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Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine
Life Sciences 69 (2001) 779–790
Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine Ian M. Coupar*, Binh L.T. Tran Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia Received 7 August 2000; accepted 22 December 2000
Abstract The aim of this study was to investigate whether the A1/A2 receptor agonist, 59-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 3 0.03 mg kg21, s.c) and CPA (4 3 0.03, 0.1 and 0.3 mg kg21, s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg21, s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg21, s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz. paw, body and ‘wet-dog’ shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg21, s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Adenosine receptors; Adenosine agonists; Adenosine antagonists; Adenosine dependence; Opiate dependence; CPA; NECA; CPDPX; DMPX
Introduction Direct and indirect evidence suggesting an interaction between opioids and adenosine has been obtained from many biological systems. Initially, this interaction was observed in mice * Corresponding author. Tel.: 61 03 9903 9567; fax: 61 03 9903 9568. E-mail address: [email protected] (I.M. Coupar) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 1 5 5 -9
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where theophylline was found to antagonise the antinociceptive action of morphine [1]. Subsequently, it was shown that morphine-induced antinociception was potentiated by adenosine receptor agonists and inhibitors of adenosine re-uptake [2–4] and attenuated by adenosine receptor antagonist [5–8]. Not surprisingly, chronic treatment of experimental animals with morphine has been shown to affect the adenosine system. For example, tolerance develops to the hypotensive and antinociceptive actions of adenosine agonists in opiate-dependent rats [9–12]. Physical dependence to adenosine agonist, as evidenced by a withdrawal (abstinence) syndrome following the termination of the drug or induced abruptly by an antagonist, was first reported by Collier & Tucker [13] in the guinea-pig ileum. The only other work to support this in vitro finding is an in vivo study by Aley et al. [9] into the peripheral antinociceptive effects of opioids and the adenosine A1 agonist, CPA, on PGE2-induced mechanical hyperalgesia of the rat hind paw. These workers also established that naloxone and the A1-adenosine receptor antagonist, PACPX, precipitated a bidirectional cross-withdrawal hyperalgesic response in the paws of rats tolerant to CPA and the m-opioid agonist, DAMGO, respectively. However, Collier & Tucker [13] in their study clearly established that cross withdrawal is not a phenomenon that occurs in the isolated guinea-pig ileum preparation. Therefore, the purpose of this study was to investigate whether antagonist-precipitated withdrawal occurs in rats treated with the mixed A1/A2 agonist NECA and the A1-selective agonist, CPA. In addition, the presence of bidirectional cross-withdrawal was also investigated. Methods Animals and dosing procedures All experiments were carried out using male and female Hooded Wistar rats (weight ranges were 230–400g for males and 200–300g for females), such that each experimental group contained equal numbers of each gender. Animals were housed individually in North Kent Plastic breeding cages (500 mm length, 350 mm rear height, 120 mm frontal height) lined with sawdust bedding. Standard laboratory chow and tap water were provided for consumption under a 12-hr light/dark cycle in a temperature-controlled environment (maintained at 21628C). Each animal was used once only. All rats were handled for a reasonable amount of time before experimentation (daily for two days before experiment) in order to familiarise them with surroundings (transfer from animal house to laboratory), scents of the handler, handling procedures and the testing procedure for assessing withdrawal (see next section). This protocol, as demonstrated and recommended by Pierce & Raper [14] was to ensure that the effects of stress on the expression of behavioural parameters are minimised. Four doses of the test drugs (0.03, 0.1, 0.3 mg kg21 CPA or 0.03 mg kg21 NECA or 10 mg 21 kg morphine) or appropriate vehicle controls were administered individually to groups of rats. The doses were given subcutaneously into the scruff of the neck in a volume of 1 ml kg21 body weight, separated by twelve hours from the previous dose, over a 48-hour period. The dose of morphine was selected on the basis of numerous previous studies showing it to induce dependence within 48 h. The treatment period for the adenosine agonists was con-
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trolled to 48 h also, because of the evidence that adenosine receptors are involved in mediating opiate effects (see introduction). The doses of the adenosine agonists were chosen again on the basis of literature reports that the doses are pharmacological effective. Additionally, our preliminary experiments (unpublished) confirmed that the acute and sub acute doses of the agonists, including morphine, produced submaximal effects on a range of parameters (food and water consumption, faecal and urinary output, locomotor activity, body temperature). Induction of dependence and assessment of withdrawal Withdrawal (abstinence syndrome) was assessed in drug treated and vehicle treated (drug-naive) animals at the end of the 48-hour treatment period by monitoring a variety of behaviours following challenge with the antagonist or vehicle control. Each animal was placed into a clear Perspex observation box (200 mm width, 200 mm length, 350 mm height); two were used side-by-side partitioned by dark cardboard barriers between and behind them. This allowed the observer to score the behaviours of two animals simultaneously. Each observation box contained a pre-weighed paper towel for collection of dry faecal matter and estimation of diarrhoea (defined as wet faecal matter adhering to the paper towel after removal of dry faecal matter), if present. The frequency of teeth chattering (audible teeth grinding or chewing), head shakes, paw shakes, body shakes (abdomen and lower portions of body), wet-dog shakes (vigorous head and body shakes) and yawning were scored in individual subjects over a 20-minute observation period following the antagonist challenge. The quantitative assessment of physical dependence, as measured by an antagonistprecipitated withdrawal behaviour in dependent/tolerant animals is based on previously established procedures [14–16]. Withdrawal studies Effects of naloxone in morphine pre-treated rats Morphine pre-treated rats (four doses of 10 mg kg21, s.c. over a 48-hour period) and morphinenaive rats (four doses of saline, 1 ml kg21, s.c. over a 48-hour period) were challenged with the opiate antagonist, naloxone (3 mg kg21, s.c.), 12 hours after the last treatment dose. On the basis of previous experiments [14–17], no waiting period (less than 2 minutes to place the animal in the observation box) was enforced as a protocol after administering naloxone. Withdrawal behaviour was then assessed. Effects of adenosine receptor antagonists in adenosine agonist pre-treated rats CPA (4 3 0.03, 0.1 or 0.3 mg kg21, s.c. over a 48 hour period) and NECA (4 3 0.03 mg 21 kg , s.c. over a 48 hour period) pre-treated rats were challenged with the adenosine receptor antagonists, CPDPX (0.1 mg kg21, s.c.) and DMPX (0.1 mg kg21, s.c.) respectively, 12 hours after the last treatment dose. Naive animals received the vehicle of the adenosine agonist used to induce dependence followed by challenged with the adenosine receptor antagonists CPDPX (0.1 mg kg21, s.c.) or DMPX (0.1 mg kg21, s.c.). Preliminary experiments with the adenosine receptor antagonists, CPDPX and DMPX demonstrated that a waiting period of 15 minutes after a subcutaneous injection was necessary in order to see their effects.
