s i g n e d to demonstrate
OBA
in two different
species,
rat
and
mouse. MATERIALS AND METHODS
Animals Sixty-four male Sprague-Dawley rats, and 120 male CD1 mice (Charles River Canada, Inc., St. Constant, Que.) weighing 280-300 g and 25-30 g, respectively, were used. Rats and mice were housed and tested in different rooms. They were singly housed in stainless-steel cages with free access to food (Purina Lab Chow) and water. The rat diet consisted of 6 pellets (35 g) per day. This diet promotes healthy weight gain without the development of obesity. The colony room was maintained at 24 °C with a 12-h light-dark cycle (light on during experimental procedures). Surgery Rats were chronically implanted, using standard stereotaxic technique and pentobarbital (50 mg/kg) anaesthesia, with a 23gauge stainless-steel guide cannula at coordinates 0.8 mm posterior to bregma and 1.4 mm lateral to the sagittal suture. The cannula terminated 3.7 mm below the surface of the skull in the lateral ventricle. Five to 7 days was allowed for recovery before drug injections began. At the end of the experiment the rats were killed by pentobarbital overdose and an injection of dye was used to confirm the accuracy of the placement. Apparatus Analgesia was measured using a hot plate apparatus consisting of a plastic storage tub with an aluminum plate on the surface (Grant Instrument Water Bath Model AB02). The temperature of the surface was monitored continually with a thermistor (Yellow Springs Instrument Model 343 TC) and probes (Yellow Springs Banjo Surface Model 408) clamped securely to the surface of the aluminum plate. Rats were confined to the surface of the hot plate within a clear Plexiglas box (27 cm x 16.5 cm × 40 cm high) and mice within a similar Plexiglas box (27 cm × 16.5 cm × 9 cm high) with lids. Procedure Rats were randomly assigned to 4 groups and were given i.c.v.
injections of fl-FNA 10 or 3 ltg, naloxone HCL (Sigma, St. Louis) 20 pg or drug vehicle, in a 3 ~1 volume. Mice were randomly assigned to 7 groups and were given s.c. injections of fl-FNA 10, 5, 2.5 or 1.25 mg/kg, naloxone 10 or 5 mg/kg or drug vehicle, in a 10 ml/kg volume. Naloxone was dissolved in 0.9 % saline, and fl-FNA was dissolved in distilled water. The effects of fl-FNA diminish slowly over time 21 due, probably, to receptor repopulation. Therefore, to insure that p blockade was chronically maintained rats and mice in the fl-FNA groups received drug once every 48 h. Nevertheless, all animals received one injection every 24 h, and rats and mice in the fl-FNA groups received drug alternately with drug vehicle. Prior to hot plate testing all animals were given 4 acclimation trials on an ambient temperature (23 °C) plate in which they were placed individually on the plate for a duration of 60 s and returned to home cages. Animals in the fl-FNA groups received acclimation trials not less than 2 h after a vehicle injection, while animals in the naloxone groups received trials 10-15 min after a vehicle injection. Half of the controls in each experiment received trials 2 h after an injection of distilled water, and the other half received trials 10-15 min after an injection of saline. Hot plate tests were performed using the same procedure as for the acclimation trials with the exceptions that animals received drug injections according to their group prior to testing, and that the temperature of the hot plate was 52 + 0.5 °C for rats and 51 + 0.5 °C for mice. The latency to lick either a front or back paw following placement on the hot plate surface was recorded.
