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The involvement of opioid peptides in stress-induced analgesia in the slug Arion ater
Peptides, Vol. 10, pp. 9-13. ©PergamonPress plc, 1989. Printedin the U.S.A.
0196-9781/89 $3.00 + .00
The Involvement of Opioid Peptides in Stress-Induced Analgesia in the Slug A r i o n ater L. M. D A L T O N A N D P. S. W I D D O W S O N 1
Anglo-European College o f Chiropractic, 13-15 P a r k w o o d R o a d Boscombe, Bournemouth, Dorset, B H 5 2DF, U.K. Received 3 J u n e 1988 DALTON, L. M. AND P. S. WIDDOWSON. The involvement of opioid peptides in stress-induced analgesia in the slug Arion ater. PEPTIDES 10(1) 9-13, 1989.--Application of tail-pinch stress to the terrestrial slug, Arion ater, produced a significant increase in the response time when tested on the hot-plate for foot-lifting response. The analgesia was completely reversed by injections of the opiate antagonists, naltrexone and ICI 174864, in a dose-dependent manner. Analgesia could also be elicited by the injection into the foot of 3-endorphin and the enkephalin analogues, DAGO and DADLE. No effect was seen with dynorphin A (1-8) or dynorphin A (1-17). The stress-induced analgesia disappeared after 30 minutes but could be maintained for 100 min following the injection of a mixture of bestatin and the enkephalinase inhibitor, N-carboxymethyl-L-phenylalanyl-L-leucine. This work suggests that in the slug, a physical stressor produces an analgesia which may be due to the release of endogenous opiates. Terrestrial slug
Stress-induced analgesia
Endogenous opiates
THE study of invertebrate systems has enabled neuroscientists to investigate simple nervous pathways in great detail. Investigations using molluscs such as Aplesia and Hermissendra, both marine gastropods, have revealed valuable information about neural circuits involved in a variety of simple behaviors. There is growing evidence to suggest that in invertebrate species, opiate systems are closely involved in the regulation of behavior and fundamental physiological processes (16, 18, 21). In molluscs, there is biochemical and behavioral evidence for an active opiate system which may serve to modulate other neurochemical systems in a manner similar to that seen in vertebrate species (25, 26, 31, 38). Two classes of opiate binding sites have been found in the pedal ganglia of the marine mollusc, Mytilus edulis (23,41). In addition, immunoreactive 3-endorphin has been found in the earthworm, Lumbricus terrestris (1) and Met- and Leu-enkephalin-like material has been reported in the ganglia of Lymnea stagnalis (25) and in Mytilus pedal ganglia (26). The presence of enkephalin degrading enzymes in some invertebrates is further evidence that endogenous opiates may be important in their physiology (6). Exogenous administration of opiates has been shown to alter dopamine levels in Mytilus (37, 40, 41), the land snail Helix pomatia and the freshwater mussel, Anodonta cygnea (38). Morphine has been demonstrated to modulate the acetylcholine-induced contracture of Mytilus anterior byssus rector muscle (3). Opiates have been implicated in behavioral thermoregulation in the terrestrial snail, Cepaea nemoralis (16) and in states of rigidity and immobility in Helix pomatia (39). Levels
of feeding in the slug Limax maximus have been reported to increase following administration of the opiate agonists, morphine, ketocyclozocine and U-50,488H, which could be antagonised by naloxone (18,19). This indicates that #- and K-opiate receptors may be involved in feeding in the slug, however the tS-opiate receptor antagonist, ICI 154,129, antagonised the morphine-induced increase in feeding (19), suggesting that morphine may be acting through tS- rather than/z-type receptors. It has been well documented that in vertebrate experimental animals, various physical stressful procedures such as footshock (15), immobilisation (2,15) and forced swimming (4,32) can induce analgesic effects. These changes in analgesia have been shown to be divided into two types, opioid- and nonopioid-mediated effects depending on their cross-tolerance with morphine or reversal with naloxone (13, 15, 27, 28). As in vertebrate systems, stress-induced analgesia in Cepaea has been divided into opiate and nonopiate components (22). Warming the snails resulted in an increased thermal nociceptive threshold which could be reversed by naloxone, the tS-opiate receptor antagonist, ICI 154,129 and with pretreatment with the irreversible opiate antagonist 3-funaltrexamine. In contrast, coldstress was unaffected by any of the opiate antagonists (22). It is apparent that agonists and antagonists to the/z-, tS- and r-opiate systems can affect invertebrate behavior. This paper set out to investigate the opiate-induced analgesic response to the application of a physical stressor in the terrestrial slug. Also, the experiments were designed to determine which opiate receptor subtype mediates the analgesic response and whether
~Requests for reprints should be addressed to Peter S. Widdowson, Ph.D., Department of Psychiatry, Case Western Reserve University, Cleveland, OH 44109.
