Peptides,Vol. 13, pp. 1123-1126, 1992
0196-9781/92$5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
Spinal Antinociceptive Effects of [D-Ala2]Deltorphin II, a Novel and Highly Selective Delta-Opioid Receptor Agonist G. I M P R O T A 1 A N D M. B R O C C A R D O Institute o f Pharmacology III, University "La Sapienza, "" P. le A. Moro 5-00185 Rome, Italy Received 28 F e b r u a r y 1992 IMPROTA, G. AND M. BROCCARDO. Spinal antinociceptive effects of[D-Ala2]deltorphin II, a novel and highly selective deltaopioid receptor agonist. PEPTIDES 13(6) 1123-1126, 1992.--Pharmacological assays in isolated tissues and binding tests have recently shown that two peptides, with the sequence Tyr-D-Ala-Phe-Asp-(orGIu)-VaI-VaI-GIy-NH2,isolated from skin extracts of Phyllomedusa bicolor and named [D-Ala2]deltorphinI and II, respectively, possess a higher affinity and selectivityfor deltaopioid receptors than any other known natural compound. Since much evidence supports the role of spinal delta-opioid sites in producing antinociceptive effects, we investigatedwhether analgesia might be detected by direct spinal cord administration of [DAla2]deltorphin II (DADELT II) in the rat. The thermal antinociceptive effects of intrathecal DADELT II and dermorphin, a potent mu-selective agonist, were compared at different postinjection times by means of the tail-flick test. The DADELT II produced a dose-related inhibition of the tail-flick response, which lasted 10-60 min depending on the dose and appeared to be of shorter duration than the analgesia produced in rats after intrathecai injection of dermorphin (20-120 rain). The analgesic effect of infused or injected DADELT II was completely abolished by naltrindole, the highly selective delta antagonist. These results confirm the involvement of delta receptors in spinal analgesicactivity in the rat. Deltorphin
Delta receptor stimulation
Spinal analgesia
ISOLATED from skin extracts of frogs belonging to the genus
Phyllomedusa, deltorphins are linear heptapeptides that have a higher affinity and selectivity for delta-opioid binding sites than any other natural compound known (11). Recently, two deltorphins, one with the sequence Tyr-t>-Ala-Phe-Asp-Val-Val-GlyNH2 ([D-Ala2]deltorphin I) and the other with the sequence TyrD-AIa-Phe-GIu-Val-VaI-Gly-NH2 ([D-Ala2]deltorphin II), have been isolated from skin extracts of Phyllomedusa bicolor (7). Pharmacological assays in isolated tissues and binding assays have shown that these peptides possess a higher affinity for delta receptors than the previously characterized deltorphin, which contains D-Met as the second amino acid. These new deltorphins contain the N-terminal sequence Tyr-D-Ala-Phe. This sequence is present in another constituent of Phyllomedusa skin, dermorphin (1), which is highly selective for mu-opioid receptors. Pharmacological evidence has been accumulated strongly implicating both mu- and delta-receptor agonists in spinal antinociception in the rat (6,15,17,18). The superficial dorsal horn of the spinal cord is rich in mu receptors. Although less abundant, delta sites are present in the substantia gelatinosa of the spinal cord (9,16). Processing of nociceptive transmission in the dorsal horn of the spinal cord can be modulated by the opiate receptor system. Yet the role of different subtypes of opioid receptors remains controversial, principally owing to the lack of highly Requests for reprints should to be addressed to Prof. G. Improta.
1123
selective agonists and antagonists. Because the recently available peptide [D-Ala2]deltorphin II (DADELT II) is the most selective delta agonist known to date, it should help to provide further insight into the pharmacological and physiological role of the delta-opioid receptor system. Thus, in the present study we have evaluated and compared DADELT II with dermorphin (DER), after direct intrathecal administration, in an antinociceptive assay in vivo in the rat, to increase our understanding of delta-opioid receptor involvement in mediating spinal analgesia. METHOD
Animal Model Male Sprague-Dawley rats (300-350 g) were chronically implanted with a lumbar intrathecal catheter (PE-10, 0.75 mm in diameter, 12 cm in length) according to Yaksh and Rudy (19). Briefly, in animals lightly anesthetized with ether, a slit was made in the atlanto-occipital membrane and the catheter was inserted to the level of the lumbar cord (8.5 cm). The other end of the catheter was externalized between the eyes and plugged with a steel wire stylet. After 7 days recovery, the animals were used to determine the effect of peptide injection or infusion on a thermal test (tail-flick test).