Test of K agonist-induced analgesia On the day after the 4th hot plate test, mice from all groups were tested for analgesia produced by the selective r receptor agonist, U50-488H. Half of the mice in each group were given a 5 mg/kg s.c. injection of U50-488H in an injection volume of 1 ml/kg. The other half of the mice in each group were given a l ml/kg s.c. injection of 0.9% saline. Thirty rain after the injections each mouse was tested on the hot plate as described above. RESULTS A total of 11 rats (3 f r o m c o n t r o l , 3 f r o m 3 p g f l - F N A , 4 f r o m 10/~g f l - F N A , and 1 f r o m n a l o x o n e g r o u p ) w e r e r e m o v e d f r o m the e x p e r i m e n t d u e to d a m a g e d or m i s p l a c e d cannulas. D a t a f r o m t h e s e animals w e r e not i n c l u d e d in analyses. Fig. l a depicts t h e m e a n (_+ S . E . M . ) P L L for the rat groups t r e a t e d with f l - F N A o v e r t h e 4 test days. A n a l y s i s of v a r i a n c e r e v e a l e d significant effects o f f l - F N A treatm e n t (F2,33 -= 4.08, P < 0.02) a n d test day (F3.99 = 15.80, P < 0.001). Fig. l b depicts the m e a n ( + S . E . M . ) P L L for the rats t r e a t e d with n a l o x o n e o v e r t h e 4 test days. A n a l y s i s of v a r i a n c e r e v e a l e d significant effects of n a l o x o n e treatm e n t (F1,21 = 4.83, P < 0.03) and test day (F3.63 = 6.90, P < 0.001). Since s u b s e q u e n t analysis r e v e a l e d no significant diff e r e n c e b e t w e e n the f l - F N A and n a l o x o n e v e h i c l e - t r e a t e d control g r o u p s t h e y w e r e c o m p i l e d in o r d e r to p e r f o r m an overall analysis of v a r i a n c e on all of the rat g r o u p s in this e x p e r i m e n t . T h e o v e r a l l analysis r e v e a l e d significant effects of drug t r e a t m e n t
(F3.49 = 4.51, P < 0.007), test
d a y (F3,147 = 33.53, P < 0.001) and d r u g x day i n t e r a c t i o n (F9,147 = 2.45, P < 0.01). Post h o c T u k e y tests
a)
b)
PJ,PPJ,,'TOF CHRONIC/I-FNA TREAllME)ff ON THE DEVELOPMENT OF ANALGESIA IN THE RAT
40.
40.
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30
~
,~.~
~
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ETREEr Of" CHRONIC N/COXONE TRF.AIMENT ON 11.1E DEVELOPMENT OF A N A L ~ IN 114E RAT
20
•/./1 ----o-------~ i
(')
20
"&
i
/
l
v
10
10-
I 1
I 2
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TEST DAY TEST DAY Fig. 1. a: mean (+ S.E.M.) PLL of rats pre-treated with i.c.v, microinjections of 0 (O), 3 (A) or 10 (F1)/~g fl-FNA, b: mean (+ S.E.M.) PLL of rats pre-treated with i.c.v, microinjections of 0 (0) or 20 (A) #g naloxone. Measurements of PLL for each animal were made during daily hot plate tests over 4 consecutive days.
believed to be a form of stress-induced analgesia 3. Fig. 2a depicts the mean (_+ S.E.M.) P L L for the mouse groups treated with fl-FNA. Analysis of variance revealed significant effects of fl-FNA treatment (F4,7o = 5.18, P < 0.001), test day (F3,21o = 3.41, P < 0.01) and fl-FNA treatment x day interaction (Fi2,21o = 5.53, P 0.001). Fig. 2b depicts the mean (_+ S.E.M.) P L L for the mouse groups treated with naloxone. Analysis of variance revealed significant effects of naloxone treatment (Fz,a2 = 7.52, P < 0.002), test day (F3,126 = 11.44, P < 0.001) and naloxone treatment x day interaction (F6jz6 = 7.47, P < 0.001).
(P < 0.05) indicated that the 10 # g fl-FNA and naloxone groups displayed significantly longer P L L than the controls on days 2 - 4 while the 3 /.tg fl-FNA group displayed significantly longer P L L than the controls on days 3 and 4. In this experiment a P L L increase was also observed for vehicle-treated rats. This increase began on the second trial and reached a peak on the third trial. As described above, the analgesic response of control rats was of significantly lower magnitude than that of the fl-FNA- or naloxone-treated rats. A hot plate temperature-dependent P L L increase in saline-treated rats has also been observed by Greeley and Westbrook 3 and is
o) 30.
pil
,',-P¢.C1"OF CHRONICp-F'NA TREATMENT ON ~ DL~ME~FI" OF ANALGESIA IN MICE
0 - - 0 conf.'~l ~
1.,2am
O
b) 30. l l ~ I
kg
PJ-i-l:.GTOF' CHRONIC NN.OXONETREATME~'T' ON THE DEVELOPMENT OF N~N.GESIA IN MICE control 5 rno/
f
T
/ll'~'--A~
20.