10
DALTON AND WIDDOWSON
changes seen in the vertebrate were similar to those reported in vertebrates. METHOD
Animals Garden black slugs, Arion ater (5-20 g), were collected locally and housed in a glass tank (30x40x50 cm) at constant temperature (19-22°C). The atmosphere was maintained at 8090°7o humidity and the animals subjected to a normal photoperiod (14 hour light followed by 10 hours dark). They were allowed free access to water and lettuce leaves.
Stress Treatment and Injections The animals were randomised into treatment groups according to their weights and injected with 25 #I of saline, peptide or drugs intramuscular into the foot with a microsyringe. Animals receiving two treatments were first injected with the antagonist followed 1 min later by the injection of the agonist. One minute following the injection, the slugs were stressed by gently pinching the tail for a 2 min period with a pair of blunt forceps. They were then hydrated for 10 min in separate glass sealed containers (10 cm diameter) in which the humidity approached 95%. This is because the response time on the hotplate for terrestrial snails has been shown to be dependent on the levels of hydration (35). Thirteen minutes after the injection, the slugs were transferred to a hot plate (40+0.5°C) and the time recorded for the animals to exhibit a stereotypic lifting of the anterior portion of the extended foot. The footlifting response was not observed in animals subjected to ambient temperatures. The time from placing the animals on the warm surface to lifting their foot was recorded as the latency of response. The animals were then returned to the glass tank following the experiment. The animals received no more than 3 injections each.
secretion was not observed in unstressed animals or in those receiving saline, peptide or drug administration, also suggesting that the injection procedure was not in itself stressful. The opiate antagonists, naltrexone (1 mg/kg) and ICI 174864 (1 mg/kg) (9) were both able to significantly antagonise the stress-induced increase in the response time. The injection of either opiate antagonist alone without the application of stress did not alter the response (Fig. 1). Both naltrexone and the ICI 174864 were able to reverse the stress-induced changes in a dose-dependent manner, the naltrexone being approximately five times more potent (Fig. 2).
Time Course of Analgesia To investigate how long the analgesia persisted following the stressor application, the slugs were placed on the hot-plate after varying times. There was a return to control levels after 30 min and they remained at this level for up to 100 min (Fig. 3). Injection of a mixture of the enzyme inhibitor, bestatin (2 mg/kg), and the specific inhibitor of enkephalinase, N-carboxymethylL-phenylanalyl-L-leucine(2 mg/kg), l0 min before the stressor
No-stress 35
t
3O
2S
20 t
tS
Drugs and Peptides All the drugs and peptides were diluted in sterile 0.9% saline solution and injected in a total volume of 25 tA. The naltrexone, [D-AlaZ,D-Leu5] enkephalin (DADLE), dynorphin A (117), dynorphin A (1-8), N-carboxymethyl-L-phenylalanyl-Lleucine and bestatin HC1 were obtained from Sigma Chemicals, Poole, Dorset, U.K. The/3-endorphin, [D-Ala2,NMePhe4,Glyol 5] enkephalin (DAGO) and (N,N-diallyl-Tyr-Aib-Aib-PheLeu-OH) ICI 174864 were obtained from Cambridge Research Biochemicals Ltd., Cambridge, U.K.
Stress
f
A (n) (7)
B (6)
C (7)
D {6)
(7)
C ~7)
D (7)
FIG. 1. The latency of response on the hot-plate before foot-lifting with and without stress exposure. A=control, B=25 #1 saline injection, C=naltrexone (1 mg/kg), D=ICI 174864 (1 mg/kg). **p<0.01 compared with controls, tp<0.05, ~tp<0.01 compared with saline-injected stressed animals.