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Bidirectional cross-withdrawal studies Effects of naloxone in adenosine agonist pre-treated rats CPA (4 3 0.03, 0.1 or 0.3 mg kg21, s.c. over a 48 hour period) and NECA (4 3 0.03 mg 21 kg , s.c. over a 48 hour period) pre-treated rats were challenged with naloxone (3 mg kg21, s.c.) 12 hours after the last treatment dose. Naive animals received the vehicle of the adenosine agonist used to induce dependence followed by challenge with naloxone. Withdrawal behaviour was then assessed. Effects of adenosine receptor antagonists in morphine pre-treated rats Morphine pre-treated rats (four doses of 10 mg kg21, s.c. over a 48 hour period) and morphinenaive rats (four doses of saline, 1 ml kg21, s.c. over a 48 hour period) were challenged with the adenosine receptor antagonists, CPDPX (0.1 mg kg21, s.c.) or DMPX (0.1 mg kg21, s.c.), 12 hours after the last treatment dose. The waiting period between post adenosine antagonist challenge and assessment of withdrawal behaviour was 15 min. Experiments were carried out in accordance with the NH&MRC “Australian code of practice for the care and use of animals for scientific purposes” 1997. Drugs The drugs used were N6-cyclopentyladenosine (CPA); 59-N-ethylcarboxamidoadenosine (NECA); 8-cyclopentyl-1,3-dipropylxanthine (CPDPX); 3,7-dimethyl-1-propargylxanthine (DMPX) [Research Biochemicals Inc., Natick USA.]; morphine hydrochloride [GlaxoWellcome, Melbourne, Australia] and naloxone hydrochloride [Du Pont Merck, Wilmington, USA]. CPA, NECA, morphine, DMPX and naloxone were dissolved in normal saline (0.9% wv21 NaCl) at the appropriate concentrations. CPDPX was dissolved in 0.75% v v21 1M NaOH / 1% v v21 DMSO and diluted to the required concentration in normal saline. Statistical analysis One-way ANOVA was used to test the significance of differences in withdrawal signs between different dose groups. Comparisons between individual dosage groups and vehicle and/or antagonist controls were made using the Dunnett’s t-test. Student’s unpaired t-test was used to analyse the significance of difference in withdrawal signs between vehicle controls and morphine and NECA treated groups. In all experimental protocols, P , 0.05 was selected as the criterion of statistical difference. Statistical analysis was performed using the computer program SigmaStat® 2.0 (Jandel Scientific Software, Erkrath, Germany) and graphics by GraphPad Prism® (GraphPad Software Inc., San Diego, U. S. A.). Results Effects of naloxone in morphine pre-treated rats The morphine treatment induced a physical dependence as revealed by a characteristic withdrawal syndrome (paw shakes, body shakes, ‘wet-dog’ shakes, teeth chattering and diarrhoea) soon after administering naloxone (P , 0.01, n 5 5–6; Figs. 1–3, data for diarrhoea not shown). The expression of these withdrawal signs are similar to the abstinence syndrome
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Fig. 1. Incidence of withdrawal paw shakes and head shakes. The three sets of data on the left show withdrawal induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.) and naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively. Injections of vehicle acted as controls to the agonists (saline or DMSO/saline). The three sets of data on the right show cross-withdrawal reponses. The doses of the agonists as indicated and the vehicle controls were administered s.c. twice-daily for two days, 12 hours apart. Columns show the mean incidence of the behaviours during the 20 min observation period (6s.e. mean, n 5 5–6). Asterisks indicate that the frequency of the sign is significantly greater than the basal level in vehicleinjected animals challenged with the antagonists (P, 0.05, CPA Dunnett’s t test, NECA and morphine unpaired t test).
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Fig. 2. Incidence of body shakes and wet-dog shakes induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.), or naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively and cross-withdrawal responses (n 5 5–6). Other details as Fig. 1.
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Fig. 3. Incidence of withdrawal teeth-chattering and yawning induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.) or naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively and cross-withdrawal responses (n 5 5–6). Other details as Fig. 1.
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found in other studies utilising slow release morphine preparations, indicating that this treatment paradigm induced a dependent state in rats. Effects of adenosine receptor antagonists in adenosine agonist pre-treated rats A state of physical dependence was induced in animals treated with the non-selective adenosine A1/A2 receptor agonist, NECA as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, DMPX (P , 0.05, n 5 5–6; Figs. 1–3). Similarly, the adenosine A1 receptor antagonist, CPDPX also precipitated a significant dose-dependent withdrawal syndrome in animals pre-treated with the adenosine A1 receptor agonist, CPA (P , 0.05, n 5 5–6; Fig. 1–3). The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz. paw, body and ‘wet-dog’ shakes, but with the additional behaviours of head shaking (Fig. 1B) and yawning (Fig. 3B). In further contrast to the opiate withdrawal syndrome, there was no diarrhoea in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists (P . 0.05, n 5 5–6). Bidirectional cross-withdrawal studies Effects of naloxone in adenosine agonist pre-treated rats Naloxone precipitated a similar abstinence syndrome to the adenosine receptor antagonist, CPDPX in adenosine agonist pre-treated animals. Figs. 1 and 3 show significant dose-dependent increases in the incidence of paw shakes, head shakes, teeth chattering and yawning behaviour were observed in animals pre-treated with the adenosine agonist, CPA (P , 0.05, n 5 5–6). Significant withdrawal body shakes and ‘wet-dog’ shakes were only evident in animals pretreated with the highest dose of CPA (0.3 mg kg21, s.c.; P , 0.05, n 5 5–6) (Fig. 2). Naloxone did not induce diarrhoea at any dose of CPA as a pre-treatment (P . 0.05, n 5 5–6). Effects of adenosine receptor antagonists in morphine pre-treated rats The incidence of shaking behaviours (paw, head, body and ‘wet-dog’ shakes) and teeth chattering were all significantly increased in animals pre-treated with morphine and challenged with the adenosine receptor antagonists, CPDPX or DMPX with respect to naive animals subjected to the same antagonist challenge (P , 0.05, n 5 5–6) (Figs. 1, 2 & 3A). However, neither yawning behaviour nor diarrhoea were induced in the groups of morphine-dependent animals given the adenosine antagonists, CPDPX, and DMPX (P . 0.05, n 5 5–6; Fig. 3B, data for diarrhoea not given). Discussion This study has established that repeated administration of the adenosine agonists, CPA and NECA leads to a form of physical dependence. In addition, bidirectional cross-withdrawal syndromes were demonstrated in groups of animals pre-treated with adenosine agonists and morphine. Development of physical dependence on adenosine agonists Non-motor stimulant doses of the adenosine antagonists were used to precipitate withdrawal, as have been used previously in other behavioural studies [18, 19] and vehicle/antagonist
