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Exploring δ-receptor function using the selective opioid antagonist naltrindole
Neuropharmacologl! Vo1.28,No.12, pp.1427-1430, 1989 Printedin GreatBritain
0028-3908/89 $3.00+0.00 Pergamon Press plc
EXPLORING C-RECEPTOR FUNCTION USING THE SELECTIVE OPIOID ANTAGONIST NALTRINDOLE Helen C. Jackson, Tamzin L. Ripley and D.J. Nutt Reckitt and Colman PsychopharmacologyUnit, School of Medical Sciences, University Walk, Bristol, BS8 lTD, England. [Accepted 19
Octobm 19B91
SUMMARY
Until recently the only pharmacological probes for 6receptors have been peptide enkephalin analogues. These suffer from a number of limitations includinghigh cost, partial agonist effects and a propensity for neurotoxicity.A stable non-peptide antagonist, naltrindole, has recently become available. We have explored its intrinsic actions and found that it attenuated swim stress-induced antinociception,amodel for endogenous C-receptor activation. Naltrindole may therefore be a useful alternative to presently available C-receptor antagonists. Key words: naltrindole, analgesia, stress-induced antinociception, C-receptors For a number of years there has been interest in the activity of endogenous ligands for 6-receptors especially in the area of analgesia. Until recently the only selective antagonists available were vevtides such as ICI 154,129 and ICI 174,864 (Gormley, Morley, hriestley, Shaw, Turnbuil and Wheeler, 1682; Shaw, Miller, Turnbull, Gormley and Morley, 1982; Cotton, Giles, Miller, Shaw and Timms, 1984): These suifered from a number of disadvantages; they were expensive and generally had to be given by the i.c.v. route (except in mice and young rats); their selectivity was questioned after p-agonist actions were reported in some studies; they were shown to act as partial C-agonists; and they had a vrovensitv to neurotoxicitv (Dray and Nunan. 1984: Cohen, Shuman,-Osborne &d Gesellchen, 1988; Lcander, Ges&lchen and Mendelsohn, 1988; Long, Petras and Holaday, 1988). Recently naltrindole, a.stablenon-peptide selective 6-recepto; antagonisf. of high affinity has been synthesized. It was shown to be active in vitro and a very high dose (20 mgfkg s.c.) was reported to antagonize the antinociceptive effects of a b-selective agonist in mice without activity against p- or k- agonists (Portoghese, Sultana and Takemori, 1988). However, there have been no reports of either its intrinsic nociceptive activity or of its actions situations of altered endogenous enkephalin tone. in Consequently we have assessed the effects of naltrindole on tail immersion latencies in rats and then used it to evaluate the potential6-receptor component of stress-inducedantinociception, a phenomenon that has been suggested to be in part mediated through these receptors (Jackson and Kitchen, 1989). METHODS Male Wistar rats (Bantin and Kingman) in the weight range 130-150g (45-50 days old) were used in all experiments. All procedures were carried out between 13.00-17.00h in a quiet, airconditioned laboratory (23f2'C) and animals were acclimatized for two hours to these conditions before use. To explore the effects of naltrindole on antinociceptiongroups of 8 rats were subdivided into 4 groups and injected i.p. with either 0.9% saline or naltrindole (0.3, 1 and 10 mg/kg; synthesized at Reckitt & Colman, Hull) using a blind procedure and a dose volume of Sml/kg. Four groups of eight rats were used so that each 1427
Preliminary Notes
1428
treatment group contained eight animals. Antinociception was after drug assessed, before and at several time points administration, using the tail immersion test, in which rats were hand-held over a hot water bath with the terminal 2-3 cm portion of their tails in water at a temperature of 55OC. This noxious stimulus produced a distinct tail flick response and tail immersion latencies were determined to the nearest 0.1 set using a stop-watch. In the studies of stress-induced antinociception, four groups of eight rats were used again. Two animals in each group were injected i.p. with 0.9% saline and remained in the home cage. The remaining six animals were treated with either saline or naltrindole (0.3 or 1 mg/kg ) using a blind procedure, and ten minutes later placed individually in 25OC water for 3 minutes. The water was contained in buckets 29 cm in diameter and 32 cm high. At the end of the swimming period rats were