-
Statistical Analyses
tS)
z.O (7l
All results were expressed as mean+SEM for groups of between 6 and 7 animals. Statistical differences between groups of differently treated animals were assessed with the Wilcoxon rank sums test.
30
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RESULTS
Effects of Stress
16}
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The delay for the foot-lifting in response to the noxious heat stimulus was about 8-10 seconds and was not affected by the saline injection. When the animals were stressed, the latency in the response rose to around 30 sec, which again showed no change upon injection of the 25 #l saline. It was noticed that there was a stress-related increase in mucus secretion during and after the tail-pinching procedure. This increased mucus
{61 S
SaLine 0,01
0,05 0.1 [Na[trexone ] mg/Kg
1,0
Saline 0.01
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0,5 1,0
2,5
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FIG. 2. Dose-response curves for the ability of naltrexone and ICI 174864 to inhibit the stress-induced analgesia on the hot-plate test. *p<0.05, **p<0.01 compared with saline controls.
STRESS-INDUCED A N A L G E S I A IN SLUGS
11
was able to maintain a degree of stress-induced analgesia for up to 100 min (Fig. 3).
Administration of Opiate Agonists Agonists directed towards the ~t-, 6- and r-opiate receptors were injected into the slugs to determine which opiate receptors were involved in the response to stress. The injection of the specific/~-opiate receptor agonist, DAGO (0.1-2.5 mg/kg), produced a significant, dose-dependent increase in the response time as did injections of ~-endorphin (0.1-2.5 mg/kg) (Fig. 4A and B). The ~/-endorphin response (1 mg/kg) could be prevented by the pretreatment with naltrexone (1 mg/kg), showing that the peptide was acting at opiate receptors. In addition, the t%opiate receptor agonist, DADLE (14) also produced a dose-dependent, significant increase in the response time, which could be prevented by the pretreatment with ICI 174864. Injections of the r-opiate agonists, dynorphin A (1-17) (2.5 mg/kg) (5) or dynorphin A (1-8) (2.5 mg/kg (7,42) failed to alter the response time for foot-lifting (Fig. 5B).
These findings agree well with the results obtained in the slug Limax maximus (20) and in Cepaea nemoralis (22). In Cepaea, both/~- and &opiate antagonists were able to reduce the warm stress-induced analgesia but were ineffective against cold-induced stress analgesia (22). This suggests that both mechanical, i.e., tail-pinch, and warm-induced stress are both opiate dependent whilst cold-induced analgesia is independent of endogenous opiate influences. Reports in vertebrate species have shown that physical stressors, such as immobilisation and forced swimming, can produce opiate-dependent analgesia (15,30) whilst cold exposure stress is independent of the opiate system
(15,3o). The time course for the stress-induced analgesia was rapid, the latency for response returning to control values after 30
!
~.Or
~o 35
DISCUSSION
The application of the tail-pinch to the slugs was able to increase up to three-fold the latency of response to foot-lifting when placed on the hot-plate. The technique of tail-pinch was observed to be an adequate method of stressing the animals since mucus secretion was markably enhanced during and following the procedure. Mucus production is associated with skin irritation and serves to reduce tissue damage. Injection of saline alone was not associated with either increased mucus secretion or a significant change of the latency of response on the hotplate, showing that injections alone were not stressful. The ability of both naltrexone and ICI 174864 to completely reverse the analgesia in a dose-dependent manner suggests that application of the stressor induces the release of endogenous opiates which produces analgesia. The naltrexone was slightly more potent in its ability to reverse the stress-induced analgesia, suggesting that #-opioid receptor activity might predominate in the analgesic response. However, ICI 174864, which has shown to a specific 6-opioid receptor antagonist (9), indicates that there must be some involvement with 6-receptors. Both receptor antagonists, injected alone, failed to change the response time, showing that they do not act as partial agonists at their respective receptors.
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3o
E ZO
20 15
10 5
A
¢
B
A
B
E
E
F
FIG. 4. (A) Dose-response of the analgesic effect of injections of DAGO. A=saline control, B=DAGO (0.1 mg/kg), C=DAGO (0.5 mg/kg), D=DAGO (1 mg/kg), E=DAGO (2.5 mg/kg). **p<0.01, n=6. (B) Dose-response of the analgesic effect of ~-endorphin injections. A=saline control, B=~-endorphin (0.1 mg/kg), C=~-endorphin (0.5 mg/kg), D=/3-endorphin (1 mg/kg), E=/3-endorphin (2.5 mg/kg), F=~/-endorphin (l mg/kg) + naltrexone (1 mg/kg). *p<0.05, **p<0.01, n=6.
