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The κ-opioid receptor agonist U50,488H induces acute physical dependence in guinea-pigs
European Journal of Pharmacology, 241 (1993) 149-156
149
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53254
The K-opioid receptor agonist U50,488H induces acute physical dependence in guinea-pigs P a u l J. B r e n t , Loris A. Chahl, P.A. C a n t a r e l l a a n d C. K a v a n a g h Neuropharmacology Laboratory, Faculty of Medicine, University of Newcastle, Newcastle, NSW 2308, Australia
Received 18 February 1993, revised MS received 16 June 1993,accepted 22 June 1993
The present study was undertaken to determine whether acute physical dependence occurred in guinea-pigs in vivo and guinea-pig isolated ileum following a single dose of the K-opioid receptor agonist U50,488H. Administration of naloxone hydrochloride, 15 and 30 mg/kg s.c., to guinea-pigs treated 1 h before with U50,488H, 10 mg/kg s.c., induced increased locomotor activity accompanied by behavioural responses which differed from those previously found in this species with morphine withdrawal. Nor-binaltorphimine, 10 mg/kg s.c., given 1 h after administration of U50,488H, 10 mg/kg s.c., produced a small but significant increase in locomotor activity but no other withdrawal behaviours. The morphine withdrawal response was not significantly affected by U50,488H, 1 or 10 mg/kg s.c. On the guinea-pig isolated ileum, nor-binaltorphimine, 1 ~M, produced a withdrawal contracture following 2 min contact of the ileum with U50,488H 1 /~M. U50,488H, 1 /~M, abolished the [MetS]enkephalin withdrawal response of the ileum. It is concluded that dependence occurs following activation of K-opioid receptors, which is largely non-morphine-like in the central nervous system, but which is morphine-like in the enteric nervous system. U50,488H; Nor-binaltorphimine; Naloxone; Opioid withdrawal; Locomotor activity; (Guinea-pig)
I. Introduction
It is well established that opioid receptors can be classified into three major types, /~, ~ and K. The discovery of opioid receptor types has raised the possibility of developing opioid analgesic drugs devoid of the undesirable properties of abuse potential and respiratory depression of opioids in current use. K-Opioid receptor agonists are of particular interest since they provide acceptable analgesia, with less effect on respiration and gastrointestinal transit. Although K-opioid receptor agonists of the benzomorphan class elicit unpleasant dysphoric states in humans (Pfeiffer et al., 1986), non-benzomorphan K-opioid receptor agonists, the prototype of which is U50,488H (Von Voigtlander et al., 1983; James and Goldstein, 1984; Lahti et al., 1985), are more promising. Studies on the dependence liability of U50,488H have shown marked species variation. Little evidence of dependence was observed with U50,488H administered in multiple doses either systematically or cen-
Correspondence to: P.J. Brent, Faculty of Medicine, University of Newcastle, Newcastle, NSW 2308, Australia.
trally to rats or mice (Cowan et al., 1988; Cowan and Murray, 1989). However, rhesus monkeys could be made dependent on U50,488H following chronic administration, although the dependence was milder and different in character from that induced by morphine (see Gmerek et al., 1987 for references). Earlier investigations of K-opioid receptor mechanisms were hindered by the lack of a selective antagonist for the K-opioid receptor. However, the availability of the selective K-opioid receptor antagonist nor-binaltorphimine (Portoghese et al., 1987; Takemori et al., 1988), has facilitated these studies. A number of previous reports showing that withdrawal occurred following a single dose of morphine in guinea-pigs in vivo (Chahl and Thornton, 1987; Brent et al., 1987; Brent and Chahl, 1989; Johnston and Chahl, 1991) and in the guinea-pig ileum in vitro (Chahl, 1986, 1990) offered simple models for the study of the pharmacology of the morphine withdrawal response. However, no investigations have so far been undertaken using these models to study dependence on U50,488H. Guinea-pigs are especially suitable for the study of K-opioid receptor agonists, since the relative density of K-opioid receptors in the central nervous system of this species resembles that of primates (Mansour et al., 1988), and high-affinity binding sites
150 for K-opioid receptors have been demonstrated in the guinea-pig ileum (Dissanayake et al., 1990). The present study examined firstly, whether behavioural evidence of withdrawal could be induced by administration of naloxone or nor-binaltorphimine following a single dose of U50,488H in guinea-pigs. Secondly, the effect of U50,488H on the locomotor response to morphine withdrawal induced by naloxone was investigated. Finally, the interaction between U50,488H and nor-binaltorphimine was investigated on the guinea-pig isolated ileum.
2. Materials and methods
2.1. Drug treatment of guinea-pigs In all experiments, adult coloured guinea-pigs of either sex, weighing 300-600 g were used. Before and after the experiments they were housed in a room maintained on a 12 h light-dark cycle at 20°C and were allowed water and food ad libitum. In the first series of experiments, separate groups of guinea-pigs were placed in activity cages and locomotor activity was measured after subcutaneous (s.c.) injection of U50,488H (10 m g / k g ) followed 1 h later by either naloxone hydrochloride (15 or 30 m g / k g s.c., n = 6), nor-binaltorphimine (10 m g / k g s.c., n = 4) or nor-binaltorphimine (50 nmol into each lateral ventricle, total dose 100 nmol, n = 5). Control animals (n = 5) received saline instead of U50,488H. Locomotor activity was recorded in another group of guinea-pigs after intracerebroventricular (i.c.v.) injection of U50,488H (50 nmol into each ventricle, total dose 100nmol, n = 4) followed 0.5 h later by nor-binaltorphimine (50 nmol into each ventricle, total dose 100 nmol). Control animals received saline i.c.v. (n = 4) followed by nor-binaltorphimine i.c.v. In the second series of experiments, locomotor activity was recorded for 1 h before and 2 h after injection of morphine sulphate (15 m g / k g s.c.), and for 1 h after injection of naloxone hydrochloride (15 m g / k g s.c.). U50,488H (1 and 10 m g / k g s.c.) was injected 0.5 h before naloxone in five animals respectively. Control animals (n = 5) received saline instead of U50,488H. The above doses of morphine, naloxone and U50,488H were chosen since they have been shown to produce consistent behavioural effects in many previous studies in guinea-pigs (Chahl and Thornton, 1987; Brent et al., 1987; Brent and Chahl, 1989; Johnston and Chahl, 1991; Brent and Chahl, 1991; Brent and Bot, 1992).
2.2. l.c.v, injection of drugs I.c.v. injections were carried out as described previously (Brent et al., 1988; Johnston and Chahl, 1991).
Briefly, guinea-pigs were anaesthetized with ketamine hydrochloride (Ketalar, 40 m g / k g s.c.) and xylazine (Rompun, 4 m g / k g s.c.) and stainless steel guide cannulas (i.d. 0.3 mm, o.d. 0.5 mm, 6 mm in length) were inserted bilaterally 2 mm caudal and 2.5 mm lateral to bregma and to a depth of 1 mm and glued to the skull. The animals were then allowed to recover consciousness. After surgery the guinea-pigs were housed in individual cages for 3 days to recover. For i.c.v, injections guinea-pigs were restrained by gently wrapping them in a cotton towel with their heads exposed. On the day of experiment (at least 3 days after surgery), injections of opioids and saline (controls) were made with a 25 /xl Hamilton syringe with plastic tubing attached. The needle was sleeved to restrict the depth of injection to within the lateral ventricle.