1124
IMPROTA AND BROCCARDO
100"
50-
60 IJJ e,
40 O"
20 n
0
0
30
-5O
•
60 90 120 POSTINJECTION TIME (rain)
150
FIG. 1. Time course of the analgesia produced by graded IT doses of injected DADELT II (l pg/rat, A; 10 pg/rat, O; 25 pg/rat, m) and DER (10 ng/rat, [~). Each point represents the mean _+ SEM of the MPE determined in seven rats per group.
J
0
1'0
i
,
u
,
i
.
20 30 40 50 POSTINJECTION TIME (mln)
i
60
•
u
70
FIG. 2. Effect ofSC naloxone (1 mg/kg, • and 10 mg/kg, O) and SC naltrindole (l mg/kg, • and l0 mg/kg, El) on the inhibition of tail flick by IT-injected DADELT II (10 ug/rat, A). Each point represents the mean + SEM of the MPE determined in five rats per group. Asterisks indicate significantdifference from DADELT II group at each time point (p < 0.001, Dunnett's t-test).
Drug Administration Injections. Drugs for intrathecal (IT) injections, DADELT II (1, 10, and 25 pg/rat), and DER (10 ng/rat) were administered in a 10 #1 volume followed by t0 pl sterile and distilled water to flush the catheter. Subcutaneous (SC) naloxone (NLX), the mu-preferring antagonist, or naitrindole (NLD), the delta-selective antagonist (13), were given 5 rain before the peptide injections. Infusions. Continuous IT infusions of DADELT II at different doses (0.1, 1, and 3 pg/pl/min) were administered for a period of 120 rain, with a Hamilton microliter syringe connected to a constant infusion Harvard pump. During this experimental session, animals were restrained in Plexiglas tubes from which their tail extended; they were never disconnected from the apparatus. In rats treated with opioid antagonists, DADELT II (0.5 #g/pl/ min) was infused continuously for 210 rain and NLD (1 mg/ kg) and NLX (1 and 10 mg/kg) were injected SC 90 rain after the start of the peptide infusion. Analgesia was tested at 30-rain intervals throughout DADELT II infusion. Analgesic Assay The analgesic effect of IT drugs was examined on the tailflick test (TF) (5). The TF response was evoked by placing the rat's tail under an infrared source (100 W bulb), whose radiant energy is focused onto a photo cell, by an aluminized parabolic mirror (Socrel Tail-flick Unit, BRA, Comerio, Varese, Italy). The response latency was taken as the time required for the rat to vigorously flick its tail and was determined, via a suitable electronic circuit, with a second counter connected to the TF unit. Animals not flicking their tails within 5 s were discarded. To prevent tissue damage, the cutofflatency for the TF response was 10 s. The TF latency thus obtained was expressed as maximum percent effect (MPE), calculated as follows:
RESULTS
Injections Figure l shows the dose- and time-related inhibition of the TF response induced by IT injections ofDADELT II compared with the TF response induced by DER. The maximal antinociceptive response occurred 5-l 0 min after DADELT II administration and 30-60 min after DER injection. The DADELT II analgesia lasted 10-60 min, depending on the dose, and appeared to be of shorter duration than the analgesia produced in rats after IT injection of DER (20-120 min). The effect of l0 pg DADELT II on the TF response was partially and completely abolished by pretreatment with SC NLD at l and l0 mg/kg, respectively (Fig. 2). When given subcutaneously at a dose of 1 mg/kg, 5 min before the administration ofDADELT II, NLX failed to modify the analgesic response. At a tenfold higher dose (t0 mg/kg), although unable to antagonize DADELT II-induced analgesia completely, NLX reduced the intensity and duration of the effect.
Infusions Figure 3 shows the antinociceptive time-response curves of graded IT doses (0.1,0.5, and 3 pg/pl/min) ofinfused DADELT
INFUISlON
_|.