6
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(min.)
FIG. 3. Time course of the analgesic response following exposure to the tail-pinch stressor. (o)=control, (e)=following the injection of peptidase inhibitors, bestatin (2 mg/kg) and N-carboxymethyl-L phenylalanyl L-leucine (2 mg/kg) in 25 ~l saline.
B
C
F
A
B
c
FIG. 5. (A) Dose-response of the analgesic effect of DADLE injections. A=saline control, B=DADLE (0.1 mg/kg), C=DADLE (0.5 mg/kg), D=DADLE (1 mg/kg), E=DADLE (2.5 mg/kg), F=DADLE (2.5 mg/kg) + ICI 174864 (2.5 mg/kg). *o<0.05, **p<0.01, n=6. (B) Effect of dynorphin A injections on the analgesic response on the hotplate test. A=saline control, B=dynorphin A (1-17) (2.5 mg/kg), C=dynorphin A (1-8) (2.5 mg/kg), n=6.
12
DALTON AND WIDDOWSON
min. This suggests that the application of a stressor causes the release of endogenous opiates for only a few minutes and that the peptides must be then rapidly removed by degradative peptidases which have been shown to be present in invertebrates (6). Inhibition of peptidase activity, however, with a mixture of bestatin and the specific enkephalinase inhibitor, N-carboxyL-phenylalanyl-L-leucine (12) was able to maintain analgesia for up to 100 min. The persistent effect, long after the stressor is removed, indicates that the rapid degradation of the opiate peptides is primarily responsible for the cessation o f the analgesia coupled with a reduction in their release. Injection of #-opioid receptor agonists, D A G O and /3endorphin (33) produced a significant dose-dependent increase in the latency of response. This is evidence for a/~-opioid receptor c o m p o n e n t to the analgesic response and agrees with the finding that morphine, also acting on #-opiate receptors, produces analgesia. In addition, the administration of the b-opioid receptor agonist, D A D L E (8,14), also significantly increased the latency of response in a dose-dependent manner. Neither dynorphin A (1-8), nor dynorphin A (1-17), which have been reported to act on the K-opioid receptors (7,17), were able to significantly alter the response time. This indicates that perhaps K-opioid systems are not involved in analgesia in the invertebrate system. However, kappa opiate agonists have been reported to alter feeding in Limax maximus (19) showing that there may be an active K-mechanism underlying some behavior, but not stress-induced analgesia. Alternatively, there may be species differences between invertebrates as to the role of Kopiate systems in stress-induced behaviors. The ability of both naltrexone and ICI 174864 to reverse the stress-induced analgesia suggests that two classes of endogenous opiates are involved in the response. One class interacts with a
#-type receptor and the other with the b-receptor. Receptor binding work in the marine mollusc, Mytilus edulis, showed two classes of opiate receptors (23), but were suggested to be pre- and postsynaptic sites or perhaps interaction with an enzymatic site rather than the/x- and b-opiate receptors described in vertebrate preparations. It is quite possible that only one opiate receptor exists in slugs which exhibits binding characteristics of both/z- and b-type opiate receptors. This early evolutionary receptor may have then developed into the various categories of opiate binding sites characterised in m a m m a l i a n species (36). The structure of the few receptors which have been successfully sequenced, such as the muscarinic (24), substance K (29), adrenergic receptors (10) and the rhodopsin molecule (11), are remarkably similar in their protein structure and conformation across the membrane. This suggests that at some stage, all of these receptors, and possibly the family of opiate sites, may have evolved from one ancestor. This idea is further strengthened by the difficulty of molecular biologists to isolate the /~-opiate receptor type from the b-type (34,43). Even the binding characteristics between #- and b-types are similar; agonists and antagonists often show activity at both sites but at different potencies (43). It is possible, then, that the endogenous t3-endorphin and enkephalin like-immunoreactive material reported in Lumbricus terrestris (1), Mytilus edulis (26) and Lymnea stagnalis (25) all bind to one class of opiate receptor which may be post- or presynaptic. ACKNOWLEDGEMENTS The authors would like to acknowledge the assistance of Mr. G. Watson for help in preparing the illustrations and Mr. M. Marler for help with the statistical analysis.