2.3. Measurement of locomotor activity and behavioural responses Experiments were performed between 9:00 a.m. and 6:00 p.m. and equal numbers of control and treated guinea-pigs were used in the mornings and afternoon of each day to avoid diurnal bias. Guinea-pigs were habituated to the cages for 1 h before commencement of the 1 h control period. On the day of experiment (at least 3 days after insertion of guide cannulas in animals which were to receive i.c.v, injections of drugs), locomotor activity was measured as described previously (e.g. Brent et al., 1987, 1988) in two cages each fitted with an infra-red photocell and detector, so that every crossing of the beam 1.5 s apart was recorded simultaneously on a digital counter and as a single pulse on a chart recorder. This time delay was chosen so as to avoid measurement of small body movements. Test cages contained shavings on the floor similar to home cages. Activity scores were obtained by adding the total number of counts over successive 10 min periods. In addition, the presence or absence of observable behaviours was evaluated and recorded by a trained observer using a behavioural checklist (Brent, 1991a, b), which included behaviours such as response to handling (for evidence of limb rigidity, flaccidity or catalepsy), sedation, flattened body posture, rearing, circling, grooming, sniffing, chewing, licking, digging, h e a d / b o d y shaking and ataxia. Behavioural observations were made every 1 min for a 5 s period throughout the experiments.
2.4. Guinea-pig ileum experiments Guinea-pigs were killed by a blow to the head and 2 cm segments of distal ileum were suspended in organ baths under 1 g tension in oxygenated Tyrode solution at 37°C. Responses were recorded isometrically using a force transducer and Grass polygraph. At the com-
151
mencement of each experiment the sensitivity of the recording was adjusted so that the response to a high concentration of acetylcholine (5 ~M) giving a maximal response (100%) was the full width of the chart recorder, and all responses were expressed as % of the acetylcholine maximum taken as 100%. Repeated submaximal responses to acetylcholine (0.1 /xM) were obtained at the commencement of each experiment until the responses were reproducible. Responses to U50,488H, 1 izM, followed 2 min later by naloxone or nor-binaltorphimine, 1 or 5 IzM, and the effect of U50,488H, 1 tzM, on responses to [MetS]enkephalin, 1 /xM, followed 2 min later by washout or naloxone, 1 #M, were tested. The effect of tetrodotoxin, 1/.,M, on the response to nor-binaltorphimine, 5 /xM, was also investigated. The numbers of experiments are shown in Results.
2.5. Statistical analysis Analysis of variance for repeated measures was used to test the significance of each treatment on locomotor activity. Student's t-tests were used to compare the means for other behavioural responses as well as for heights of ileum contractions.
2. 6. Drugs and solutions The following drugs were used: acetylcholine chloride (Sigma Chemical Company, USA); ketamine hydrochloride (Ketalar, 100 mg/ml, Parke Davis, Australia); morphine sulphate (30 mg/ml, David Bull Laboratories, Australia); naloxone hydrochloride (Sigma); nor-binaltorphimine (17,17'-bis(cyclopropylmethyl)6,6 ',7,7 '-tetrahydro-4,5,4',5 '-diepoxy-6,6'-(imino)[7,7 'bimorphinan]-3,3',14,14'-tetrol dihydrochloride) (Research Biochemicals Inc., USA); tetrodotoxin (Sigma); U50,488H (trans-(_+)-3,4-dichloro-N-methyl-N(2-(1pyrrolidinyl)cyclohexyl)-benzeneacetamide methanesulphonate) (Upjohn, USA); xylazine (Rompun, 20 mg/ml, Bayer, Germany). Naloxone, U50,488H and nor-binaltorphimine were dissolved in 0.9% saline. The composition of the Tyrode solution used for the ileum experiments was (mM): NaC1 136.9; KCI 2.7; MgCI 2 1.05; CaCI 2 1.8; NaH2PO 4 0.42; NaHCO 3 11.9; and glucose 5.5.
3. Results
3.1. Effects of naloxone and nor-binaltorphimine on behauiour of guinea-pigs treated with U50,488H As previously reported (Brent and Bot, 1992; Brent, 1993), U50,488H (10 mg/kg s.c.) produced abnormal dystonic-like movements with hindlimbs hyperextended
40-
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0 30 t i m e (min)
60 ~ Saline
90
120 150 f Naloxone (N)
180
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Fig. 1. Locomotor responses of guinea-pigs treated with saline (N = 5) or U50,488H, 10 m g / k g s.c., and 1 h later with naloxone hydrochloride, 15 or 30 m g / k g s.c. (N = 5 and 6 respectively). Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means, and bars, S.E. U50,488H significantly increased the locomotor responses to naloxone, 30 m g / k g s.c. (analysis of variance for repeated measures, P = 0.027).
caudally while the animal continued to pull itself forward with the functional forelimbs. More severely affected animals were immobilised, in a twisted, flattened posture with both hindlimbs and forelimbs extended tonically forward, and had copious eye mucous and pronounced head and neck torticollis. The U50,488H-induced dystonic-like movements were severe by 12 rain after injection, and remained severe until 1 h after injection of U50,488H (responses not quantified in this study). Administration of naloxone hydrochloride (15 and 30 mg/kg s.c.) to animals treated 1 h before with U50,488H, reversed the abnormal dystonic-like posture and induced withdrawal behaviours lasting approximately 0.5 h, characterized by significantly increased locomotor activity (P = 0.005 and P = 0.027 for 15 and 30 mg/kg doses of naloxone hydrochloride respectively compared with controls given saline followed by naloxone hydrochloride 30 mg/kg, analysis of variance for repeated measures) (fig. 1), and other behaviours including circling, rearing, shivering, teeth chattering and vocalising (see table I for levels of significance). Following the withdrawal phase, animals backed into a corner of the cage and adopted a frozen, hunched posture and exhibited exopthalmos. Guinea-pigs were extremely reactive to any movement or noise near the cage after adoption of this 'defence-like' posture. The withdrawal induced by naloxone in U50,488H-treated animals was apparently dose-related (fig. 1), although the response induced by the 30 mg/kg dose of naloxone was not significantly greater than that produced by the 15 mg/kg dose. Furthermore, the withdrawal behaviour produced by
152 TABLE 1
20
Effects of naloxone (15 and 30 m g / k g s.c.) on withdrawal behaviours in guinea-pigs treated 1 h before with U50,488H (10 m g / k g s.c.). Data are m e a n s + S . E , of five animals in each group for each behaviour over a 1 h time period after injection of naloxone. Behaviour
Saline ~
0 u
10
Dose of naloxone
Circling a,b Rearing ~'~ Digging ~ Chewing ~ Shivering b Teeth chatter b Vocalising b Frozen posture b Grooming ~ H e a d / b o d y shaking ~
15 m g / k g
30 m g / k g
10.4 +_4,0 ~ 9.4+_3.8 c 4.8 +_ 1,7 ~ 7.4 +_4.4 5.0 +- 1.4 d 0.0 _+0.0 6.2 +_2.0 c Present Absent Absent
12.2 + 3.5 d 8.6+_2.5 ~ 1,0 +_0.13 1.0 +- 0.13 7.0 +_2,6 c 2.4 +_0.5 e 4.0 +_0.5 c Present Absent Absent
¢)
4-I o o u
0 0
l
1 6 b
l
' 90
'
120
150
180
small but significant increase in locomotor activity (P = 0.021) (fig. 2) but no other withdrawal behaviours. Nor-binaltorphimine (10(3 nmol total dose i.c.v.) administered 0.5 h after U50,488H (10 mg/kg s.c.) reversed the dystonic-like movements produced by U50,488H. However, withdrawal responses were too variable to produce significance, since increased locomotor activity (fig. 3) and other behaviours, such as circling and head/body shaking were produced in only two of the five animals tested (data not shown). Nor-bi-
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Saline
"~ 5 0 40~" 3 0 -
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t i m e (min)
'
'60
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Saline
g
Norbinaltor phiraine
O
o
' 30
'~ 't Saline Norbinaltorphimine or U50,488H Fig. 3. Locomotor responses of guinea-pigs treated with saline (N = 4) or U50,488H, 10 m g / k g s.c. (N = 5), and 1 h later with norbinaltorphimiue, 100 nmol i.c.v. Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means+ and bars S.E.