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controls were performed in the same manner as the agonist/antagonist challenge. Furthermore, procedures for minimising stress-induced potentiation of withdrawal signs were also enforced as a protocol in this study, as recommended and demonstrated by Pierce & Raper [14]. The physical dependence on the adenosine agonists has aspects in common with that associated with morphine dependence. For instance, significant increases in the incidence of shaking behaviours (paw, body, head and ‘wet-dog’ shakes) and teeth-chattering were expressed in animals pre-treated with CPA or NECA and challenged with their respective adenosine receptor antagonists. Rats made dependent on morphine and withdrawn using naloxone also displayed these behaviours. However, withdrawal diarrhoea was only evident in morphine-dependent animals, whereas yawning behaviour and head shakes were only significantly expressed in adenosine agonist pre-treated animals challenged with the adenosine antagonist. The finding that the incidence of head shakes was not enhanced to a significant extent in the naloxone-challenged morphine-treated animals is in agreement with previous studies showing that head shakes is not a marker of opiate withdrawal [14, 17]. It has been demonstrated in vitro that adenosine and its more stable derivative, 2-chloroadenosine, induce dependence in the guinea-pig ileum after a 16–21 hour exposure [13]. In another study, intradermal administration of the adenosine A1 receptor antagonist, PACPX precipitated a withdrawal hyperalgesic response in rats tolerant to the adenosine A1 receptor agonist, CPA [9]. Therefore, it is not surprising that the present study has revealed adaptive changes to the adenosine agonists in response to repeated subcutaneous administration. These results are strengthened by the finding that the selective adenosine receptor agonist, CPA, had a dose-dependent effect on the development of physical dependence. Possible mechanism of adenosine agonist dependence It has been suggested that the mechanism underlying the behavioural manifestations of the opiate abstinence syndrome is due to the unopposed action of the upregulated adenylyl cyclase/cAMP system in opiate-sensitive brain regions [20, 21]. Since adenosine A1 receptors are negatively coupled to adenylyl cyclase via the inhibitory G protein, Gi/o [22, 23], as are opiate receptors, it is suggested that the behavioural signs of adenosine abstinence may also be due to similar adaptive cellular changes associated with opiate dependence and withdrawal. At present, evidence for a compensatory upregulated adenylyl cyclase/cAMP system to oppose A1 receptor mediated (Gi coupled) inhibition has only been reported in adipocytes [24, 25]. However, the present results are suggestive of the involvement of central A1 adenosine receptors, since the behavioural signs noted in the presently described work occurred as the result of pre-treating animals with the highly selective adenosine A1 agonist, CPA, and withdrawal using the highly selective adenosine A1 antagonist, CPDPX. Binding data has established CPA and CPDPX have 784 and 735 greater affinities at A1 than A2 receptors respectively [26, 27]. The possible involvement of adenosine A2 receptors was not investigated in the present study due to the lack of selective ligands. However, it appears that the A1 receptor contribution to the withdrawal behaviours is a major one, because the same withdrawal signs were induced by treating animals with the non-selective A1/A2 agonist/antagonist pair, NECA/DMPX as was produced by CPA/CPDPX.
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Adenosine and morphine interactions: bidirectional cross-withdrawal The results of the presently described cross-withdrawal studies show that the adenosine receptor antagonists, CPDPX and DMPX also precipitated an abstinence syndrome in morphine-dependent rats similar to that precipitated by naloxone itself. In comparison to the naloxone-precipitated opiate withdrawal syndrome, the adenosine receptor antagonists in morphine-dependent animals induced no significant diarrhoea. However, head shakes, noted as a marker of adenosine withdrawal, but not opiate withdrawal, was significantly expressed in opiate-dependent animals withdrawn using the adenosine receptor antagonists but not naloxone. In both the adenosine antagonist and naloxone challenge of morphine-dependent animals, withdrawal yawning behaviour, a marker of adenosine withdrawal, but not opiate withdrawal, was not expressed. The results also show that the withdrawal effects induced by adenosine antagonists in morphine-dependent rats is bidirectional, because naloxone was able to induce withdrawal signs in adenosine agonist-dependent rats. Hence, naloxone induced shaking behaviours (paw, head, body & ‘wet-dog’), teeth chattering and yawning in animals pre-treated with CPA. The cross-withdrawal responses appear to be similar to those precipitated by the adenosine receptor antagonists in the adenosine agonist pre-treated animals. Whilst withdrawal paw shakes, head shakes, teeth chattering and yawning behaviours were expressed in CPA-treated animals when challenged with naloxone, significant expression of withdrawal body shakes and ‘wet-dog’ shakes were only observed in animals pre-treated with the highest dose of CPA. Interestingly, yawning behaviour (a marker of adenosine withdrawal) was not expressed in the adenosine antagonist challenge of morphine-dependent animals, but was expressed in animals pre-treated with CPA and subjected to the naloxone challenge. Furthermore, withdrawal diarrhoea was not significantly precipitated by naloxone in CPA pre-treated animals. Possible mechanisms of cross withdrawals The presence of cross-withdrawal syndromes provides further evidence for the association between opiate and adenosine mechanisms. Kaplan et al. [28] have shown that cortical A1 receptors are up-regulated in morphine-dependent mice. Hence, it is possible that the cross-withdrawal response shown in the present study is due to blockade by CPDPX of the inhibitory ‘tone’ exerted by endogenous adenosine acting at these upregulated inhibitory A1 receptors as a homeostatic response to modulate opiate dependence and withdrawal. The recent finding of Salem & Hope [29] that the levels of the adenosine metabolites, hypoxanthine and inosine, are elevated in the nucleus accumbens of rats undergoing withdrawal from morphine supports this view. The finding that naloxone precipitated a cross-withdrawal response in animals dependent on CPA suggest the possible involvement of endogenous opioids, similar to the role of endogenous adenosine discussed earlier, in modulating the cellular changes associated with sub-chronic adenosine agonist treatment. For instance, the adaptive increase in cAMP (in response to a decrease in cAMP induced by repeated administration of CPA) could be modulated by endogenous opioids by their inhibitory ‘tone’, which when blocked by naloxone, could unmask this adaptive change and result in the expression of a cross-withdrawal syndrome. However, there appear to be no reports to date in the literature supporting this suggestion.