gently dried and placed together in a clean cage. Nociceptive responses and rectal temperatures (measured with a digital thermometer; Model BAT-12, Sensortek) were determined immediately before injection and at various time intervals after the swimming stress. Doses of naltrindole (0.3 and 1 mg/kg), having no effect on tail immersion latencies, were chosen from the previous study. In all treatment experiments means (n=8; 4 cages) were group statistically compared using analysis of variance and Dunnett's test. RESULTS In the first experiment naltrindole had no overt behavioural effects. Only at the highest dose was there an increase in tail immersion latencies which was significant at two time points In the second experiment swim-stress produced a (Fig. 1.). significant antinociceptive response which emerged within 1 min and was still apparent 15 min later (Fig 2.). Pretreatment with naltrindole attenuated the swim stress-induced antinociception in a dose-related manner. Hence, tail immersion latencies of animals treated with the highest dose of the opioid antagonist were reduced to baseline values at the 10 and 15 min readings, i.e. 23 and 28 min after administration of naltrindole. These findings, taken with the observation that antinociception developed 30 min after administration of a high dose of naltrindole, suggest that it may be more appropriate to a use a slightly longer pretreatment time (e.g 20 instead of 10 min preswim) in future studies. 5z $ -
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2-
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15 Time
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60
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Fig. 1. Tail immersion latencies in the rat following administration of naltrindole. Groups of 8 rats were injected i.p. with either saline (open circles) or naltrindole : 0.3 mg/kg (closed circles); 1 mg/kg (open squares) or 10 mg/kg (closed squares) Significant differences from the saline-treated controls are denoted by ‘P-zO.05.
Preliminary
Notes
1429
654’ 321'
-10
151015
Time
30
45
60
(min)
Fig. 2. Effects of naltrindole on antinociception induced by a 3 min swim in rats. Values represent means for groups of 8 animals, error bars have been omitted from the graph for clarity. Treatment groups were: saline, unstressed (open circles); saline, swim-stressed (closed circles); naltrindole 0.3 mg/kg, swim-stressed (open squares); naltrindole 1 mg/kg. swim stressed (closed squares). Significant differences from unstressed controls are denoted by * PcO.05 and from swim-stressed controls by t P
A 3 min swim also produced marked hypothermia (30.8f0.4 compared with 36.9+0.2'C in control animals) although body temperatures of the stressed animals had normalised within an hour. This hypothermic response was not altered by treatment with naltrindole (data not shown) nor was it accompanied by any signs of behavioural depression. DISCUSSION These results demonstrate that on systemic administration doses of up to 1 mg/kg naltrindole have no antinociceptive However, these doses attenuated stress-induced effects. antinociception in young rats in a dose-dependent manner. This confirms the earlier reports obtained using non-selective antagonists such as naloxone and the peptide antagonist ICI 174,864 (Jackson and Kitchen, 1989). It suggests that a significant component of swim stress-induced antinociception is mediated by the release of endogenous 6-receptor agonists, presumably enkephalins. In contrastpretreatmentwith naltrindole had no effect on the hypothermia produced by this procedure. Since previous studies have demonstrated even high dose naloxone to be ineffective against this variable (Jackson and Kitchen, 1989) it is probably not an opioid-mediated effect.
At the doses used in the present study naltrindole alone had no overt behavioural effects. However, at the high dose of 10 mg/kg it did produce a small increase in tail immersion latency. The reasons for this are unclear but could include some partial agonist activity at opioid receptors or the production of an active metabolite. The fact that a dose of 1 mg/kg attenuated stress-induced increases in b-receptor function but did not alter control responses suggests an absence of endogenous tone at these This supports the view that endogenous opioid receptors. processes subserve phasic ratherthantonic functions in the CNS.