20
oU
'
t i m e (min)
U50,488H was non-morphine-like, since behaviours characteristic of morphine withdrawal in guinea-pigs, such as digging, chewing, grooming and head-body shaking (Brent et al., 1987; Chahl and Thornton, 1987; Johnston and Chahl, 1991), were infrequent or absent (table 1), whereas behaviours such as vocalising, shivering, teeth chatter and circling, which are not characteristic of the morphine withdrawal response in guineapigs, predominated. Nor-binaltorphimine (10 mg/kg s.c.), given 1 h after administration of U50,488H, 10 mg/kg s.c., produced a
4J O
,i
o
Behaviours representative of morphine withdrawal, b Behaviours representative of U50,488H withdrawal, c p < 0.05. d p < 0.01. ~ P < 0.001 Student's t-test. All behaviours were absent in control animals (saline-naloxone).
4-1 U
U50,488H
' 9 0 "~'
~' U50,488H
i+j[io
'120
i~0
180
'~ Saline or Norbinaltorphimine
Fig. 2. Locomotor responses of guinea-pigs treated with U50,488H, 10 m g / k g s.c., and 1 h later with saline or norbinaltorphimine, 10 m g / k g s.c. (N = 5 and 4 respectively). Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means, and bars, S.E. Locomotor responses were significantly increased to nor-binaltorphimine (analysis of variance for repeated measures, P = 0.021)•
~ U S O , 4 8 8 H 1 mg/kg .U50,d88H
10 mg/kg
4-'
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time (min) '
.~.~,~.-.
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Morphine
~.~.,~.'-,
120
150
180
.....
210
240
~
Saline Naloxone or USO,4BBH
Fig. 4. Effect of U50,488H, l or 10 mg/kg s.c. (both N = 5), on the morphine locomotor withdrawal response of guinea-pigs treated with morphine sulphate, 15 m g / k g s.c., and 2 h later with naloxone hydrochloride, 15 m g / k g s.c. Saline or U50,488H was injected 0.5 h before naloxone. Locomotor activity was expressed as n u m b e r of counts over successive 10 rain intervals. Histograms represent means, and bars S.E.
153
naltorphimine (100 nmol i.c.v.) produced no response in animals treated with saline (fig. 3). U50,488H (100 nmol total dose i.c.v.) significantly increased locomotor activity (P=0.0004; data not shown) and other behaviours such as head/body shaking, circling, rearing, chewing and head-down sniffing, compared to saline i.c.v., similar to a previous recent report (Bot et al., 1992). Injection of nor-binaltorphimine (100 nmol total dose i.c.v.) 0.5 h after treatment of guinea-pigs with U50,488H (100 nmol i.c.v.) produced a small, transient increase in locomotor activity (P = 0.031; data not shown), followed by piloerection and inactivity. This response cannot be interpreted as a withdrawal response since the excitatory response to U50,488H might have affected the response to subsequent i.c.v, injections.
3.2. Effect of U50,488H on the morphine withdrawal response in guinea-pigs As shown in previous experiments (Brent et al., 1987; Chahl and Thornton, 1987; Johnston and Chahl, 1991), guinea-pigs treated with morphine sulphate (15 mg/kg s.c.) followed 2 h later by naloxone hydrochloride (15 mg/kg s.c.) exhibited withdrawal behaviours characterized by increased locomotor activity (fig. 4) and other behaviours such as rearing, digging and head/body shaking typical of the morphine withdrawal response (see above) (data not shown). The morphine withdrawal response was not significantly affected by a dose of either 1 or 10 mg/kg s.c. of U50,488H (fig. 4). However some of the U50,488H withdrawal behaviours induced by naloxone in the absence of morphine (e.g.
^
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Fig. 5. Withdrawal responses of guinea-pig isolated ileum to U50,488H (U50), 1 /.LM, induced by nor-binaltorphimine (nBNI), 1 /zM. (A and B) Each show responses of two preparations from different animals to norbinaltorphimine alone and following 2 rain contact of the ileum with U50,488H. Responses to acetylcholine (ACH) are also shown. (C) Shows two withdrawal responses to U50,488H induced by nor-binaltorphimine on the same preparation. Note that the response is not reproducible.
154
vocalization) were reduced or absent following morphine treatment (see table 1). This experiment also showed that morphine given 1.5 h before did not affect the dystonic-like movements produced by U50,488H (10 mg/kg s.c.), whereas naloxone given 0.5 h after U50,488H, reversed the dystonic posture produced by U50,488H.
3.3. Effect of nor-binaltorphimine on the response of guinea-pig isolated ileum to U50,488H U50,488H 1 /zM produced relaxation of all 18 preparations tested (fig. 5), but since the preparations had low tone this was not quantified. Addition of naloxone, 1 or 5 /zM after addition of U50,488H, 1 /zM, did not produce contractions of the ileum (5 preparations), but addition of nor-binaltorphimine, 1 and 5 ~M, 2 min after addition of U50,488H did produce contractions (fig. 5). Interpretation of these results was complicated by the fact that nor-binaltorphimine also produced contractions on untreated preparations. On five preparations nor-binaltorphimine 5 / z M alone produced a maximal contracture of the preparations. However, a concentration of nor-binaltorphimine, 1 /xM, produced a mean contracture (±S.E.) on eight preparations of 16 + 5% of acetylcholine maximum, whereas following U50,488H 1 /zM the mean response on 10 preparations was 36 + 6% which was significantly greater than the response produced by nor-binaltorphimine alone (P = 0.025, Student's t-test). Thus nor-binaltorphimine induced a withdrawal contracture following addition of U50,488H. Although the contracture produced by nor-binaltorphimine waned after 5-10 min the blocking effect was prolonged since the relaxation response of the ileum to U50,488H did not return up to 2 h later despite washing (see fig. 5C for response after 1 h). Thus only one response could be obtained on each preparation. The contracture produced by nor-binaltorphimine (5 /xM) was completely abolished by tetrodotoxin 1/~M (n = 5). As previously shown (Chahl, 1983) a withdrawal response of the ileum could be induced by washout or addition of naloxone following 2 min contact with [MetS]enkephalin. U50,488H, 1/zM, added 30 s before washout or addition of naloxone, 1 /xM, abolished the withdrawal responses of ileum following 2 min contact with [MetS]enkephalin, 1 ~M ([MetS]enkephalin washout withdrawal, 27 +_4% n = 5; [MetS]enkephalin naloxone withdrawal, 56 + 7% n = 8; both reduced to 0 _+0; P = 0.0002 and P < 0.0001 respectively). 4. Discussion
The present study has shown that in guinea-pigs a single systemic dose of U50,488H produced acute phys-
ical dependence which was revealed as a nonmorphine-like withdrawal response on injection of naloxone. This is the first demonstration of marked acute physical dependence to U50,488H in a nonprimate species. This result contrasts with the previous work reported for rats in which physical dependence resulting from prolonged systemic (Von Voigtlander et al., 1983) or central (Cowan et al., 1988) administration of U50,488H produced very mild withdrawal. Thus it would appear that the guinea-pig is similar to the monkey in exhibiting dependence to K-opioids (Gmerek and Woods, 1986; Gmerek et al., 1987), possibly reflecting the greater proportion of K-opioid receptors in guinea-pig and primate brain compared with that in rat brain (Mansour et al., 1988). The results from the present study were also in agreement with previous findings in monkeys (Gmerek et al., 1987) in that the withdrawal response to U50,488H in guinea-pigs, although opioid in nature since it was precipitated by naloxone, was non-morphine-like, the animals exhibiting behaviours which were qualitatively different from morphine withdrawal behaviours observed in this and previous studies (Chahl and Thornton, 1987; Brent et al., 1987; Johnston and Chahl, 1991). As reported in previous studies (Brent and Bot, 1992; Brent, 1993), systemic administration of U50,488H induced dystonic-like movements in guineapigs. Since this response to U50,488H was reversed by systematically administered naloxone and by nor-binaltorphimine administration (i.c.v.), and was unaffected by prior treatment with morphine, it was apparently mediated via K-opioid receptors. In a previous study it has also been shown that the increased locomotor activity produced by administration of U50,488H 100 nmol by the i.c.v, route in guinea-pigs was attenuated by nor-binaltorphimine given i.c.v. 20 min before U50,488H (Bot et al., 1992), indicating that this response to U50,488H was also mediated by K-opioid receptors. In the present experiments the excitatory effects of U50,488H administered i.c.v, precluded clear demonstration of withdrawal responses induced by subsequent nor-binaltorphimine administration. Following systemic administration of U50,488H, nor-binaltorphimine administered systemically produced only a mild withdrawal response compared with that produced by naloxone. The most likely explanation for this finding is that the dose of nor-binaltorphimine was inadequate. Alternatively, the kinetics of its onset of action might have been such that sufficient neurones did not withdraw synchronously to produce overt changes in behaviour following a single dose of U50,488H. This latter explanation would agree with the reported slow onset of action of nor-binaltorphimine (Endoh et al., 1990). The observation that nor-binaltorphimine i.c.v, produced mild withdrawal signs in two guinea-pigs given U50,488H s.c.