REFERENCES
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0196-9781/89 $3.00 + .00
The Involvement of Opioid Peptides in Stress-Induced Analgesia in the Slug A r i o n ater L. M. D A L T O N A N D P. S. W I D D O W S O N 1
Anglo-European College o f Chiropractic, 13-15 P a r k w o o d R o a d Boscombe, Bournemouth, Dorset, B H 5 2DF, U.K. Received 3 J u n e 1988 DALTON, L. M. AND P. S. WIDDOWSON. The involvement of opioid peptides in stress-induced analgesia in the slug Arion ater. PEPTIDES 10(1) 9-13, 1989.--Application of tail-pinch stress to the terrestrial slug, Arion ater, produced a significant increase in the response time when tested on the hot-plate for foot-lifting response. The analgesia was completely reversed by injections of the opiate antagonists, naltrexone and ICI 174864, in a dose-dependent manner. Analgesia could also be elicited by the injection into the foot of 3-endorphin and the enkephalin analogues, DAGO and DADLE. No effect was seen with dynorphin A (1-8) or dynorphin A (1-17). The stress-induced analgesia disappeared after 30 minutes but could be maintained for 100 min following the injection of a mixture of bestatin and the enkephalinase inhibitor, N-carboxymethyl-L-phenylalanyl-L-leucine. This work suggests that in the slug, a physical stressor produces an analgesia which may be due to the release of endogenous opiates. Terrestrial slug
Stress-induced analgesia
Endogenous opiates
THE study of invertebrate systems has enabled neuroscientists to investigate simple nervous pathways in great detail. Investigations using molluscs such as Aplesia and Hermissendra, both marine gastropods, have revealed valuable information about neural circuits involved in a variety of simple behaviors. There is growing evidence to suggest that in invertebrate species, opiate systems are closely involved in the regulation of behavior and fundamental physiological processes (16, 18, 21). In molluscs, there is biochemical and behavioral evidence for an active opiate system which may serve to modulate other neurochemical systems in a manner similar to that seen in vertebrate species (25, 26, 31, 38). Two classes of opiate binding sites have been found in the pedal ganglia of the marine mollusc, Mytilus edulis (23,41). In addition, immunoreactive 3-endorphin has been found in the earthworm, Lumbricus terrestris (1) and Met- and Leu-enkephalin-like material has been reported in the ganglia of Lymnea stagnalis (25) and in Mytilus pedal ganglia (26). The presence of enkephalin degrading enzymes in some invertebrates is further evidence that endogenous opiates may be important in their physiology (6). Exogenous administration of opiates has been shown to alter dopamine levels in Mytilus (37, 40, 41), the land snail Helix pomatia and the freshwater mussel, Anodonta cygnea (38). Morphine has been demonstrated to modulate the acetylcholine-induced contracture of Mytilus anterior byssus rector muscle (3). Opiates have been implicated in behavioral thermoregulation in the terrestrial snail, Cepaea nemoralis (16) and in states of rigidity and immobility in Helix pomatia (39). Levels
of feeding in the slug Limax maximus have been reported to increase following administration of the opiate agonists, morphine, ketocyclozocine and U-50,488H, which could be antagonised by naloxone (18,19). This indicates that #- and K-opiate receptors may be involved in feeding in the slug, however the tS-opiate receptor antagonist, ICI 154,129, antagonised the morphine-induced increase in feeding (19), suggesting that morphine may be acting through tS- rather than/z-type receptors. It has been well documented that in vertebrate experimental animals, various physical stressful procedures such as footshock (15), immobilisation (2,15) and forced swimming (4,32) can induce analgesic effects. These changes in analgesia have been shown to be divided into two types, opioid- and nonopioid-mediated effects depending on their cross-tolerance with morphine or reversal with naloxone (13, 15, 27, 28). As in vertebrate systems, stress-induced analgesia in Cepaea has been divided into opiate and nonopiate components (22). Warming the snails resulted in an increased thermal nociceptive threshold which could be reversed by naloxone, the tS-opiate receptor antagonist, ICI 154,129 and with pretreatment with the irreversible opiate antagonist 3-funaltrexamine. In contrast, coldstress was unaffected by any of the opiate antagonists (22). It is apparent that agonists and antagonists to the/z-, tS- and r-opiate systems can affect invertebrate behavior. This paper set out to investigate the opiate-induced analgesic response to the application of a physical stressor in the terrestrial slug. Also, the experiments were designed to determine which opiate receptor subtype mediates the analgesic response and whether
~Requests for reprints should be addressed to Peter S. Widdowson, Ph.D., Department of Psychiatry, Case Western Reserve University, Cleveland, OH 44109.