Naltrindole promises to be a valuable new drug with which to explore b-receptor function, and will facilitate our understanding of the circumstances under which these receptors are activated.
1430
Preliminary Notes
REFERENCES Cohen M. L., Shuman R. T., Osborne J. J. and Gesellchen P. D. (1986) Opioid agonist activity of ICI 174864 and its carboxypeptidasedegradation product, LY281217. J. Pharmac. exp. Ther. 238: 769-772. Cotton R., Giles M. G., Miller L., Shaw J. S. and Timms, D.(1984) ICI 174,864: a highly selective antagonist for the opioid d-receptor. Eur. J. Pharmac. 97: 331-332. Dray, A. and Nunan, L. (1984) Selective 6-opioid receptor antagonism by ICI 174,864 in the central nervous system. Peptides 5: 1015-1016. Gormley J. J., Morley J. S., Priestley T., Shaw J. S., Turnbull M. J. and Wheeler, H. (1982) In vivo evaluation of the opiate delta receptor antagonist ICI 154,129. Life Sci. 31: 1263-1266. Jackson Ii. C. and Kitchen I. (1989) Swim-stress-induced antinociception in young rats. Br. J. Pharmac. 96: 617622. Leander J. D., Gesellchen P. D. and Mendelsohn L. G. (1988) antinociceptive Opioid effects of delta-receptor antagonists. Pharmac. Biochem. Behav. 29: 351-355. Lone J. B., Petras J. M. and deficits and neuronal nonopioid actions of the ICI 174864. J. Pharmac.
Holadav, J. W. (1988) Neurolosic injury--in rats. resulting from delta opioid receptor antagonist exp. Ther. 244: 1169-1177.
Portoghese P. S., Sultana M. and Takemori A. E. (1988) Naltrindole, a highly selective and potent non-peptide 6 opioid receptor antagonist. Eur. J. Pharmac. 146: 185186. Shaw J. S., Miller L., Turnbull M. J., Gormley J. J. and Morley J. S. (1982) Selective antagonists at the opiate delta receptor. Life Sci. 31: 1259-1262.
0028-3908/89 $3.00+0.00 Pergamon Press plc
EXPLORING C-RECEPTOR FUNCTION USING THE SELECTIVE OPIOID ANTAGONIST NALTRINDOLE Helen C. Jackson, Tamzin L. Ripley and D.J. Nutt Reckitt and Colman PsychopharmacologyUnit, School of Medical Sciences, University Walk, Bristol, BS8 lTD, England. [Accepted 19
Octobm 19B91
SUMMARY
Until recently the only pharmacological probes for 6receptors have been peptide enkephalin analogues. These suffer from a number of limitations includinghigh cost, partial agonist effects and a propensity for neurotoxicity.A stable non-peptide antagonist, naltrindole, has recently become available. We have explored its intrinsic actions and found that it attenuated swim stress-induced antinociception,amodel for endogenous C-receptor activation. Naltrindole may therefore be a useful alternative to presently available C-receptor antagonists. Key words: naltrindole, analgesia, stress-induced antinociception, C-receptors For a number of years there has been interest in the activity of endogenous ligands for 6-receptors especially in the area of analgesia. Until recently the only selective antagonists available were vevtides such as ICI 154,129 and ICI 174,864 (Gormley, Morley, hriestley, Shaw, Turnbuil and Wheeler, 1682; Shaw, Miller, Turnbull, Gormley and Morley, 1982; Cotton, Giles, Miller, Shaw and Timms, 1984): These suifered from a number of disadvantages; they were expensive and generally had to be given by the i.c.v. route (except in mice and young rats); their selectivity was questioned after p-agonist actions were reported in some studies; they were shown to act as partial C-agonists; and they had a vrovensitv to neurotoxicitv (Dray and Nunan. 