155
indicated that norbinaltorphimine administered i.c.v. was also potentially capable of inducing withdrawal following U50,488H administration. Further studies on K-opioid withdrawal using higher doses of norbinaltorphimine a n d / o r varying time intervals following administration of K-opioid receptor agonists are required. Dynorphin-(1-13) has been reported to suppress morphine withdrawal signs (Aceto et al., 1982; Green and Lee, 1988). This is not unexpected since, although dynorphin is considered to be the naturally occurring ligand for the K-opioid receptor, it is not highly selective and has ~-opioid receptor agonist activity. However, U50,488H is 1300-fold more selective for K-opioid receptors than for tz-opioid receptors, and 12000-fold more selective for K-opioid receptors than for 8-opioid receptors in guinea-pig brain (James and Goldstein, 1984). In the present study, U50,488H did not inhibit any of the behaviours associated with the morphine withdrawal response. This finding supported previous studies (Fukagawa et al., 1989), that tz- and x-opioid receptor agonists do not produce cross-dependence in the central nervous system. However some behavioural responses to U50,488H withdrawal, e.g. vocalization, were apparently attenuated by morphine. Thus in the pathways involved in these latter responses, Iz- and K-opioid receptors might be present on the same neurones, whereas in the pathways involved in behavioural responses such as locomotor activity, they might be present on different neurones. In agreement with previous studies on the guinea-pig isolated ileum (Chahl, 1990) naloxone in the doses used did not induce a withdrawal contracture on preparations pretreated with U50,488H. Experiments with nor-binaltorphimine on the guinea-pig ileum were complicated by the marked contracture produced by nor-binaltorphimine alone. However, since the response to nor-binaltorphimine 1 /zM was significantly greater following U50,488H treatment of the ileum than that obtained in the absence of U50,488H, it is concluded that nor-binaltorphimine produced a withdrawal contracture on preparations treated with U50,488H. The conclusion that U50,488H induces dependence is in agreement with the findings of Valeri et al. (1992) on rabbit jejunum. The contracture of untreated ileum produced by nor-binaltorphimine might represent withdrawal from an endogenous K-opioid receptor tone which has been reported to regulate peristalsis in the ileum (Johnson et al., 1988; Kromer, 1988, 1990; Waterman et al., 1992). It is unlikely that the contracture produced by nor-binaltorphimine was due to a non-specific effect since it was inhibited by tetrodotoxin indicating that action potentials were involved. In agreement with previous studies on the ileum (Seidl and Schulz, 1983), withdrawal from U50,488H was inhibited by [MetS]enkephalin, which acts mainly
on iz-opioid receptors in the ileum. Thus cross-dependence occurred in the ileum between /x- and K-opioid receptor agonists. It has also been shown previously that U50,488H inhibits the withdrawal response of the ileum to [MetS]enkephalin (Chahl, 1990). In conclusion, the present study has shown that dependence occurs following activation of x-opioid receptors, which is largely non-morphine-like in the central nervous system, but which is morphine-like in the enteric nervous system.
References Aceto, M.D., W.L. Dewey, J.-K. Chang and N.M. Lee, 1982, Dynorphin-(1-13): Effects in nontolerant and morphine-dependent rhesus monkeys, Eur. J. Pharmacol. 83, 139. Bot, G., L.A. Chahl, P.J. Brent and P.A. Johnston, 1992, Effects of intracerebroventricularly administered mu-, delta- and kappaopioid agonists on locomotor activity of the guinea pig and the pharmacology of the locomotor responses to U50,488H, Neuropharmacology 31, 825. Brent, P.J., 1991a, Behavioural effects of selective dopamine D-1 and D-2 agonists and antagonists in guinea-pigs, Psychopharmacology 104, 201. Brent, P.J., 1991b, Similar behavioural effects of sigma agonists and PCP-like non-competitive NMDA antagonists in guinea-pigs, Psychopharmacology 105, 421. Brent, P.J., 1993, Behavioural effect of pretreatment with opioid antagonists and sigma binding site ligands on the abnormal motor response produced by the kappa opioid agonist U50,488H in guinea-pigs, Neuropharmacology (in press). Brent, P.J. and G. Bot, 1992, Morphine, D-Pen 2, D-Pen 5 enkephalin and U50,488H differentially affect the locomotor activity and behaviours induced by quinpirole in guinea-pigs, Psychopharmacology 107, 581. Brent, P.J. and L.A. Chahl, 1989, The effects of selective dopamine D-1 and D-2 antagonists on the locomotor response and brain monoamine changes induced by naloxone-precipitated morphine withdrawal in guinea-pigs, in: Progress in Clinical and Biological Research, Vol. 328, eds. R. Quirion, K. Jhamandas and C. Gianoulakis (A.R. Liss, New York) p. 515. Brent, P.J. and L.A. Chahl, 1991, Effects of naloxone-precipitated withdrawal after a single dose of morphine on catecholamine concentrations in guinea-pig brain, Neurochem. Res. 16, 525. Brent, P.J., P.A. Johnston and L.A. Chahl, 1987, Plasma catecholamine concentrations during morphine withdrawal in conscious guinea-pigs, Clin. Exp. Pharmacol. Physiol. 14, 623. Chahl, L.A., 1983, Contracture of guinea-pig ileum on withdrawal of methionineS-enkephalin is mediated by substance P, Br. J. Pharmacol. 80 741. Chahl, L.A., 1986, Withdrawal responses of guinea-pig isolated ileum following brief exposure to opiates and opioid peptides, NaunynSchmiedeb. Arch. Pharmacol. 333, 387. Chahl, L.A., 1990, Effects of putative neurotransmitters and related drugs on withdrawal contractures of guinea-pig isolated ileum following brief contact with [MetS]enkephalin, Br. J. Pharmacol. 101,908. Chahl, L.A. and C. Thornton, 1987, Locomotor activity and contracture of isolated ileum precipitated by naloxone following treatment of guinea-pigs with a single dose of morphine, J. Pharm. Pharmacol. 39, 52. Cowan, A. and C.W. Murray, 1989, Effect of nor-binaltorphimine on the behaviour of mice and rats receiving multiple injections o|
156 U50,488H, in: Progress in Clinical and Biological Research, Vol. 328, eds. R. Quirion, K. Jhamandas and C. Gianoulakis (A.R. Liss, New York) p. 303. Cowan, A., X.Z. Zhu, H.I. Mosberg, J.R. Omnas and F. Porreca, 1988, Direct dependence studies in rats with agents selective for different types of opioid receptors, J. Pharmacol. Exp. Ther. 246, 950. Dissanayake, V.U.K., J.C. Hunter, R.G. Hill and J. Hughes, 1990, Characterization of r-opioid receptors in the guinea-pig ileum, Eur. J. Pharmacol. 182, 73. Endoh, T., H. Koike, H. Matsuura and H. Nagase, 1990, Nor-Binaltorphimine (nor-BNI); a potent and selective kappa-opioid receptor antagonist with ultra-long-lasting activity in vivo, in: New Leads in Opioid Research, eds. J.M. Van Ree, A.H. Mulder, V.M. Wiegant and T.B. Van Wimersma Greidanus (Excerpta Medica, Amsterdam) p. 82. Fukagawa, Y., J.L. Katz and T. Suzuki, 1989, Effects of a selective K-opioid agonist, U50,488H on morphine dependence in rats, Eur. J. Pharmacol. 170, 47. Gmerek, D.E. and J.H. Woods, 1986, Kappa receptor mediated opioid dependence in rhesus monkeys, Life Sci. 39, 69. Gmerek, D.E., L.A. Dykstra and J.H. Woods, 1987, Kappa opioids in rhesus monkeys. III. Dependence associated with chronic administration, J. Pharmacol. Exp. Ther. 242, 428. Green, P.G. and N.M. Lee, 1988, Dynorphin A-(l-13) attenuates withdrawal in morphine-dependent rats: effect of route of administration, Eur. J. Pharmacol. 145, 267. James, I.F. and A. Goldstein, 1984, Site-directed alkylation of multiple opioid receptors. I. Binding selectivity, Mol. Pharmacol. 25, 337. Johnson, S.M., M. Costa and C.M.S. Humphreys, 1988, Opioid mu and kappa receptors on axons of cholinergic excitatory motor neurons supplying the circular muscle of guinea-pig ileum, Naunyn-Schmiedeb. Arch. Pharmacol. 338, 397. Johnston, P.A. and L.A. Chahl, 1991, Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs, NaunynSchmiedeb. Arch. Pharmacol. 343, 283.