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Conclusion The present study has emphasised the underlying complexity of opiate dependence. In particular it illustrates the close adaptive changes that occur between opiate and adenosine systems that commonly influence the neuronal circuits that control certain behaviours. A greater understanding of these systems will be relevant to future studies of opiate addiction and hopefully improving its treatment. Acknowledgments We wish to thank Mr Steven Haas for carrying out some of the pilot experiments. References 1. Ho IK, Loh HH, Way, EL. Cyclic adenosine monophosphate antagonism of morphine analgesia. J. Pharmacol. Exp. Ther. 1973; 185 336–346 2. Ahlijanian MK, Takemori AE. Effects of (-)-N6-(R-phenyl-isopropyl)-adenosine (PIA) and caffeine on nociception and morphine induced analgesia, tolerance and dependence in mice. Eur. J. Pharmacol. 1985; 112 171–179 3. Contreras E, Germany A, Villar M. Effects of some adenosine analogs on morphine induced analgesia and tolerance. General Pharmacol. 1990; 21 763–767 4. DeLander G.E, Hopkins CJ. Involvement of A2a adenosine receptors in spinal mechanisms of antinoception. Eur. J. Pharmacol. 1987; 139, 215–223 5. DeLander G.E, Hopkins CJ. Spinal adenosine modulates descending antinociceptive pathways stimulated by morphine. Pharmacol. Exp. Ther. 1986; 239, 88–93 6. DeLander GE, Mosberg HI, Porreca FJ. Involvement of adenosine in antinociception produced by spinal or supraspinal receptor-selective opioid agonists: dissociation from gastrointestinal effects in mice. Pharmacol. Exp. Ther. 1992; 263 1097–1104 7. Sawynok J, Espey MJ, Reid A. 8-Phenytheophylline reverses the antinociceptive action of morphine in the periaqueductal gray. Neuropharmacol. 1991; 30 871–877 8. Sweeney MI, White TD, Sawynok J. Morphine, capsaicin and K1release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord. Pharmacol. Exp. Ther. 1987; 243 657–665 9. Aley KO, Green PG, Levine JD. Opioid and adenosine peripheral antinociception are subject to tolerance and withdrawal. J. Neurosci. 1995; 15 8031–8038 10. Tao PL, Liu CF, Tsai HC. Chronic intracerebroventricular administration of morphine down regulates spinal adenosine A1 receptors in rats. Eur. J. Pharmacol. 1995; 278 233–237 11. Tao PL, Lui, CF. Chronic morphine treatment causes down regulation of spinal adenosine A1 receptors in rats. Eur. J. Pharmaol. 1992; 215 301–304 12. White PJ, Rose’Meyer RB, Hope W. Changes in adenosine receptors mediating hypotension in morphinedependent rats. Eur. J. Pharmacol. 1995; 294 215–220 13. Collier HOJ, Tucker JF. Novel form of drug dependence on adenosine in guinea-pig ileum. Nature, 1983; 302 618–621 14. Pierce TL, Raper, CJ. The effects of laboratory handling procedures on naloxone-precipitated withdrawal behaviour in morphine dependent rats. Pharmacol. Toxicol. Methods 1995; 34 149–155 15. Dionyssopoulos T, Hope W, Coupar IM. Effect of adenosine analogues on the expression of opiate withdrawal in rats. Pharmacol. Biochemi. Behav. 1992; 42 201–206 16. Salem A, Hope W. Effect of adenosine receptor agonists and antagonist on the expression of opiate withdrawal in rats. Pharmacol. Biochem. Behav. 1997; 54, 671–679 17. Collier HOJ, Francis DL, Schneider C. Modification of morphine withdrawal by drugs interacting with humoral mechanisms: some contradictions and their interpretations. Nature, 1972; 237 220–223
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18. Griebel G, Saffroy-Spittler M, Misslin R, Remmy D, Vogel E, Bourguignon JJ. Comparison of behavioural effects of an adenosine A1/A2-receptor antagonist, CGS-15943A, and an A1-selective antagonist, DPCPX. Psychopharmacol. 1991; 103 541–544 19. Seale TW, Abla KA, Shamim MT, Carney JM, Daly JW. 3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs. Life Sci. 1988; 43 1671–1684 20. Collier HOJ, Francis DL. Morphine abstinence is associated with increased brain cyclic AMP. Nature 1975; 255 159–162 21. Nestler EJ. Molecular mechanisms of drug addiction. J. Neurosci. 1992; 12 2439–2450 22. van Caulker D, Muller M, Hamprecht BJ. Adenosine regulates two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. Neurochem. 1979; 33 999–1005 23. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol. Rev. 1998; 50 415–475 24. Hoffman BB, Chang H, Dall’Aglio E, Reaven GM. Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes. J. Clin. Invest. 1986; 78 185–190 25. Longabaugh JP, Didsbury J, Spiegel A, Stiles GL. Modification of rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist: alterations in the quantity of Gsa and Gia are not associated with changes in their mRNAs. Mol. Pharmacol. 1989; 36 681–688 26. Bruns RF, Lu GH, Pugsley TA. Characterisation of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol. Pharmacol. 1986; 29 331–346 27. Bruns RF, Fergus JH, Badger EW, Bristol JA, Santay LA, Hartman JD, Hays SJ, Huang CC. Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes NaunynSchmiedeberg’s Arch. Pharmacol. 1987; 335 59–63 28. Kaplan GB, Leite-Morris KA, Sears MT. Alterations of adenosine A1 receptors in morphine dependence. Brain Research 1994; 657 347–350 29. Salem A, Hope W. Role of endogenous adenosine in the expression of opiate withdrawal in rats. Eur. J. Pharmacol. 1999; 39 39–42
Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine Ian M. Coupar*, Binh L.T. Tran Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia Received 7 August 2000; accepted 22 December 2000
Abstract The aim of this study was to investigate whether the A1/A2 receptor agonist, 59-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 3 0.03 mg kg21, s.c) and CPA (4 3 0.03, 0.1 and 0.3 mg kg21, s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg21, s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg21, s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz. paw, body and ‘wet-dog’ shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg21, s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Adenosine receptors; Adenosine agonists; Adenosine antagonists; Adenosine dependence; Opiate dependence; CPA; NECA; CPDPX; DMPX
Introduction Direct and indirect evidence suggesting an interaction between opioids and adenosine has been obtained from many biological systems. Initially, this interaction was observed in mice * Corresponding author. Tel.: 61 03 9903 9567; fax: 61 03 9903 9568. E-mail address: [email protected] (I.M. Coupar) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 1 5 5 -9