1984: Cohen, Shuman,-Osborne &d Gesellchen, 1988; Lcander, Ges&lchen and Mendelsohn, 1988; Long, Petras and Holaday, 1988). Recently naltrindole, a.stablenon-peptide selective 6-recepto; antagonisf. of high affinity has been synthesized. It was shown to be active in vitro and a very high dose (20 mgfkg s.c.) was reported to antagonize the antinociceptive effects of a b-selective agonist in mice without activity against p- or k- agonists (Portoghese, Sultana and Takemori, 1988). However, there have been no reports of either its intrinsic nociceptive activity or of its actions situations of altered endogenous enkephalin tone. in Consequently we have assessed the effects of naltrindole on tail immersion latencies in rats and then used it to evaluate the potential6-receptor component of stress-inducedantinociception, a phenomenon that has been suggested to be in part mediated through these receptors (Jackson and Kitchen, 1989). METHODS Male Wistar rats (Bantin and Kingman) in the weight range 130-150g (45-50 days old) were used in all experiments. All procedures were carried out between 13.00-17.00h in a quiet, airconditioned laboratory (23f2'C) and animals were acclimatized for two hours to these conditions before use. To explore the effects of naltrindole on antinociceptiongroups of 8 rats were subdivided into 4 groups and injected i.p. with either 0.9% saline or naltrindole (0.3, 1 and 10 mg/kg; synthesized at Reckitt & Colman, Hull) using a blind procedure and a dose volume of Sml/kg. Four groups of eight rats were used so that each 1427
Preliminary Notes
1428
treatment group contained eight animals. Antinociception was after drug assessed, before and at several time points administration, using the tail immersion test, in which rats were hand-held over a hot water bath with the terminal 2-3 cm portion of their tails in water at a temperature of 55OC. This noxious stimulus produced a distinct tail flick response and tail immersion latencies were determined to the nearest 0.1 set using a stop-watch. In the studies of stress-induced antinociception, four groups of eight rats were used again. Two animals in each group were injected i.p. with 0.9% saline and remained in the home cage. The remaining six animals were treated with either saline or naltrindole (0.3 or 1 mg/kg ) using a blind procedure, and ten minutes later placed individually in 25OC water for 3 minutes. The water was contained in buckets 29 cm in diameter and 32 cm high. At the end of the swimming period rats were gently dried and placed together in a clean cage. Nociceptive responses and rectal temperatures (measured with a digital thermometer; Model BAT-12, Sensortek) were determined immediately before injection and at various time intervals after the swimming stress. Doses of naltrindole (0.3 and 1 mg/kg), having no effect on tail immersion latencies, were chosen from the previous study. In all treatment experiments means (n=8; 4 cages) were group statistically compared using analysis of variance and Dunnett's test. RESULTS In the first experiment naltrindole had no overt behavioural effects. Only at the highest dose was there an increase in tail immersion latencies which was significant at two time points In the second experiment swim-stress produced a (Fig. 1.). significant antinociceptive response which emerged within 1 min and was still apparent 15 min later (Fig 2.). Pretreatment with naltrindole attenuated the swim stress-induced antinociception in a dose-related manner. Hence, tail immersion latencies of animals treated with the highest dose of the opioid antagonist were reduced to baseline values at the 10 and 15 min readings, i.e. 23 and 28 min after administration of naltrindole. These findings, taken with the observation that antinociception developed 30 min after administration of a high dose of naltrindole, suggest that it may be more appropriate to a use a slightly longer pretreatment time (e.g 20 instead of 10 min preswim) in future studies. 5z $ -
4-
0" s 5 -
3-
: s E 2
2-
a ,
I
0
30
15 Time
45
60
(min)
Fig. 1. Tail immersion latencies in the rat following administration of naltrindole. Groups of 8 rats were injected i.p. with either saline (open circles) or naltrindole : 0.3 mg/kg (closed circles); 1 mg/kg (open squares) or 10 mg/kg (closed squares) Significant differences from the saline-treated controls are denoted by ‘P-zO.05.