10
DALTON AND WIDDOWSON
changes seen in the vertebrate were similar to those reported in vertebrates. METHOD
Animals Garden black slugs, Arion ater (5-20 g), were collected locally and housed in a glass tank (30x40x50 cm) at constant temperature (19-22°C). The atmosphere was maintained at 8090°7o humidity and the animals subjected to a normal photoperiod (14 hour light followed by 10 hours dark). They were allowed free access to water and lettuce leaves.
Stress Treatment and Injections The animals were randomised into treatment groups according to their weights and injected with 25 #I of saline, peptide or drugs intramuscular into the foot with a microsyringe. Animals receiving two treatments were first injected with the antagonist followed 1 min later by the injection of the agonist. One minute following the injection, the slugs were stressed by gently pinching the tail for a 2 min period with a pair of blunt forceps. They were then hydrated for 10 min in separate glass sealed containers (10 cm diameter) in which the humidity approached 95%. This is because the response time on the hotplate for terrestrial snails has been shown to be dependent on the levels of hydration (35). Thirteen minutes after the injection, the slugs were transferred to a hot plate (40+0.5°C) and the time recorded for the animals to exhibit a stereotypic lifting of the anterior portion of the extended foot. The footlifting response was not observed in animals subjected to ambient temperatures. The time from placing the animals on the warm surface to lifting their foot was recorded as the latency of response. The animals were then returned to the glass tank following the experiment. The animals received no more than 3 injections each.
secretion was not observed in unstressed animals or in those receiving saline, peptide or drug administration, also suggesting that the injection procedure was not in itself stressful. The opiate antagonists, naltrexone (1 mg/kg) and ICI 174864 (1 mg/kg) (9) were both able to significantly antagonise the stress-induced increase in the response time. The injection of either opiate antagonist alone without the application of stress did not alter the response (Fig. 1). Both naltrexone and the ICI 174864 were able to reverse the stress-induced changes in a dose-dependent manner, the naltrexone being approximately five times more potent (Fig. 2).
Time Course of Analgesia To investigate how long the analgesia persisted following the stressor application, the slugs were placed on the hot-plate after varying times. There was a return to control levels after 30 min and they remained at this level for up to 100 min (Fig. 3). Injection of a mixture of the enzyme inhibitor, bestatin (2 mg/kg), and the specific inhibitor of enkephalinase, N-carboxymethylL-phenylanalyl-L-leucine(2 mg/kg), l0 min before the stressor
No-stress 35
t
3O
2S
20 t
tS
Drugs and Peptides All the drugs and peptides were diluted in sterile 0.9% saline solution and injected in a total volume of 25 tA. The naltrexone, [D-AlaZ,D-Leu5] enkephalin (DADLE), dynorphin A (117), dynorphin A (1-8), N-carboxymethyl-L-phenylalanyl-Lleucine and bestatin HC1 were obtained from Sigma Chemicals, Poole, Dorset, U.K. The/3-endorphin, [D-Ala2,NMePhe4,Glyol 5] enkephalin (DAGO) and (N,N-diallyl-Tyr-Aib-Aib-PheLeu-OH) ICI 174864 were obtained from Cambridge Research Biochemicals Ltd., Cambridge, U.K.
Stress
f
A (n) (7)
B (6)
C (7)
D {6)
(7)
C ~7)
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FIG. 1. The latency of response on the hot-plate before foot-lifting with and without stress exposure. A=control, B=25 #1 saline injection, C=naltrexone (1 mg/kg), D=ICI 174864 (1 mg/kg). **p<0.01 compared with controls, tp<0.05, ~tp<0.01 compared with saline-injected stressed animals.