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(.)
C=) 10-
0
10-
I 1
I 2
I 3
TEST DAY
I 4
I
0
I 1
I 2
I 3
I 4.
TEST DAY
Fig. 2. a: mean (_+ S.E.M.) PLL of mice pre-treated with s.c. injections of 0 (O), 1.25 (A), 2.5 (n), 5 (~7) or 10 (~) mg/kg fl-FNA, b: mean (+ S.E.M.) PLL of mice pre-treated with s.c. injections of 0 (O), 5 (1) or 10 (A) mg/kg naloxone. Measurements of PLL for each animal were made during daily hot plate tests over 4 consecutive days.
184 a)
60-
ANALGESICrJ-I-P.CTSOF U50-4.88H IN MICE RECEIVING CHRONIC PRE-TREATMENTWITH/Y-F'NA
b)
60-
n~a U S O ~ - t r e o t e d i--I vohl~e-b'etfl:e(l
50-
ANALGESICEFFECI~ OF USO-488H IN MICE RECEIVING CHRONIC PRE-TREATME1'~TWITH NALOXONE USO-4.88H-treated r'--I vehlcle-b'eatod
lib
50-
PLL 40
PLL 40
(,)
(s)
30
30
20
20
10
10
O
0 O
1.25 2.5 5 10 ~-FNA PRE-TREATMENTDOSE (mg/kg)
0
5
10
NALOXONE PRE-TREATMENT DOSE (mg/kg)
Fig. 3. a: mean (+ S.E.M.) PLL of mice treated chronically with 0, 1.25, 2.5, 5 or 10 mg/kg fl-FNA, b: mean (+ S.E.M.) PLL of mice treated chronically with 0, 5 or 10 mg/kg naloxone. Half of the animals from each fl-FNA and naloxone dose group received a s.c. injection of U50-488H (5 mg/kg) and the other half received a s.c. injection of vehicle 30 min prior to hot plate testing.
As in the rat experiment, subsequent analysis revealed no significant difference between the fl-FNA and naloxone vehicle-treated controls and, therefore, they were compiled in order to perform an overall analysis of variance on all the data. The overall analysis of variance revealed significant effects of drug t r e a t m e n t (F6,113 = 6.53, P < 0.001), test day (F3,339 = 15.95, P < 0.001) and drug × day interaction (F18,339 ~-- 6.95, P < 0.001). Post hoc Tukey tests (P < 0.05) revealed the following differences between groups on specific days. The two naloxone groups did not differ from each other over the four test days, however they displayed significantly longer PLL than the control group on days 2-4. The 10 mg/kg fl-FNA group showed PLL similar to those of the control group on the first two tests, but performed similarly to the naioxone groups on the last two trials. The three lower fl-FNA groups displayed a less pronounced effect. The 5 and 2.5 mg/kg groups did not differ from each other over the 4 test days and showed significant differences from the control group only on day 3. The 1.25 mg/kg group differed from the control only on day 4. Fig. 3a depicts mean (_+ S.E.M.) PLL for mice pre-treated with fl-FNA on the test of K agonist-induced analgesia. Recall that half of the animals in each group received injections of U50-488H and the other half received injections of the drug vehicle. Analysis of variance revealed significant effects of fl-FNA pretreatment (F4,65 = 5.44, P < 0.001) and U50-488H treatment (F~,65 = 30.64, P < 0.001). Post hoc Tukey tests (P < 0.05) indicated that the U50-488H-treated animals displayed significantly longer PLL than vehicle-treated animals at all pre-treatment dose levels except 1.25 mg/kg.