Kromer, W., 1988, Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion, Pharmacol. Rev. 40, 121. Kromer, W., 1990, Reflex peristalsis in the guinea-pig isolated ileum is endogenously controlled by kappa opioid receptors, NaunynSchmiedeb. Arch. Pharmacol. 341, 450. Lahti, R.A., M.M. Mickelson, J.M. McCall and P.F. Von Voigtlander, 1985, [3H]U-69,593, a highly selective ligand for the opioid x receptor, Eur. J. Pharmacol. 109, 281. Mansour, A., H. Khachaturian, M.E. Lewis, H. Akil and S.J. Watson, 1988, Anatomy of CNS opioid receptors, Trends Neurosci. 11, 308. Pfeiffer, A., V. Brantl, A. Herz and H.M. Emrich, 1986, Psychotomimesis mediated by K-opiate receptors, Science 233, 774. Portoghese, P.S., A.W. Lipkowski and A.E. Takemori, 1987, Binaltorphimine and norbinaltorphimine, potent and selective K-opioid receptor antagonists, Life Sci. 40, 1287. Seidl, E. and R. Schulz, 1983, Selective opiate tolerance in the guinea-pig ileum is not associated with selective dependence, Life Sci. 33, 357. Takemori, A.E., Y.H. Begonia, J.S. Naeseth and P.S. Portoghese, 1988, Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays, J. Pharmacol. Exp. Ther. 224, 7. Valeri, P., L.A. Morrone and L. Romanelli, 1992, Manifestations of acute opiate withdrawal contracture in rabbit jejunum after mu-, kappa- and delta-receptor agonist exposure, Br. J. Pharmacol. 106, 39. Von Voigtlander, P.F., R.A. Lahti and J.H. Ludens, 1983, U50,488H: A selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther. 225, 7. Waterman, S.A., M. Costa and M. Tonini, 1992, Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids, Br. J. Pharmacol. 106, 1004.
149
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53254
The K-opioid receptor agonist U50,488H induces acute physical dependence in guinea-pigs P a u l J. B r e n t , Loris A. Chahl, P.A. C a n t a r e l l a a n d C. K a v a n a g h Neuropharmacology Laboratory, Faculty of Medicine, University of Newcastle, Newcastle, NSW 2308, Australia
Received 18 February 1993, revised MS received 16 June 1993,accepted 22 June 1993
The present study was undertaken to determine whether acute physical dependence occurred in guinea-pigs in vivo and guinea-pig isolated ileum following a single dose of the K-opioid receptor agonist U50,488H. Administration of naloxone hydrochloride, 15 and 30 mg/kg s.c., to guinea-pigs treated 1 h before with U50,488H, 10 mg/kg s.c., induced increased locomotor activity accompanied by behavioural responses which differed from those previously found in this species with morphine withdrawal. Nor-binaltorphimine, 10 mg/kg s.c., given 1 h after administration of U50,488H, 10 mg/kg s.c., produced a small but significant increase in locomotor activity but no other withdrawal behaviours. The morphine withdrawal response was not significantly affected by U50,488H, 1 or 10 mg/kg s.c. On the guinea-pig isolated ileum, nor-binaltorphimine, 1 ~M, produced a withdrawal contracture following 2 min contact of the ileum with U50,488H 1 /~M. U50,488H, 1 /~M, abolished the [MetS]enkephalin withdrawal response of the ileum. It is concluded that dependence occurs following activation of K-opioid receptors, which is largely non-morphine-like in the central nervous system, but which is morphine-like in the enteric nervous system. U50,488H; Nor-binaltorphimine; Naloxone; Opioid withdrawal; Locomotor activity; (Guinea-pig)
I. Introduction
It is well established that opioid receptors can be classified into three major types, /~, ~ and K. The discovery of opioid receptor types has raised the possibility of developing opioid analgesic drugs devoid of the undesirable properties of abuse potential and respiratory depression of opioids in current use. K-Opioid receptor agonists are of particular interest since they provide acceptable analgesia, with less effect on respiration and gastrointestinal transit. Although K-opioid receptor agonists of the benzomorphan class elicit unpleasant dysphoric states in humans (Pfeiffer et al., 1986), non-benzomorphan K-opioid receptor agonists, the prototype of which is U50,488H (Von Voigtlander et al., 1983; James and Goldstein, 1984; Lahti et al., 1985), are more promising. Studies on the dependence liability of U50,488H have shown marked species variation. Little evidence of dependence was observed with U50,488H administered in multiple doses either systematically or cen-
Correspondence to: P.J. Brent, Faculty of Medicine, University of Newcastle, Newcastle, NSW 2308, Australia.
trally to rats or mice (Cowan et al., 1988; Cowan and Murray, 1989). However, rhesus monkeys could be made dependent on U50,488H following chronic administration, although the dependence was milder and different in character from that induced by morphine (see Gmerek et al., 1987 for references). Earlier investigations of K-opioid receptor mechanisms were hindered by the lack of a selective antagonist for the K-opioid receptor. However, the availability of the selective K-opioid receptor antagonist nor-binaltorphimine (Portoghese et al., 1987; Takemori et al., 1988), has facilitated these studies. A number of previous reports showing that withdrawal occurred following a single dose of morphine in guinea-pigs in vivo (Chahl and Thornton, 1987; Brent et al., 1987; Brent and Chahl, 1989; Johnston and Chahl, 1991) and in the guinea-pig ileum in vitro (Chahl, 1986, 1990) offered simple models for the study of the pharmacology of the morphine withdrawal response. However, no investigations have so far been undertaken using these models to study dependence on U50,488H. Guinea-pigs are especially suitable for the study of K-opioid receptor agonists, since the relative density of K-opioid receptors in the central nervous system of this species resembles that of primates (Mansour et al., 1988), and high-affinity binding sites
150 for K-opioid receptors have been demonstrated in the guinea-pig ileum (Dissanayake et al., 1990). The present study examined firstly, whether behavioural evidence of withdrawal could be induced by administration of naloxone or nor-binaltorphimine following a single dose of U50,488H in guinea-pigs. Secondly, the effect of U50,488H on the locomotor response to morphine withdrawal induced by naloxone was investigated. Finally, the interaction between U50,488H and nor-binaltorphimine was investigated on the guinea-pig isolated ileum.