-
Statistical Analyses
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All results were expressed as mean+SEM for groups of between 6 and 7 animals. Statistical differences between groups of differently treated animals were assessed with the Wilcoxon rank sums test.
30
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RESULTS
Effects of Stress
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The delay for the foot-lifting in response to the noxious heat stimulus was about 8-10 seconds and was not affected by the saline injection. When the animals were stressed, the latency in the response rose to around 30 sec, which again showed no change upon injection of the 25 #l saline. It was noticed that there was a stress-related increase in mucus secretion during and after the tail-pinching procedure. This increased mucus
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0,05 0.1 [Na[trexone ] mg/Kg
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FIG. 2. Dose-response curves for the ability of naltrexone and ICI 174864 to inhibit the stress-induced analgesia on the hot-plate test. *p<0.05, **p<0.01 compared with saline controls.
STRESS-INDUCED A N A L G E S I A IN SLUGS
11
was able to maintain a degree of stress-induced analgesia for up to 100 min (Fig. 3).
Administration of Opiate Agonists Agonists directed towards the ~t-, 6- and r-opiate receptors were injected into the slugs to determine which opiate receptors were involved in the response to stress. The injection of the specific/~-opiate receptor agonist, DAGO (0.1-2.5 mg/kg), produced a significant, dose-dependent increase in the response time as did injections of ~-endorphin (0.1-2.5 mg/kg) (Fig. 4A and B). The ~/-endorphin response (1 mg/kg) could be prevented by the pretreatment with naltrexone (1 mg/kg), showing that the peptide was acting at opiate receptors. In addition, the t%opiate receptor agonist, DADLE (14) also produced a dose-dependent, significant increase in the response time, which could be prevented by the pretreatment with ICI 174864. Injections of the r-opiate agonists, dynorphin A (1-17) (2.5 mg/kg) (5) or dynorphin A (1-8) (2.5 mg/kg (7,42) failed to alter the response time for foot-lifting (Fig. 5B).
These findings agree well with the results obtained in the slug Limax maximus (20) and in Cepaea nemoralis (22). In Cepaea, both/~- and &opiate antagonists were able to reduce the warm stress-induced analgesia but were ineffective against cold-induced stress analgesia (22). This suggests that both mechanical, i.e., tail-pinch, and warm-induced stress are both opiate dependent whilst cold-induced analgesia is independent of endogenous opiate influences. Reports in vertebrate species have shown that physical stressors, such as immobilisation and forced swimming, can produce opiate-dependent analgesia (15,30) whilst cold exposure stress is independent of the opiate system
(15,3o). The time course for the stress-induced analgesia was rapid, the latency for response returning to control values after 30
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DISCUSSION
The application of the tail-pinch to the slugs was able to increase up to three-fold the latency of response to foot-lifting when placed on the hot-plate. The technique of tail-pinch was observed to be an adequate method of stressing the animals since mucus secretion was markably enhanced during and following the procedure. Mucus production is associated with skin irritation and serves to reduce tissue damage. Injection of saline alone was not associated with either increased mucus secretion or a significant change of the latency of response on the hotplate, showing that injections alone were not stressful. The ability of both naltrexone and ICI 174864 to completely reverse the analgesia in a dose-dependent manner suggests that application of the stressor induces the release of endogenous opiates which produces analgesia. The naltrexone was slightly more potent in its ability to reverse the stress-induced analgesia, suggesting that #-opioid receptor activity might predominate in the analgesic response. However, ICI 174864, which has shown to a specific 6-opioid receptor antagonist (9), indicates that there must be some involvement with 6-receptors. Both receptor antagonists, injected alone, failed to change the response time, showing that they do not act as partial agonists at their respective receptors.
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20 15
10 5
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FIG. 4. (A) Dose-response of the analgesic effect of injections of DAGO. A=saline control, B=DAGO (0.1 mg/kg), C=DAGO (0.5 mg/kg), D=DAGO (1 mg/kg), E=DAGO (2.5 mg/kg). **p<0.01, n=6. (B) Dose-response of the analgesic effect of ~-endorphin injections. A=saline control, B=~-endorphin (0.1 mg/kg), C=~-endorphin (0.5 mg/kg), D=/3-endorphin (1 mg/kg), E=/3-endorphin (2.5 mg/kg), F=~/-endorphin (l mg/kg) + naltrexone (1 mg/kg). *p<0.05, **p<0.01, n=6.