Fig. 3b depicts the mean (+ S.E.M.) PLL for mice receiving naloxone pre-treatment on the test of r agonistinduced analgesia. Analysis of variance revealed significant effects of naloxone pre-treatment (F2.39 = 7.12, P < 0.002) and U50-488H treatment (Fi,39 = 22.07, P < 0.001). Post hoc Tukey tests (P < 0.05) indicated that the U50488H-treated mice displayed significantly longer PLL than vehicle-treated mice and all pre-treatment doses. DISCUSSION
The results of the present experiments clearly showed that chronic blockade of /~ receptors is sufficient to produce analgesia on repeated hot plate tests, and an augmented analgesic response to a r agonist. O B A can be produced when naloxone or fl-FNA are delivered via i.c.v, or s.c. routes. Finally, O B A was shown to develop in both rats and mice. As previously indicated, chronic treatment with nonselective opioid antagonists has the paradoxical effect of producing analgesia in rats 1-SA3A4. The present study demonstrates that chronic selective blockade of/~ receptors is sufficient to induce analgesia in both rats and mice. Rats treated with i.c.v, microinjections of the selective antagonist, fl-FNA, or naloxone developed similar levels of analgesia at a comparable rate. In a second experiment it was shown that treatment with fl-FNA or naloxone induced an analgesic response in mice that was similar to that observed with rats. Mice treated with the largest dose (10 mg/kg) of fl-FNA displayed an analgesic response comparable to that of the rat. In contrast, mice treated with fl-FNA doses in the range of 1.25-5 mg/kg developed an analgesic response of similar magnitude
185 which was not as great as that displayed by the highest dose group. Therefore, although this experiment did not contain enough fl-FNA dose groups to construct a proper dose response curve, there is evidence to suggest that an increase in fl-FNA treatment dose is accompanied by an increased O B A response. There is one issue that is important to discuss with regard to the analgesia produced by fl-FNA. It is highly improbable that the analgesia seen in fl-FNA-treated animals was due to agonist activity for two reasons. First, the analgesia was measured 24 h after drug administration when the agonist effects of fl-FNA are no longer present. As mentioned earlier, the agonist effects of fl-FNA occur within the first hour after administration, whereas the antagonist effects of fl-FNA are observed within an hour after administration and last for at least 4 days 2~. Second, no acute agonist effects of fl-FNA were visible in mice or rats on the first hot plate test. Accordingly, these observations constitute the first demonstration of analgesia induced by chronic selective blockade of/~ opioid receptors, and support our previous demonstrations of OBA. This study has also provided the important finding that O B A develops in the mouse as well as in the rat. Although, there were some differences with respect to the onset of OBA, the basic analgesic response to naloxone and fl-FNA treatment was very similar in rats and mice. In both species treatment with fl-FNA or naloxone resulted in the development of a similar analgesic response. Another similarity between mice and rats was observed in the test of r agonist-induced analgesia. In this experiment, mice from the naloxone pre-treatment groups that were injected with saline displayed a conditioned analgesia. Previous experiments 4'14 have clearly established that naloxone-induced analgesia reflects a conditioned analgesia in rats. Thus, the nature and effects of O B A are very similar in both species. Preceding studies 4'13"14 have demonstrated an augmented analgesic response to the analgesic effects of the /~ receptor agonist, morphine, in rats displaying OBA. In this study, an augmented analgesic response was observed to the analgesic effects of a highly selective r receptor agonist, U50-488H. This effect occurred in mice that had been previously treated with fl-FNA or naloxone. REFERENCES 1 Cappell, H., Knoke, D.M., Ld, A.D. and Poulos, C.X., Naloxone-induced analgesia: effects of the benzodiazepine antagonist Ro 15-1788, Pharmacol. Biochem. Behav., 34 (1989) 197-200, 2 Cappell, H., Poulos, C.X. and L6, A.D., Enhancement of naloxone-induced analgesia by pretreatment with morphine, Pharmacol. Biochem. Behav., 34 (1989) 425-427.