10080 'e"t
60
:E
MPE =
posttreatment latency - pretreatment latency × 100 cutoff latency - pretreatment latency
40 20
Chemicals 0
The DADELT I1 was synthesized as previously described (7), dissolved in sterile distilled water plus acetic acid, frozen in aliquots, and redissolved immediately before use. Dermorphin (Peninsula Labs), NLX (1-10 mg/kg) (Narcan, Endo Laboratories, Inc.), and NLD (1-10 mg/kg) (Research Biochemicals Inc.) were dissolved in sterile distilled water just before use.
•
0
i
1
•
i
2
•
,
3 TIME (hours)
u
4
,
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5
6
FIG. 3. Time course of the analgesia produced by graded IT doses (0.1 pg//A/min, A; 0.5 pe,/#l/min, O; 3 t~g/pl/min,B) of infused DADELT II. Each point represents the mean +_SEM of the MPE determined in six rats per group.
DELTORPHIN MEDIATION OF SPINAL ANALGESIA I1 in the rat TF test. At the smallest dose used (0.1/~g/#l/min), the peptide induced a transitory analgesia that disappeared before the end of the infusion. At the doses of 0.5 and 3 #g/#l/min an analgesic effect appeared and lasted for 90 or 180 min, respectively, after the end of the infusion. The analgesic action induced by an infusion of 0.5/~g/#l/min of DADELT II was completely antagonized by SC administration of NLD (1 mg/kg). Naloxone also antagonized DADELT II-induced analgesia. After a SC dose of 1 mg/kg the reversal lasted 90 min; after a SC dose of 10 mg/ kg the antagonistic effect lasted throughout the infusion period (Fig. 4).
1125
,~f t
These results present the first evidence for a spinal antinociceptive action in rats of DADELT lI, a naturally occurring peptide with high affinity and selectivity for delta-opioid receptors. Intrathecal injection or infusion of DADELT II produced a dose-related inhibition of the tail-flick response in rats. Pretreatment with the selective delta antagonist naltrindole completely blocked the analgesic effect of injected or infused DADELT II, indicating that this response is mediated through spinal delta receptors. The possibility that DADELT II spinally infused or injected exerts its analgesic effect through a supraspinal action was excluded by previous studies showing that intracerebroventricular injection of DADELT If, at doses up to 100 ~tg/rat, never induces analgesia (12). Although a great deal of evidence points towards a role for delta spinal receptors in the rat spinal pain processing evoked by thermal stimuli in rat, it is also true that delta agonists are generally less potent than mu agonists (10). Our study has confirmed this by showing that DADELT II is an effective analgesic, albeit considerably less potent than the mu-selective compound, dermorphin. The reason for the differing potencies might be the different rates of tissue diffusion and access to receptor sites. Alternatively, delta agonists may possess less analgesic potency because they operate through different neuronal mechanisms. The limited action of delta agonists has been correlated to the release of other peptides, such as the indirect opioid antagonist, cholecystokinin-8 (8), or substance P, which may enhance nociception (2). Arguing against this explanation are recent reports (3) indicating that IT infusions of two selective agonists ofdeltaopioid receptors significantly reduce the spinal outflow of substance P-like material. Stimulation of delta receptors might therefore exert a tonic inhibitory control of the release of substance P. However, this controversial point requires further study. In modifying the potency of opioid agonists in vitro (4), the receptor number is also an important factor and possibly also determines potency in vivo. Previous studies have shown that in the rat at supraspinal level, the selective mu agonist dermorphin alone is a potent analgesic (1), unlike DADELT II, which never induces analgesia (12). This interesting dichotomy is thought to depend on the scarcity of delta receptors in the rat substantia nigra, thalamus, and hypothalamus, supraspinal areas mediating the analgesic effect of opioids (16). At present, however, we can only say that afferent transmission of nociceptive information in the rat spinal cord could be attenuated with different potencies by a dual action at mu and delta sites. A striking difference also occurred in the temporal profiles of analgesia elicited by DADELT II and DER. This dissimilar time course suggests that different analgesic receptor mechanisms mediate the effect of the two peptides. Our study also provided another intriguing finding. The mu-
NLX or NLD s.c.
120
¥
[]
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rl
.