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FIG. 3. Time course of the analgesic response following exposure to the tail-pinch stressor. (o)=control, (e)=following the injection of peptidase inhibitors, bestatin (2 mg/kg) and N-carboxymethyl-L phenylalanyl L-leucine (2 mg/kg) in 25 ~l saline.
B
C
F
A
B
c
FIG. 5. (A) Dose-response of the analgesic effect of DADLE injections. A=saline control, B=DADLE (0.1 mg/kg), C=DADLE (0.5 mg/kg), D=DADLE (1 mg/kg), E=DADLE (2.5 mg/kg), F=DADLE (2.5 mg/kg) + ICI 174864 (2.5 mg/kg). *o<0.05, **p<0.01, n=6. (B) Effect of dynorphin A injections on the analgesic response on the hotplate test. A=saline control, B=dynorphin A (1-17) (2.5 mg/kg), C=dynorphin A (1-8) (2.5 mg/kg), n=6.
12
DALTON AND WIDDOWSON
min. This suggests that the application of a stressor causes the release of endogenous opiates for only a few minutes and that the peptides must be then rapidly removed by degradative peptidases which have been shown to be present in invertebrates (6). Inhibition of peptidase activity, however, with a mixture of bestatin and the specific enkephalinase inhibitor, N-carboxyL-phenylalanyl-L-leucine (12) was able to maintain analgesia for up to 100 min. The persistent effect, long after the stressor is removed, indicates that the rapid degradation of the opiate peptides is primarily responsible for the cessation o f the analgesia coupled with a reduction in their release. Injection of #-opioid receptor agonists, D A G O and /3endorphin (33) produced a significant dose-dependent increase in the latency of response. This is evidence for a/~-opioid receptor c o m p o n e n t to the analgesic response and agrees with the finding that morphine, also acting on #-opiate receptors, produces analgesia. In addition, the administration of the b-opioid receptor agonist, D A D L E (8,14), also significantly increased the latency of response in a dose-dependent manner. Neither dynorphin A (1-8), nor dynorphin A (1-17), which have been reported to act on the K-opioid receptors (7,17), were able to significantly alter the response time. This indicates that perhaps K-opioid systems are not involved in analgesia in the invertebrate system. However, kappa opiate agonists have been reported to alter feeding in Limax maximus (19) showing that there may be an active K-mechanism underlying some behavior, but not stress-induced analgesia. Alternatively, there may be species differences between invertebrates as to the role of Kopiate systems in stress-induced behaviors. The ability of both naltrexone and ICI 174864 to reverse the stress-induced analgesia suggests that two classes of endogenous opiates are involved in the response. One class interacts with a
#-type receptor and the other with the b-receptor. Receptor binding work in the marine mollusc, Mytilus edulis, showed two classes of opiate receptors (23), but were suggested to be pre- and postsynaptic sites or perhaps interaction with an enzymatic site rather than the/x- and b-opiate receptors described in vertebrate preparations. It is quite possible that only one opiate receptor exists in slugs which exhibits binding characteristics of both/z- and b-type opiate receptors. This early evolutionary receptor may have then developed into the various categories of opiate binding sites characterised in m a m m a l i a n species (36). The structure of the few receptors which have been successfully sequenced, such as the muscarinic (24), substance K (29), adrenergic receptors (10) and the rhodopsin molecule (11), are remarkably similar in their protein structure and conformation across the membrane. This suggests that at some stage, all of these receptors, and possibly the family of opiate sites, may have evolved from one ancestor. This idea is further strengthened by the difficulty of molecular biologists to isolate the /~-opiate receptor type from the b-type (34,43). Even the binding characteristics between #- and b-types are similar; agonists and antagonists often show activity at both sites but at different potencies (43). It is possible, then, that the endogenous t3-endorphin and enkephalin like-immunoreactive material reported in Lumbricus terrestris (1), Mytilus edulis (26) and Lymnea stagnalis (25) all bind to one class of opiate receptor which may be post- or presynaptic. ACKNOWLEDGEMENTS The authors would like to acknowledge the assistance of Mr. G. Watson for help in preparing the illustrations and Mr. M. Marler for help with the statistical analysis.
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S T R E S S - I N D U C E D A N A L G E S I A IN S L U G S
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