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where theophylline was found to antagonise the antinociceptive action of morphine [1]. Subsequently, it was shown that morphine-induced antinociception was potentiated by adenosine receptor agonists and inhibitors of adenosine re-uptake [2–4] and attenuated by adenosine receptor antagonist [5–8]. Not surprisingly, chronic treatment of experimental animals with morphine has been shown to affect the adenosine system. For example, tolerance develops to the hypotensive and antinociceptive actions of adenosine agonists in opiate-dependent rats [9–12]. Physical dependence to adenosine agonist, as evidenced by a withdrawal (abstinence) syndrome following the termination of the drug or induced abruptly by an antagonist, was first reported by Collier & Tucker [13] in the guinea-pig ileum. The only other work to support this in vitro finding is an in vivo study by Aley et al. [9] into the peripheral antinociceptive effects of opioids and the adenosine A1 agonist, CPA, on PGE2-induced mechanical hyperalgesia of the rat hind paw. These workers also established that naloxone and the A1-adenosine receptor antagonist, PACPX, precipitated a bidirectional cross-withdrawal hyperalgesic response in the paws of rats tolerant to CPA and the m-opioid agonist, DAMGO, respectively. However, Collier & Tucker [13] in their study clearly established that cross withdrawal is not a phenomenon that occurs in the isolated guinea-pig ileum preparation. Therefore, the purpose of this study was to investigate whether antagonist-precipitated withdrawal occurs in rats treated with the mixed A1/A2 agonist NECA and the A1-selective agonist, CPA. In addition, the presence of bidirectional cross-withdrawal was also investigated. Methods Animals and dosing procedures All experiments were carried out using male and female Hooded Wistar rats (weight ranges were 230–400g for males and 200–300g for females), such that each experimental group contained equal numbers of each gender. Animals were housed individually in North Kent Plastic breeding cages (500 mm length, 350 mm rear height, 120 mm frontal height) lined with sawdust bedding. Standard laboratory chow and tap water were provided for consumption under a 12-hr light/dark cycle in a temperature-controlled environment (maintained at 21628C). Each animal was used once only. All rats were handled for a reasonable amount of time before experimentation (daily for two days before experiment) in order to familiarise them with surroundings (transfer from animal house to laboratory), scents of the handler, handling procedures and the testing procedure for assessing withdrawal (see next section). This protocol, as demonstrated and recommended by Pierce & Raper [14] was to ensure that the effects of stress on the expression of behavioural parameters are minimised. Four doses of the test drugs (0.03, 0.1, 0.3 mg kg21 CPA or 0.03 mg kg21 NECA or 10 mg 21 kg morphine) or appropriate vehicle controls were administered individually to groups of rats. The doses were given subcutaneously into the scruff of the neck in a volume of 1 ml kg21 body weight, separated by twelve hours from the previous dose, over a 48-hour period. The dose of morphine was selected on the basis of numerous previous studies showing it to induce dependence within 48 h. The treatment period for the adenosine agonists was con-
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trolled to 48 h also, because of the evidence that adenosine receptors are involved in mediating opiate effects (see introduction). The doses of the adenosine agonists were chosen again on the basis of literature reports that the doses are pharmacological effective. Additionally, our preliminary experiments (unpublished) confirmed that the acute and sub acute doses of the agonists, including morphine, produced submaximal effects on a range of parameters (food and water consumption, faecal and urinary output, locomotor activity, body temperature). Induction of dependence and assessment of withdrawal Withdrawal (abstinence syndrome) was assessed in drug treated and vehicle treated (drug-naive) animals at the end of the 48-hour treatment period by monitoring a variety of behaviours following challenge with the antagonist or vehicle control. Each animal was placed into a clear Perspex observation box (200 mm width, 200 mm length, 350 mm height); two were used side-by-side partitioned by dark cardboard barriers between and behind them. This allowed the observer to score the behaviours of two animals simultaneously. Each observation box contained a pre-weighed paper towel for collection of dry faecal matter and estimation of diarrhoea (defined as wet faecal matter adhering to the paper towel after removal of dry faecal matter), if present. The frequency of teeth chattering (audible teeth grinding or chewing), head shakes, paw shakes, body shakes (abdomen and lower portions of body), wet-dog shakes (vigorous head and body shakes) and yawning were scored in individual subjects over a 20-minute observation period following the antagonist challenge. The quantitative assessment of physical dependence, as measured by an antagonistprecipitated withdrawal behaviour in dependent/tolerant animals is based on previously established procedures [14–16]. Withdrawal studies Effects of naloxone in morphine pre-treated rats Morphine pre-treated rats (four doses of 10 mg kg21, s.c. over a 48-hour period) and morphinenaive rats (four doses of saline, 1 ml kg21, s.c. over a 48-hour period) were challenged with the opiate antagonist, naloxone (3 mg kg21, s.c.), 12 hours after the last treatment dose. On the basis of previous experiments [14–17], no waiting period (less than 2 minutes to place the animal in the observation box) was enforced as a protocol after administering naloxone. Withdrawal behaviour was then assessed. Effects of adenosine receptor antagonists in adenosine agonist pre-treated rats CPA (4 3 0.03, 0.1 or 0.3 mg kg21, s.c. over a 48 hour period) and NECA (4 3 0.03 mg 21 kg , s.c. over a 48 hour period) pre-treated rats were challenged with the adenosine receptor antagonists, CPDPX (0.1 mg kg21, s.c.) and DMPX (0.1 mg kg21, s.c.) respectively, 12 hours after the last treatment dose. Naive animals received the vehicle of the adenosine agonist used to induce dependence followed by challenged with the adenosine receptor antagonists CPDPX (0.1 mg kg21, s.c.) or DMPX (0.1 mg kg21, s.c.). Preliminary experiments with the adenosine receptor antagonists, CPDPX and DMPX demonstrated that a waiting period of 15 minutes after a subcutaneous injection was necessary in order to see their effects.