0
DISCUSSION
DADELT I! I N F U S ION
30
60
90
120 150 TIME (min)
180
210
240
FIG. 4. Effect of SC injection of naloxone (NLX, 1 mg/kg, A; 10 mg/ kg, O) and naltrindole (NLD, 1 mg/kg, l ) on analgesia induced by IT infusion of DADELT I1 (0.5 ~g/ul/min, I-1). Each point represents the mean _+ SEM of the MPE in five rats per group. Asterisks indicate a significant difference from DADELT 1I alone at each time point (p < 0.001, Dunnett's t-test).
preferring antagonist naloxone partially reduced the analgesic action induced by injections of DADELT II and completely blocked analgesia induced by infusions of DADELT II. Naloxone is not selective for mu receptors and in part also occupies delta receptors. This might explain the partial antagonism obtained with high doses of naloxone (l 0 mg/kg) when DADELT II was given by injection; whereas, in contrast, after continuous infusion with DADELT II, naloxone displayed an antagonist potency equal to that of naltrindole. These equal potencies suggest that the spinal analgesic effect of DADELT II does not involve a delta component alone: a mu component also intervenes. Other authors (12) have shown that N-terminal fragments of DADELT II possess a high affinity for mu receptors. One may therefore argue that by shortening brain peptide chains, carboxypeptidases produce mu agonists, which participate in the antinociceptive effect during the infusion period. During such a long treatment period, these mu-agonist fragments might of course exert some of their effects through a direct supraspinal action or through a mu-mediated influence on spinal delta function. On the other hand, recent reports have hypothesized that delta receptors also exist in a functional or physical complex with mu receptors (14). Thus, DADELT II could produce analgesia through a delta binding site of an opioid mu/deita receptor complex. The occupation of a delta site by DADELT II may alter the conformation of the mu site and favor coupling of the mu site to the effector system. If this were so, it would explain why, when the mu site is occupied by naloxone, the message of the delta ligand is not transmitted and the analgesic effect is suppressed. Delta agonists therefore offer significant promise as modulators of the potency and efficacy of mu agonists. In conclusion, the discovery of [D-Ala2]deltorphin II, a peptide with a high degree of selectivity for delta receptors, has allowed us to confirm the involvement of delta receptors in mediating spinal analgesia in the rat and to provide further insights into the pharmacological and physiological significance of this class of opioid receptors. ACKNOWLEDGEMENTS This work was supported by the target project "Chimica Fine II" from the National Research Council. The authors wish to thank Claudio Munari for his kind technical assistance.
1126
IMPROTA AND BROCCARDO REFERENCES
1. Broceardo, M.; Erspamer, V.; Falconieri Erspamer, G.; Improta, G.; Linari, G.; Melchiorri, P.; Montecucchi, P. C. Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin. Br. J. Pharmacol. 73:625-631; 1981. 2. Cesselin, F.; Bourgoin, S.; Artaud, F.; Hamon, M. Basic and regulatory mechanisms of in vitro release of Met-enkephalin from the dorsal zone of the rat spinal cord. J. Neurochem. 43:763773; 1984. 3. Collin, E.; Mauborgue, A.; Bourgoin, S.; Hamon, M.; Cesselin, F. Endogenous opioids acting at delta receptors exert a tonic inhibitory control of substance P release from the rat spinal cord in vivo. Personal communication in the First ENC Meeting "Functional and pharmacological aspects of neuropeptides." Igls. lnnsbruck, Jan. 2326; 1991. 4. Cox, B. M.; Chavkin, C. Comparison ofdynorphyn-selective kappa receptors in mouse vas deferens and guinea pig ileum. Mol. Pharmacol. 23:36-43; 1983. 5. D'Amour, F. E.; Smith, D. L. A method of determining loss of pain sensation. J. Pharmacot. Exp. Ther. 72:74-79; 1941. 6. Dickenson, A. H.; Sullivan, A. F.; Knox, R.; Zajac, J. M.; Roques, B. P. Opioid receptor subtypes in the rat spinal cord: Electrophysiologieal studies with mu and delta-opioid receptor agonists in the control of nociception. Brain Res. 413:36-44; 1987. 7. Erspamer, V.; Melchiorri, P.; Falconieri Erspamer, G.; Negri, L.; Corsi, R.; Severini, C.; Barra, D.; Simmaco, M.; Kreil, G. Deltorphins: A family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites. Proc. Natl. Acad. Sci. USA 86:5188-5192; 1989. 8. Fails, P. L.; Komsanek, B. R.; Watkins, L. R.; Mayer, D. J. Evidence on the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 210:310-312; 1983.