2. Materials and methods
2.1. Drug treatment of guinea-pigs In all experiments, adult coloured guinea-pigs of either sex, weighing 300-600 g were used. Before and after the experiments they were housed in a room maintained on a 12 h light-dark cycle at 20°C and were allowed water and food ad libitum. In the first series of experiments, separate groups of guinea-pigs were placed in activity cages and locomotor activity was measured after subcutaneous (s.c.) injection of U50,488H (10 m g / k g ) followed 1 h later by either naloxone hydrochloride (15 or 30 m g / k g s.c., n = 6), nor-binaltorphimine (10 m g / k g s.c., n = 4) or nor-binaltorphimine (50 nmol into each lateral ventricle, total dose 100 nmol, n = 5). Control animals (n = 5) received saline instead of U50,488H. Locomotor activity was recorded in another group of guinea-pigs after intracerebroventricular (i.c.v.) injection of U50,488H (50 nmol into each ventricle, total dose 100nmol, n = 4) followed 0.5 h later by nor-binaltorphimine (50 nmol into each ventricle, total dose 100 nmol). Control animals received saline i.c.v. (n = 4) followed by nor-binaltorphimine i.c.v. In the second series of experiments, locomotor activity was recorded for 1 h before and 2 h after injection of morphine sulphate (15 m g / k g s.c.), and for 1 h after injection of naloxone hydrochloride (15 m g / k g s.c.). U50,488H (1 and 10 m g / k g s.c.) was injected 0.5 h before naloxone in five animals respectively. Control animals (n = 5) received saline instead of U50,488H. The above doses of morphine, naloxone and U50,488H were chosen since they have been shown to produce consistent behavioural effects in many previous studies in guinea-pigs (Chahl and Thornton, 1987; Brent et al., 1987; Brent and Chahl, 1989; Johnston and Chahl, 1991; Brent and Chahl, 1991; Brent and Bot, 1992).
2.2. l.c.v, injection of drugs I.c.v. injections were carried out as described previously (Brent et al., 1988; Johnston and Chahl, 1991).
Briefly, guinea-pigs were anaesthetized with ketamine hydrochloride (Ketalar, 40 m g / k g s.c.) and xylazine (Rompun, 4 m g / k g s.c.) and stainless steel guide cannulas (i.d. 0.3 mm, o.d. 0.5 mm, 6 mm in length) were inserted bilaterally 2 mm caudal and 2.5 mm lateral to bregma and to a depth of 1 mm and glued to the skull. The animals were then allowed to recover consciousness. After surgery the guinea-pigs were housed in individual cages for 3 days to recover. For i.c.v, injections guinea-pigs were restrained by gently wrapping them in a cotton towel with their heads exposed. On the day of experiment (at least 3 days after surgery), injections of opioids and saline (controls) were made with a 25 /xl Hamilton syringe with plastic tubing attached. The needle was sleeved to restrict the depth of injection to within the lateral ventricle.
2.3. Measurement of locomotor activity and behavioural responses Experiments were performed between 9:00 a.m. and 6:00 p.m. and equal numbers of control and treated guinea-pigs were used in the mornings and afternoon of each day to avoid diurnal bias. Guinea-pigs were habituated to the cages for 1 h before commencement of the 1 h control period. On the day of experiment (at least 3 days after insertion of guide cannulas in animals which were to receive i.c.v, injections of drugs), locomotor activity was measured as described previously (e.g. Brent et al., 1987, 1988) in two cages each fitted with an infra-red photocell and detector, so that every crossing of the beam 1.5 s apart was recorded simultaneously on a digital counter and as a single pulse on a chart recorder. This time delay was chosen so as to avoid measurement of small body movements. Test cages contained shavings on the floor similar to home cages. Activity scores were obtained by adding the total number of counts over successive 10 min periods. In addition, the presence or absence of observable behaviours was evaluated and recorded by a trained observer using a behavioural checklist (Brent, 1991a, b), which included behaviours such as response to handling (for evidence of limb rigidity, flaccidity or catalepsy), sedation, flattened body posture, rearing, circling, grooming, sniffing, chewing, licking, digging, h e a d / b o d y shaking and ataxia. Behavioural observations were made every 1 min for a 5 s period throughout the experiments.
2.4. Guinea-pig ileum experiments Guinea-pigs were killed by a blow to the head and 2 cm segments of distal ileum were suspended in organ baths under 1 g tension in oxygenated Tyrode solution at 37°C. Responses were recorded isometrically using a force transducer and Grass polygraph. At the com-
151
mencement of each experiment the sensitivity of the recording was adjusted so that the response to a high concentration of acetylcholine (5 ~M) giving a maximal response (100%) was the full width of the chart recorder, and all responses were expressed as % of the acetylcholine maximum taken as 100%. Repeated submaximal responses to acetylcholine (0.1 /xM) were obtained at the commencement of each experiment until the responses were reproducible. Responses to U50,488H, 1 izM, followed 2 min later by naloxone or nor-binaltorphimine, 1 or 5 IzM, and the effect of U50,488H, 1 tzM, on responses to [MetS]enkephalin, 1 /xM, followed 2 min later by washout or naloxone, 1 #M, were tested. The effect of tetrodotoxin, 1/.,M, on the response to nor-binaltorphimine, 5 /xM, was also investigated. The numbers of experiments are shown in Results.
2.5. Statistical analysis Analysis of variance for repeated measures was used to test the significance of each treatment on locomotor activity. Student's t-tests were used to compare the means for other behavioural responses as well as for heights of ileum contractions.
2. 6. Drugs and solutions The following drugs were used: acetylcholine chloride (Sigma Chemical Company, USA); ketamine hydrochloride (Ketalar, 100 mg/ml, Parke Davis, Australia); morphine sulphate (30 mg/ml, David Bull Laboratories, Australia); naloxone hydrochloride (Sigma); nor-binaltorphimine (17,17'-bis(cyclopropylmethyl)6,6 ',7,7 '-tetrahydro-4,5,4',5 '-diepoxy-6,6'-(imino)[7,7 'bimorphinan]-3,3',14,14'-tetrol dihydrochloride) (Research Biochemicals Inc., USA); tetrodotoxin (Sigma); U50,488H (trans-(_+)-3,4-dichloro-N-methyl-N(2-(1pyrrolidinyl)cyclohexyl)-benzeneacetamide methanesulphonate) (Upjohn, USA); xylazine (Rompun, 20 mg/ml, Bayer, Germany). Naloxone, U50,488H and nor-binaltorphimine were dissolved in 0.9% saline. The composition of the Tyrode solution used for the ileum experiments was (mM): NaC1 136.9; KCI 2.7; MgCI 2 1.05; CaCI 2 1.8; NaH2PO 4 0.42; NaHCO 3 11.9; and glucose 5.5.