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Bidirectional cross-withdrawal studies Effects of naloxone in adenosine agonist pre-treated rats CPA (4 3 0.03, 0.1 or 0.3 mg kg21, s.c. over a 48 hour period) and NECA (4 3 0.03 mg 21 kg , s.c. over a 48 hour period) pre-treated rats were challenged with naloxone (3 mg kg21, s.c.) 12 hours after the last treatment dose. Naive animals received the vehicle of the adenosine agonist used to induce dependence followed by challenge with naloxone. Withdrawal behaviour was then assessed. Effects of adenosine receptor antagonists in morphine pre-treated rats Morphine pre-treated rats (four doses of 10 mg kg21, s.c. over a 48 hour period) and morphinenaive rats (four doses of saline, 1 ml kg21, s.c. over a 48 hour period) were challenged with the adenosine receptor antagonists, CPDPX (0.1 mg kg21, s.c.) or DMPX (0.1 mg kg21, s.c.), 12 hours after the last treatment dose. The waiting period between post adenosine antagonist challenge and assessment of withdrawal behaviour was 15 min. Experiments were carried out in accordance with the NH&MRC “Australian code of practice for the care and use of animals for scientific purposes” 1997. Drugs The drugs used were N6-cyclopentyladenosine (CPA); 59-N-ethylcarboxamidoadenosine (NECA); 8-cyclopentyl-1,3-dipropylxanthine (CPDPX); 3,7-dimethyl-1-propargylxanthine (DMPX) [Research Biochemicals Inc., Natick USA.]; morphine hydrochloride [GlaxoWellcome, Melbourne, Australia] and naloxone hydrochloride [Du Pont Merck, Wilmington, USA]. CPA, NECA, morphine, DMPX and naloxone were dissolved in normal saline (0.9% wv21 NaCl) at the appropriate concentrations. CPDPX was dissolved in 0.75% v v21 1M NaOH / 1% v v21 DMSO and diluted to the required concentration in normal saline. Statistical analysis One-way ANOVA was used to test the significance of differences in withdrawal signs between different dose groups. Comparisons between individual dosage groups and vehicle and/or antagonist controls were made using the Dunnett’s t-test. Student’s unpaired t-test was used to analyse the significance of difference in withdrawal signs between vehicle controls and morphine and NECA treated groups. In all experimental protocols, P , 0.05 was selected as the criterion of statistical difference. Statistical analysis was performed using the computer program SigmaStat® 2.0 (Jandel Scientific Software, Erkrath, Germany) and graphics by GraphPad Prism® (GraphPad Software Inc., San Diego, U. S. A.). Results Effects of naloxone in morphine pre-treated rats The morphine treatment induced a physical dependence as revealed by a characteristic withdrawal syndrome (paw shakes, body shakes, ‘wet-dog’ shakes, teeth chattering and diarrhoea) soon after administering naloxone (P , 0.01, n 5 5–6; Figs. 1–3, data for diarrhoea not shown). The expression of these withdrawal signs are similar to the abstinence syndrome
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Fig. 1. Incidence of withdrawal paw shakes and head shakes. The three sets of data on the left show withdrawal induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.) and naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively. Injections of vehicle acted as controls to the agonists (saline or DMSO/saline). The three sets of data on the right show cross-withdrawal reponses. The doses of the agonists as indicated and the vehicle controls were administered s.c. twice-daily for two days, 12 hours apart. Columns show the mean incidence of the behaviours during the 20 min observation period (6s.e. mean, n 5 5–6). Asterisks indicate that the frequency of the sign is significantly greater than the basal level in vehicleinjected animals challenged with the antagonists (P, 0.05, CPA Dunnett’s t test, NECA and morphine unpaired t test).
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Fig. 2. Incidence of body shakes and wet-dog shakes induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.), or naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively and cross-withdrawal responses (n 5 5–6). Other details as Fig. 1.
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Fig. 3. Incidence of withdrawal teeth-chattering and yawning induced by CPDPX (0.1 mg kg21, s.c.), DMPX (0.1 mg kg21, s.c.) or naloxone (3 mg kg21, s.c.) in groups of rats pre-treated with the agonists CPA, NECA and morphine respectively and cross-withdrawal responses (n 5 5–6). Other details as Fig. 1.
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found in other studies utilising slow release morphine preparations, indicating that this treatment paradigm induced a dependent state in rats. Effects of adenosine receptor antagonists in adenosine agonist pre-treated rats A state of physical dependence was induced in animals treated with the non-selective adenosine A1/A2 receptor agonist, NECA as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, DMPX (P , 0.05, n 5 5–6; Figs. 1–3). Similarly, the adenosine A1 receptor antagonist, CPDPX also precipitated a significant dose-dependent withdrawal syndrome in animals pre-treated with the adenosine A1 receptor agonist, CPA (P , 0.05, n 5 5–6; Fig. 1–3). The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz. paw, body and ‘wet-dog’ shakes, but with the additional behaviours of head shaking (Fig. 1B) and yawning (Fig. 3B). In further contrast to the opiate withdrawal syndrome, there was no diarrhoea in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists (P . 0.05, n 5 5–6). Bidirectional cross-withdrawal studies Effects of naloxone in adenosine agonist pre-treated rats Naloxone precipitated a similar abstinence syndrome to the adenosine receptor antagonist, CPDPX in adenosine agonist pre-treated animals. Figs. 1 and 3 show significant dose-dependent increases in the incidence of paw shakes, head shakes, teeth chattering and yawning behaviour were observed in animals pre-treated with the adenosine agonist, CPA (P , 0.05, n 5 5–6). Significant withdrawal body shakes and ‘wet-dog’ shakes were only evident in animals pretreated with the highest dose of CPA (0.3 mg kg21, s.c.; P , 0.05, n 5 5–6) (Fig. 2). Naloxone did not induce diarrhoea at any dose of CPA as a pre-treatment (P . 0.05, n 5 5–6). Effects of adenosine receptor antagonists in morphine pre-treated rats The incidence of shaking behaviours (paw, head, body and ‘wet-dog’ shakes) and teeth chattering were all significantly increased in animals pre-treated with morphine and challenged with the adenosine receptor antagonists, CPDPX or DMPX with respect to naive animals subjected to the same antagonist challenge (P , 0.05, n 5 5–6) (Figs. 1, 2 & 3A). However, neither yawning behaviour nor diarrhoea were induced in the groups of morphine-dependent animals given the adenosine antagonists, CPDPX, and DMPX (P . 0.05, n 5 5–6; Fig. 3B, data for diarrhoea not given). Discussion This study has established that repeated administration of the adenosine agonists, CPA and NECA leads to a form of physical dependence. In addition, bidirectional cross-withdrawal syndromes were demonstrated in groups of animals pre-treated with adenosine agonists and morphine. Development of physical dependence on adenosine agonists Non-motor stimulant doses of the adenosine antagonists were used to precipitate withdrawal, as have been used previously in other behavioural studies [18, 19] and vehicle/antagonist