There are two possible explanations for this effect. First, it is possible that supersensitivity to opioid agonists is related to up-regulation of opioid receptors after chronic treatment with antagonists. Functional supersensitivity to opiates after chronic treatment with opioid antagonists has been reliably demonstrated by various investigators 9'~8'22'23. In addition, an increase in opioid receptor density has been observed in many studies following chronic treatment with naloxone or naltrexone 1°'22'23. There are, however, some important differences with respect to these studies and the present findings. Specifically, previous demonstrations of opiate supersensitivity and receptor up-regulation have followed treatment with doses of naloxone or naltrexone which are considerably higher than those used in this study 8-~1'~7. Furthermore, these studies have always involved chronic, continuous administration of drug 9A°'22'23. In this study the fl-FNA groups would constitute a continuous treatment condition, however, it is clear that this is not the case with naloxone. It should be noted, that unlike the present study, previous studies were performed in the absence of repeated stressful or painful stimulation. Therefore, the question remains as to whether chronic treatment with lower doses of naloxone in the presence of such stimuli results in opiate supersensitivity. A second possible explanation of this finding involves the observation that mice from the naloxone pretreatment groups displayed a conditioned analgesia after an injection of saline on the test of r agonist-induced analgesia. It has been clearly established that naloxoneinduced analgesia reflects a conditioned analgesia 4"14, accordingly, an augmented response to ~ and r receptor agonists may reflect summation between their analgesic effects and this conditioned analgesia. On the basis of the present data, however, it is impossible to choose which of these explanations truly underlies the increased opioid agonist-induced analgesia observed in animals displaying OBA. Acknowledgements. This study was funded by National Science and Engineering Council of Canada Grant 3-640-118-80 to H.C. and C.X.P.M.J.K.W. was supported by an Addiction Research Foundation Scholarship. We are grateful to the Upjohn Company for the generous donation of U50-488H. We would also like to thank Ms. Annette Goebel and Ms. Chris Cotie for their excellent technical assistance.
3 Greeley, J.D. and Westbrook, R.E, Some effects of exposure to a heat stressor upon the rat's subsequent reactions to that stressor, Quart. J. Exp. Psychol. B, 42 (1990) 1-40. 4 Greeley, J.D., Conditioned Inhibition in a Homeostatic Response System: Evidence from Pharmacological Conditioning with Naloxone and Morphine, A thesis submitted in conformity with the
requirements of the degree of Doctor of Philosophy, Department of Psychology, University of Toronto, Toronto, 1987. 5 Greeley, J.D., L~, A.D., Poulos, C.X. and CappeU, H.,
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6
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"Paradoxical' analgesia induced by naloxone and naltrexone, Psychopharmacology, 96 (1988) 36-39. Jaffe, J.H. and Martin, W.R., Opioid analgesics and antagonists. In A.G. Gilman, L.S. Goodman, T.W. Rail and E Murad (Eds.), Goodman and Gilman's, The Pharmacological Basis of Therapeutics, MacMillan, New York, 1985, pp. 491-531. Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J., Anatomy of CNS opioid receptors, Trends. Neurosci., 11 (1988) 308-316. Martin, W.R., Pharmacology of opioids, Pharmacol. Rev., 35 (1984) 283-323. Millan, M.J., Morris, B.J. and Herz, A., Antagonist-induced opioid receptor up-regulation. I. Characterization of supersensitivity to selective mu and kappa agonists, J. Pharmacol. Exp. Ther., 247 (1988) 721-728. Morris, B.J., Millan, M.J. and Herz, A., Antagonist-induced opioid receptor up-regulation. II. Regionally specific modulation of mu, delta and kappa binding sites in rat brain revealed by quantitative autoradiography, J. Pharmacol. Exp. Ther., 247 (1988) 729-736. Pfeiffer, A., Pfeiffer, D.G., Feuerstein, G., Faden, A.I. and Kopin, I.J., An increase in opiate receptor-sites is associated with enhanced cardiovascular depressant, but not respiratory depressant action of morphine, Brain Research, 296 (1984) 305-311. Portoghese, P.S., Larson, D.L., Sayer, L.M., Fries, D.S. and Takemori, A.E., A novel opioid receptor site directed alkylating agent with irreversible narcotic antagonistic and reversible agonistic activities, J. Med. Chem. 23 (1980) 233-234. Pouios, C.X., Knoke, D.M., L6, A.D. and Cappell, H., Naloxone-induced analgesia and morphine supersensitivity effects are contingent upon prior exposure to analgesic testing,
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