3. Results
3.1. Effects of naloxone and nor-binaltorphimine on behauiour of guinea-pigs treated with U50,488H As previously reported (Brent and Bot, 1992; Brent, 1993), U50,488H (10 mg/kg s.c.) produced abnormal dystonic-like movements with hindlimbs hyperextended
40-
Saline + N 30 mg/kg
o~ 30-
0 v
~U •
I0 mg/kg + N 15 mg/kg
O I0 mg/kg + N 30 mg/kg
200 m
0
10-
0 0
o
0 30 t i m e (min)
60 ~ Saline
90
120 150 f Naloxone (N)
180
or
USO,488H (U)
Fig. 1. Locomotor responses of guinea-pigs treated with saline (N = 5) or U50,488H, 10 m g / k g s.c., and 1 h later with naloxone hydrochloride, 15 or 30 m g / k g s.c. (N = 5 and 6 respectively). Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means, and bars, S.E. U50,488H significantly increased the locomotor responses to naloxone, 30 m g / k g s.c. (analysis of variance for repeated measures, P = 0.027).
caudally while the animal continued to pull itself forward with the functional forelimbs. More severely affected animals were immobilised, in a twisted, flattened posture with both hindlimbs and forelimbs extended tonically forward, and had copious eye mucous and pronounced head and neck torticollis. The U50,488H-induced dystonic-like movements were severe by 12 rain after injection, and remained severe until 1 h after injection of U50,488H (responses not quantified in this study). Administration of naloxone hydrochloride (15 and 30 mg/kg s.c.) to animals treated 1 h before with U50,488H, reversed the abnormal dystonic-like posture and induced withdrawal behaviours lasting approximately 0.5 h, characterized by significantly increased locomotor activity (P = 0.005 and P = 0.027 for 15 and 30 mg/kg doses of naloxone hydrochloride respectively compared with controls given saline followed by naloxone hydrochloride 30 mg/kg, analysis of variance for repeated measures) (fig. 1), and other behaviours including circling, rearing, shivering, teeth chattering and vocalising (see table I for levels of significance). Following the withdrawal phase, animals backed into a corner of the cage and adopted a frozen, hunched posture and exhibited exopthalmos. Guinea-pigs were extremely reactive to any movement or noise near the cage after adoption of this 'defence-like' posture. The withdrawal induced by naloxone in U50,488H-treated animals was apparently dose-related (fig. 1), although the response induced by the 30 mg/kg dose of naloxone was not significantly greater than that produced by the 15 mg/kg dose. Furthermore, the withdrawal behaviour produced by
152 TABLE 1
20
Effects of naloxone (15 and 30 m g / k g s.c.) on withdrawal behaviours in guinea-pigs treated 1 h before with U50,488H (10 m g / k g s.c.). Data are m e a n s + S . E , of five animals in each group for each behaviour over a 1 h time period after injection of naloxone. Behaviour
Saline ~
0 u
10
Dose of naloxone
Circling a,b Rearing ~'~ Digging ~ Chewing ~ Shivering b Teeth chatter b Vocalising b Frozen posture b Grooming ~ H e a d / b o d y shaking ~
15 m g / k g
30 m g / k g
10.4 +_4,0 ~ 9.4+_3.8 c 4.8 +_ 1,7 ~ 7.4 +_4.4 5.0 +- 1.4 d 0.0 _+0.0 6.2 +_2.0 c Present Absent Absent
12.2 + 3.5 d 8.6+_2.5 ~ 1,0 +_0.13 1.0 +- 0.13 7.0 +_2,6 c 2.4 +_0.5 e 4.0 +_0.5 c Present Absent Absent
¢)
4-I o o u
0 0
l
1 6 b
l
' 90
'
120
150
180
small but significant increase in locomotor activity (P = 0.021) (fig. 2) but no other withdrawal behaviours. Nor-binaltorphimine (10(3 nmol total dose i.c.v.) administered 0.5 h after U50,488H (10 mg/kg s.c.) reversed the dystonic-like movements produced by U50,488H. However, withdrawal responses were too variable to produce significance, since increased locomotor activity (fig. 3) and other behaviours, such as circling and head/body shaking were produced in only two of the five animals tested (data not shown). Nor-bi-
6O
-ff
Saline
"~ 5 0 40~" 3 0 -
10
++i++++,+,,
°o
•
'30
t i m e (min)
'
'60
'
~
Saline
g
Norbinaltor phiraine
O
o
' 30
'~ 't Saline Norbinaltorphimine or U50,488H Fig. 3. Locomotor responses of guinea-pigs treated with saline (N = 4) or U50,488H, 10 m g / k g s.c. (N = 5), and 1 h later with norbinaltorphimiue, 100 nmol i.c.v. Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means+ and bars S.E.
20
oU
'
t i m e (min)
U50,488H was non-morphine-like, since behaviours characteristic of morphine withdrawal in guinea-pigs, such as digging, chewing, grooming and head-body shaking (Brent et al., 1987; Chahl and Thornton, 1987; Johnston and Chahl, 1991), were infrequent or absent (table 1), whereas behaviours such as vocalising, shivering, teeth chatter and circling, which are not characteristic of the morphine withdrawal response in guineapigs, predominated. Nor-binaltorphimine (10 mg/kg s.c.), given 1 h after administration of U50,488H, 10 mg/kg s.c., produced a
4J O
,i
o
Behaviours representative of morphine withdrawal, b Behaviours representative of U50,488H withdrawal, c p < 0.05. d p < 0.01. ~ P < 0.001 Student's t-test. All behaviours were absent in control animals (saline-naloxone).
4-1 U
U50,488H
' 9 0 "~'
~' U50,488H
i+j[io
'120
i~0
180
'~ Saline or Norbinaltorphimine
Fig. 2. Locomotor responses of guinea-pigs treated with U50,488H, 10 m g / k g s.c., and 1 h later with saline or norbinaltorphimine, 10 m g / k g s.c. (N = 5 and 4 respectively). Locomotor activity was expressed as n u m b e r of counts over successive 10 min intervals. Histograms represent means, and bars, S.E. Locomotor responses were significantly increased to nor-binaltorphimine (analysis of variance for repeated measures, P = 0.021)•
~ U S O , 4 8 8 H 1 mg/kg .U50,d88H
10 mg/kg
4-'
o0 . . , 30 ~..J
60
time (min) '
.~.~,~.-.
90
Morphine
~.~.,~.'-,
120
150
180
.....
210
240
~
Saline Naloxone or USO,4BBH
Fig. 4. Effect of U50,488H, l or 10 mg/kg s.c. (both N = 5), on the morphine locomotor withdrawal response of guinea-pigs treated with morphine sulphate, 15 m g / k g s.c., and 2 h later with naloxone hydrochloride, 15 m g / k g s.c. Saline or U50,488H was injected 0.5 h before naloxone. Locomotor activity was expressed as n u m b e r of counts over successive 10 rain intervals. Histograms represent means, and bars S.E.
153
naltorphimine (100 nmol i.c.v.) produced no response in animals treated with saline (fig. 3). U50,488H (100 nmol total dose i.c.v.) significantly increased locomotor activity (P=0.0004; data not shown) and other behaviours such as head/body shaking, circling, rearing, chewing and head-down sniffing, compared to saline i.c.v., similar to a previous recent report (Bot et al., 1992). Injection of nor-binaltorphimine (100 nmol total dose i.c.v.) 0.5 h after treatment of guinea-pigs with U50,488H (100 nmol i.c.v.) produced a small, transient increase in locomotor activity (P = 0.031; data not shown), followed by piloerection and inactivity. This response cannot be interpreted as a withdrawal response since the excitatory response to U50,488H might have affected the response to subsequent i.c.v, injections.
3.2. Effect of U50,488H on the morphine withdrawal response in guinea-pigs As shown in previous experiments (Brent et al., 1987; Chahl and Thornton, 1987; Johnston and Chahl, 1991), guinea-pigs treated with morphine sulphate (15 mg/kg s.c.) followed 2 h later by naloxone hydrochloride (15 mg/kg s.c.) exhibited withdrawal behaviours characterized by increased locomotor activity (fig. 4) and other behaviours such as rearing, digging and head/body shaking typical of the morphine withdrawal response (see above) (data not shown). The morphine withdrawal response was not significantly affected by a dose of either 1 or 10 mg/kg s.c. of U50,488H (fig. 4). However some of the U50,488H withdrawal behaviours induced by naloxone in the absence of morphine (e.g.
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Fig. 5. Withdrawal responses of guinea-pig isolated ileum to U50,488H (U50), 1 /.LM, induced by nor-binaltorphimine (nBNI), 1 /zM. (A and B) Each show responses of two preparations from different animals to norbinaltorphimine alone and following 2 rain contact of the ileum with U50,488H. Responses to acetylcholine (ACH) are also shown. (C) Shows two withdrawal responses to U50,488H induced by nor-binaltorphimine on the same preparation. Note that the response is not reproducible.