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controls were performed in the same manner as the agonist/antagonist challenge. Furthermore, procedures for minimising stress-induced potentiation of withdrawal signs were also enforced as a protocol in this study, as recommended and demonstrated by Pierce & Raper [14]. The physical dependence on the adenosine agonists has aspects in common with that associated with morphine dependence. For instance, significant increases in the incidence of shaking behaviours (paw, body, head and ‘wet-dog’ shakes) and teeth-chattering were expressed in animals pre-treated with CPA or NECA and challenged with their respective adenosine receptor antagonists. Rats made dependent on morphine and withdrawn using naloxone also displayed these behaviours. However, withdrawal diarrhoea was only evident in morphine-dependent animals, whereas yawning behaviour and head shakes were only significantly expressed in adenosine agonist pre-treated animals challenged with the adenosine antagonist. The finding that the incidence of head shakes was not enhanced to a significant extent in the naloxone-challenged morphine-treated animals is in agreement with previous studies showing that head shakes is not a marker of opiate withdrawal [14, 17]. It has been demonstrated in vitro that adenosine and its more stable derivative, 2-chloroadenosine, induce dependence in the guinea-pig ileum after a 16–21 hour exposure [13]. In another study, intradermal administration of the adenosine A1 receptor antagonist, PACPX precipitated a withdrawal hyperalgesic response in rats tolerant to the adenosine A1 receptor agonist, CPA [9]. Therefore, it is not surprising that the present study has revealed adaptive changes to the adenosine agonists in response to repeated subcutaneous administration. These results are strengthened by the finding that the selective adenosine receptor agonist, CPA, had a dose-dependent effect on the development of physical dependence. Possible mechanism of adenosine agonist dependence It has been suggested that the mechanism underlying the behavioural manifestations of the opiate abstinence syndrome is due to the unopposed action of the upregulated adenylyl cyclase/cAMP system in opiate-sensitive brain regions [20, 21]. Since adenosine A1 receptors are negatively coupled to adenylyl cyclase via the inhibitory G protein, Gi/o [22, 23], as are opiate receptors, it is suggested that the behavioural signs of adenosine abstinence may also be due to similar adaptive cellular changes associated with opiate dependence and withdrawal. At present, evidence for a compensatory upregulated adenylyl cyclase/cAMP system to oppose A1 receptor mediated (Gi coupled) inhibition has only been reported in adipocytes [24, 25]. However, the present results are suggestive of the involvement of central A1 adenosine receptors, since the behavioural signs noted in the presently described work occurred as the result of pre-treating animals with the highly selective adenosine A1 agonist, CPA, and withdrawal using the highly selective adenosine A1 antagonist, CPDPX. Binding data has established CPA and CPDPX have 784 and 735 greater affinities at A1 than A2 receptors respectively [26, 27]. The possible involvement of adenosine A2 receptors was not investigated in the present study due to the lack of selective ligands. However, it appears that the A1 receptor contribution to the withdrawal behaviours is a major one, because the same withdrawal signs were induced by treating animals with the non-selective A1/A2 agonist/antagonist pair, NECA/DMPX as was produced by CPA/CPDPX.
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Adenosine and morphine interactions: bidirectional cross-withdrawal The results of the presently described cross-withdrawal studies show that the adenosine receptor antagonists, CPDPX and DMPX also precipitated an abstinence syndrome in morphine-dependent rats similar to that precipitated by naloxone itself. In comparison to the naloxone-precipitated opiate withdrawal syndrome, the adenosine receptor antagonists in morphine-dependent animals induced no significant diarrhoea. However, head shakes, noted as a marker of adenosine withdrawal, but not opiate withdrawal, was significantly expressed in opiate-dependent animals withdrawn using the adenosine receptor antagonists but not naloxone. In both the adenosine antagonist and naloxone challenge of morphine-dependent animals, withdrawal yawning behaviour, a marker of adenosine withdrawal, but not opiate withdrawal, was not expressed. The results also show that the withdrawal effects induced by adenosine antagonists in morphine-dependent rats is bidirectional, because naloxone was able to induce withdrawal signs in adenosine agonist-dependent rats. Hence, naloxone induced shaking behaviours (paw, head, body & ‘wet-dog’), teeth chattering and yawning in animals pre-treated with CPA. The cross-withdrawal responses appear to be similar to those precipitated by the adenosine receptor antagonists in the adenosine agonist pre-treated animals. Whilst withdrawal paw shakes, head shakes, teeth chattering and yawning behaviours were expressed in CPA-treated animals when challenged with naloxone, significant expression of withdrawal body shakes and ‘wet-dog’ shakes were only observed in animals pre-treated with the highest dose of CPA. Interestingly, yawning behaviour (a marker of adenosine withdrawal) was not expressed in the adenosine antagonist challenge of morphine-dependent animals, but was expressed in animals pre-treated with CPA and subjected to the naloxone challenge. Furthermore, withdrawal diarrhoea was not significantly precipitated by naloxone in CPA pre-treated animals. Possible mechanisms of cross withdrawals The presence of cross-withdrawal syndromes provides further evidence for the association between opiate and adenosine mechanisms. Kaplan et al. [28] have shown that cortical A1 receptors are up-regulated in morphine-dependent mice. Hence, it is possible that the cross-withdrawal response shown in the present study is due to blockade by CPDPX of the inhibitory ‘tone’ exerted by endogenous adenosine acting at these upregulated inhibitory A1 receptors as a homeostatic response to modulate opiate dependence and withdrawal. The recent finding of Salem & Hope [29] that the levels of the adenosine metabolites, hypoxanthine and inosine, are elevated in the nucleus accumbens of rats undergoing withdrawal from morphine supports this view. The finding that naloxone precipitated a cross-withdrawal response in animals dependent on CPA suggest the possible involvement of endogenous opioids, similar to the role of endogenous adenosine discussed earlier, in modulating the cellular changes associated with sub-chronic adenosine agonist treatment. For instance, the adaptive increase in cAMP (in response to a decrease in cAMP induced by repeated administration of CPA) could be modulated by endogenous opioids by their inhibitory ‘tone’, which when blocked by naloxone, could unmask this adaptive change and result in the expression of a cross-withdrawal syndrome. However, there appear to be no reports to date in the literature supporting this suggestion.