Preliminary
Notes
1429
654’ 321'
-10
151015
Time
30
45
60
(min)
Fig. 2. Effects of naltrindole on antinociception induced by a 3 min swim in rats. Values represent means for groups of 8 animals, error bars have been omitted from the graph for clarity. Treatment groups were: saline, unstressed (open circles); saline, swim-stressed (closed circles); naltrindole 0.3 mg/kg, swim-stressed (open squares); naltrindole 1 mg/kg. swim stressed (closed squares). Significant differences from unstressed controls are denoted by * PcO.05 and from swim-stressed controls by t P
A 3 min swim also produced marked hypothermia (30.8f0.4 compared with 36.9+0.2'C in control animals) although body temperatures of the stressed animals had normalised within an hour. This hypothermic response was not altered by treatment with naltrindole (data not shown) nor was it accompanied by any signs of behavioural depression. DISCUSSION These results demonstrate that on systemic administration doses of up to 1 mg/kg naltrindole have no antinociceptive However, these doses attenuated stress-induced effects. antinociception in young rats in a dose-dependent manner. This confirms the earlier reports obtained using non-selective antagonists such as naloxone and the peptide antagonist ICI 174,864 (Jackson and Kitchen, 1989). It suggests that a significant component of swim stress-induced antinociception is mediated by the release of endogenous 6-receptor agonists, presumably enkephalins. In contrastpretreatmentwith naltrindole had no effect on the hypothermia produced by this procedure. Since previous studies have demonstrated even high dose naloxone to be ineffective against this variable (Jackson and Kitchen, 1989) it is probably not an opioid-mediated effect.
At the doses used in the present study naltrindole alone had no overt behavioural effects. However, at the high dose of 10 mg/kg it did produce a small increase in tail immersion latency. The reasons for this are unclear but could include some partial agonist activity at opioid receptors or the production of an active metabolite. The fact that a dose of 1 mg/kg attenuated stress-induced increases in b-receptor function but did not alter control responses suggests an absence of endogenous tone at these This supports the view that endogenous opioid receptors. processes subserve phasic ratherthantonic functions in the CNS.
Naltrindole promises to be a valuable new drug with which to explore b-receptor function, and will facilitate our understanding of the circumstances under which these receptors are activated.
1430
Preliminary Notes
REFERENCES Cohen M. L., Shuman R. T., Osborne J. J. and Gesellchen P. D. (1986) Opioid agonist activity of ICI 174864 and its carboxypeptidasedegradation product, LY281217. J. Pharmac. exp. Ther. 238: 769-772. Cotton R., Giles M. G., Miller L., Shaw J. S. and Timms, D.(1984) ICI 174,864: a highly selective antagonist for the opioid d-receptor. Eur. J. Pharmac. 97: 331-332. Dray, A. and Nunan, L. (1984) Selective 6-opioid receptor antagonism by ICI 174,864 in the central nervous system. Peptides 5: 1015-1016. Gormley J. J., Morley J. S., Priestley T., Shaw J. S., Turnbull M. J. and Wheeler, H. (1982) In vivo evaluation of the opiate delta receptor antagonist ICI 154,129. Life Sci. 31: 1263-1266. Jackson Ii. C. and Kitchen I. (1989) Swim-stress-induced antinociception in young rats. Br. J. Pharmac. 96: 617622. Leander J. D., Gesellchen P. D. and Mendelsohn L. G. (1988) antinociceptive Opioid effects of delta-receptor antagonists. Pharmac. Biochem. Behav. 29: 351-355. Lone J. B., Petras J. M. and deficits and neuronal nonopioid actions of the ICI 174864. J. Pharmac.
Holadav, J. W. (1988) Neurolosic injury--in rats. resulting from delta opioid receptor antagonist exp. Ther. 244: 1169-1177.
Portoghese P. S., Sultana M. and Takemori A. E. (1988) Naltrindole, a highly selective and potent non-peptide 6 opioid receptor antagonist. Eur. J. Pharmac. 146: 185186. Shaw J. S., Miller L., Turnbull M. J., Gormley J. J. and Morley J. S. (1982) Selective antagonists at the opiate delta receptor. Life Sci. 31: 1259-1262.