154
vocalization) were reduced or absent following morphine treatment (see table 1). This experiment also showed that morphine given 1.5 h before did not affect the dystonic-like movements produced by U50,488H (10 mg/kg s.c.), whereas naloxone given 0.5 h after U50,488H, reversed the dystonic posture produced by U50,488H.
3.3. Effect of nor-binaltorphimine on the response of guinea-pig isolated ileum to U50,488H U50,488H 1 /zM produced relaxation of all 18 preparations tested (fig. 5), but since the preparations had low tone this was not quantified. Addition of naloxone, 1 or 5 /zM after addition of U50,488H, 1 /zM, did not produce contractions of the ileum (5 preparations), but addition of nor-binaltorphimine, 1 and 5 ~M, 2 min after addition of U50,488H did produce contractions (fig. 5). Interpretation of these results was complicated by the fact that nor-binaltorphimine also produced contractions on untreated preparations. On five preparations nor-binaltorphimine 5 / z M alone produced a maximal contracture of the preparations. However, a concentration of nor-binaltorphimine, 1 /xM, produced a mean contracture (±S.E.) on eight preparations of 16 + 5% of acetylcholine maximum, whereas following U50,488H 1 /zM the mean response on 10 preparations was 36 + 6% which was significantly greater than the response produced by nor-binaltorphimine alone (P = 0.025, Student's t-test). Thus nor-binaltorphimine induced a withdrawal contracture following addition of U50,488H. Although the contracture produced by nor-binaltorphimine waned after 5-10 min the blocking effect was prolonged since the relaxation response of the ileum to U50,488H did not return up to 2 h later despite washing (see fig. 5C for response after 1 h). Thus only one response could be obtained on each preparation. The contracture produced by nor-binaltorphimine (5 /xM) was completely abolished by tetrodotoxin 1/~M (n = 5). As previously shown (Chahl, 1983) a withdrawal response of the ileum could be induced by washout or addition of naloxone following 2 min contact with [MetS]enkephalin. U50,488H, 1/zM, added 30 s before washout or addition of naloxone, 1 /xM, abolished the withdrawal responses of ileum following 2 min contact with [MetS]enkephalin, 1 ~M ([MetS]enkephalin washout withdrawal, 27 +_4% n = 5; [MetS]enkephalin naloxone withdrawal, 56 + 7% n = 8; both reduced to 0 _+0; P = 0.0002 and P < 0.0001 respectively). 4. Discussion
The present study has shown that in guinea-pigs a single systemic dose of U50,488H produced acute phys-
ical dependence which was revealed as a nonmorphine-like withdrawal response on injection of naloxone. This is the first demonstration of marked acute physical dependence to U50,488H in a nonprimate species. This result contrasts with the previous work reported for rats in which physical dependence resulting from prolonged systemic (Von Voigtlander et al., 1983) or central (Cowan et al., 1988) administration of U50,488H produced very mild withdrawal. Thus it would appear that the guinea-pig is similar to the monkey in exhibiting dependence to K-opioids (Gmerek and Woods, 1986; Gmerek et al., 1987), possibly reflecting the greater proportion of K-opioid receptors in guinea-pig and primate brain compared with that in rat brain (Mansour et al., 1988). The results from the present study were also in agreement with previous findings in monkeys (Gmerek et al., 1987) in that the withdrawal response to U50,488H in guinea-pigs, although opioid in nature since it was precipitated by naloxone, was non-morphine-like, the animals exhibiting behaviours which were qualitatively different from morphine withdrawal behaviours observed in this and previous studies (Chahl and Thornton, 1987; Brent et al., 1987; Johnston and Chahl, 1991). As reported in previous studies (Brent and Bot, 1992; Brent, 1993), systemic administration of U50,488H induced dystonic-like movements in guineapigs. Since this response to U50,488H was reversed by systematically administered naloxone and by nor-binaltorphimine administration (i.c.v.), and was unaffected by prior treatment with morphine, it was apparently mediated via K-opioid receptors. In a previous study it has also been shown that the increased locomotor activity produced by administration of U50,488H 100 nmol by the i.c.v, route in guinea-pigs was attenuated by nor-binaltorphimine given i.c.v. 20 min before U50,488H (Bot et al., 1992), indicating that this response to U50,488H was also mediated by K-opioid receptors. In the present experiments the excitatory effects of U50,488H administered i.c.v, precluded clear demonstration of withdrawal responses induced by subsequent nor-binaltorphimine administration. Following systemic administration of U50,488H, nor-binaltorphimine administered systemically produced only a mild withdrawal response compared with that produced by naloxone. The most likely explanation for this finding is that the dose of nor-binaltorphimine was inadequate. Alternatively, the kinetics of its onset of action might have been such that sufficient neurones did not withdraw synchronously to produce overt changes in behaviour following a single dose of U50,488H. This latter explanation would agree with the reported slow onset of action of nor-binaltorphimine (Endoh et al., 1990). The observation that nor-binaltorphimine i.c.v, produced mild withdrawal signs in two guinea-pigs given U50,488H s.c.
155
indicated that norbinaltorphimine administered i.c.v. was also potentially capable of inducing withdrawal following U50,488H administration. Further studies on K-opioid withdrawal using higher doses of norbinaltorphimine a n d / o r varying time intervals following administration of K-opioid receptor agonists are required. Dynorphin-(1-13) has been reported to suppress morphine withdrawal signs (Aceto et al., 1982; Green and Lee, 1988). This is not unexpected since, although dynorphin is considered to be the naturally occurring ligand for the K-opioid receptor, it is not highly selective and has ~-opioid receptor agonist activity. However, U50,488H is 1300-fold more selective for K-opioid receptors than for tz-opioid receptors, and 12000-fold more selective for K-opioid receptors than for 8-opioid receptors in guinea-pig brain (James and Goldstein, 1984). In the present study, U50,488H did not inhibit any of the behaviours associated with the morphine withdrawal response. This finding supported previous studies (Fukagawa et al., 1989), that tz- and x-opioid receptor agonists do not produce cross-dependence in the central nervous system. However some behavioural responses to U50,488H withdrawal, e.g. vocalization, were apparently attenuated by morphine. Thus in the pathways involved in these latter responses, Iz- and K-opioid receptors might be present on the same neurones, whereas in the pathways involved in behavioural responses such as locomotor activity, they might be present on different neurones. In agreement with previous studies on the guinea-pig isolated ileum (Chahl, 1990) naloxone in the doses used did not induce a withdrawal contracture on preparations pretreated with U50,488H. Experiments with nor-binaltorphimine on the guinea-pig ileum were complicated by the marked contracture produced by nor-binaltorphimine alone. However, since the response to nor-binaltorphimine 1 /zM was significantly greater following U50,488H treatment of the ileum than that obtained in the absence of U50,488H, it is concluded that nor-binaltorphimine produced a withdrawal contracture on preparations treated with U50,488H. The conclusion that U50,488H induces dependence is in agreement with the findings of Valeri et al. (1992) on rabbit jejunum. The contracture of untreated ileum produced by nor-binaltorphimine might represent withdrawal from an endogenous K-opioid receptor tone which has been reported to regulate peristalsis in the ileum (Johnson et al., 1988; Kromer, 1988, 1990; Waterman et al., 1992). It is unlikely that the contracture produced by nor-binaltorphimine was due to a non-specific effect since it was inhibited by tetrodotoxin indicating that action potentials were involved. In agreement with previous studies on the ileum (Seidl and Schulz, 1983), withdrawal from U50,488H was inhibited by [MetS]enkephalin, which acts mainly
on iz-opioid receptors in the ileum. Thus cross-dependence occurred in the ileum between /x- and K-opioid receptor agonists. It has also been shown previously that U50,488H inhibits the withdrawal response of the ileum to [MetS]enkephalin (Chahl, 1990). In conclusion, the present study has shown that dependence occurs following activation of x-opioid receptors, which is largely non-morphine-like in the central nervous system, but which is morphine-like in the enteric nervous system.
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