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Conclusion The present study has emphasised the underlying complexity of opiate dependence. In particular it illustrates the close adaptive changes that occur between opiate and adenosine systems that commonly influence the neuronal circuits that control certain behaviours. A greater understanding of these systems will be relevant to future studies of opiate addiction and hopefully improving its treatment. Acknowledgments We wish to thank Mr Steven Haas for carrying out some of the pilot experiments. References 1. Ho IK, Loh HH, Way, EL. Cyclic adenosine monophosphate antagonism of morphine analgesia. J. Pharmacol. Exp. Ther. 1973; 185 336–346 2. Ahlijanian MK, Takemori AE. Effects of (-)-N6-(R-phenyl-isopropyl)-adenosine (PIA) and caffeine on nociception and morphine induced analgesia, tolerance and dependence in mice. Eur. J. Pharmacol. 1985; 112 171–179 3. Contreras E, Germany A, Villar M. Effects of some adenosine analogs on morphine induced analgesia and tolerance. General Pharmacol. 1990; 21 763–767 4. DeLander G.E, Hopkins CJ. Involvement of A2a adenosine receptors in spinal mechanisms of antinoception. Eur. J. Pharmacol. 1987; 139, 215–223 5. DeLander G.E, Hopkins CJ. Spinal adenosine modulates descending antinociceptive pathways stimulated by morphine. Pharmacol. Exp. Ther. 1986; 239, 88–93 6. DeLander GE, Mosberg HI, Porreca FJ. Involvement of adenosine in antinociception produced by spinal or supraspinal receptor-selective opioid agonists: dissociation from gastrointestinal effects in mice. Pharmacol. Exp. Ther. 1992; 263 1097–1104 7. Sawynok J, Espey MJ, Reid A. 8-Phenytheophylline reverses the antinociceptive action of morphine in the periaqueductal gray. Neuropharmacol. 1991; 30 871–877 8. Sweeney MI, White TD, Sawynok J. Morphine, capsaicin and K1release purines from capsaicin-sensitive primary afferent nerve terminals in the spinal cord. Pharmacol. Exp. Ther. 1987; 243 657–665 9. Aley KO, Green PG, Levine JD. Opioid and adenosine peripheral antinociception are subject to tolerance and withdrawal. J. Neurosci. 1995; 15 8031–8038 10. Tao PL, Liu CF, Tsai HC. Chronic intracerebroventricular administration of morphine down regulates spinal adenosine A1 receptors in rats. Eur. J. Pharmacol. 1995; 278 233–237 11. Tao PL, Lui, CF. Chronic morphine treatment causes down regulation of spinal adenosine A1 receptors in rats. Eur. J. Pharmaol. 1992; 215 301–304 12. White PJ, Rose’Meyer RB, Hope W. Changes in adenosine receptors mediating hypotension in morphinedependent rats. Eur. J. Pharmacol. 1995; 294 215–220 13. Collier HOJ, Tucker JF. Novel form of drug dependence on adenosine in guinea-pig ileum. Nature, 1983; 302 618–621 14. Pierce TL, Raper, CJ. The effects of laboratory handling procedures on naloxone-precipitated withdrawal behaviour in morphine dependent rats. Pharmacol. Toxicol. Methods 1995; 34 149–155 15. Dionyssopoulos T, Hope W, Coupar IM. Effect of adenosine analogues on the expression of opiate withdrawal in rats. Pharmacol. Biochemi. Behav. 1992; 42 201–206 16. Salem A, Hope W. Effect of adenosine receptor agonists and antagonist on the expression of opiate withdrawal in rats. Pharmacol. Biochem. Behav. 1997; 54, 671–679 17. Collier HOJ, Francis DL, Schneider C. Modification of morphine withdrawal by drugs interacting with humoral mechanisms: some contradictions and their interpretations. Nature, 1972; 237 220–223
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18. Griebel G, Saffroy-Spittler M, Misslin R, Remmy D, Vogel E, Bourguignon JJ. Comparison of behavioural effects of an adenosine A1/A2-receptor antagonist, CGS-15943A, and an A1-selective antagonist, DPCPX. Psychopharmacol. 1991; 103 541–544 19. Seale TW, Abla KA, Shamim MT, Carney JM, Daly JW. 3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs. Life Sci. 1988; 43 1671–1684 20. Collier HOJ, Francis DL. Morphine abstinence is associated with increased brain cyclic AMP. Nature 1975; 255 159–162 21. Nestler EJ. Molecular mechanisms of drug addiction. J. Neurosci. 1992; 12 2439–2450 22. van Caulker D, Muller M, Hamprecht BJ. Adenosine regulates two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. Neurochem. 1979; 33 999–1005 23. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol. Rev. 1998; 50 415–475 24. Hoffman BB, Chang H, Dall’Aglio E, Reaven GM. Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes. J. Clin. Invest. 1986; 78 185–190 25. Longabaugh JP, Didsbury J, Spiegel A, Stiles GL. Modification of rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist: alterations in the quantity of Gsa and Gia are not associated with changes in their mRNAs. Mol. Pharmacol. 1989; 36 681–688 26. Bruns RF, Lu GH, Pugsley TA. Characterisation of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol. Pharmacol. 1986; 29 331–346 27. Bruns RF, Fergus JH, Badger EW, Bristol JA, Santay LA, Hartman JD, Hays SJ, Huang CC. Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes NaunynSchmiedeberg’s Arch. Pharmacol. 1987; 335 59–63 28. Kaplan GB, Leite-Morris KA, Sears MT. Alterations of adenosine A1 receptors in morphine dependence. Brain Research 1994; 657 347–350 29. Salem A, Hope W. Role of endogenous adenosine in the expression of opiate withdrawal in rats. Eur. J. Pharmacol. 1999; 39 39–42