9. Gonarderes, C.; Cros, J.; Quirion, R. Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea pig spinal cord. Neuropeptides 6:331-342; 1985. 10. Heyman, J. S.; Vaught, J. L.; Baffa, R. B.; Porreca, F. Can supraspinal delta-opioid receptors mediate antinociception? Trends Pharmacol. Sci. 9:134-138; 1988. 11. Kreil, G.; Barra, D.; Simmaco, M.; Erspamer, V.; Falconieri Erspamer, G.; Negri, L.; Severini, C.; Corsi, R.; Melchiorri, P. Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors. Eur. J. Pharmacol. 162:123-128; 1989. 12. Negri, L.; Noviello, V.; Angelucci, F. Behavioural effects of deltorphins in rats. Eur. J. Pharmacol. 209:163-168; 1991. 13. Portoghese, P. S.; Sultana, M.; Takemori, A. E. Naltrindole, a highly selective and potent nonpeptide delta-opioid receptor antagonist. Eur. J. Pharmacol. 146:185-186; 1988. 14. Rothman, R. B.; Jacobson, A. E.; Rice, K. C.; Herkenham, M. M. Autoradiographic evidence for two classes of mu opioid binding sites in rat brain. Peptides 8:1015-1019; 1987. 15. Schmauss, C.; Yaksh, T. L. In vivo studies on spinal opiate receptor system mediating antinociception. J. Pharmacol. Exp. Ther. 228:111; 1984. 16. Sharif, N. A.; Hughes, J. Discrete mapping of brain mu and delta opioid receptors using selective peptides: Quantitative autoradiography, species differences and comparison with kappa receptors. Peptides 10:499-522; 1989. 17. Stevens, C.; Yaksh, T. L. Spinal action ofdermorphin, an extremely potent opioid peptide from frog skin. Brain Res. 385:300-304; 1986. 18. Tung, A. S.; Yaksh, T. In vivo evidence for multiple opiate receptors mediating analgesia in the rat spinal cord. Brain Res. 247:75-83; 1982. 19. Yaksh, T. L.; Rudy, T. A. Chronic catheterization of the spinal subarachnoid space. Physiol. Behav. 17:1031-1036; 1976.
0196-9781/92$5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
Spinal Antinociceptive Effects of [D-Ala2]Deltorphin II, a Novel and Highly Selective Delta-Opioid Receptor Agonist G. I M P R O T A 1 A N D M. B R O C C A R D O Institute o f Pharmacology III, University "La Sapienza, "" P. le A. Moro 5-00185 Rome, Italy Received 28 F e b r u a r y 1992 IMPROTA, G. AND M. BROCCARDO. Spinal antinociceptive effects of[D-Ala2]deltorphin II, a novel and highly selective deltaopioid receptor agonist. PEPTIDES 13(6) 1123-1126, 1992.--Pharmacological assays in isolated tissues and binding tests have recently shown that two peptides, with the sequence Tyr-D-Ala-Phe-Asp-(orGIu)-VaI-VaI-GIy-NH2,isolated from skin extracts of Phyllomedusa bicolor and named [D-Ala2]deltorphinI and II, respectively, possess a higher affinity and selectivityfor deltaopioid receptors than any other known natural compound. Since much evidence supports the role of spinal delta-opioid sites in producing antinociceptive effects, we investigatedwhether analgesia might be detected by direct spinal cord administration of [DAla2]deltorphin II (DADELT II) in the rat. The thermal antinociceptive effects of intrathecal DADELT II and dermorphin, a potent mu-selective agonist, were compared at different postinjection times by means of the tail-flick test. The DADELT II produced a dose-related inhibition of the tail-flick response, which lasted 10-60 min depending on the dose and appeared to be of shorter duration than the analgesia produced in rats after intrathecai injection of dermorphin (20-120 rain). The analgesic effect of infused or injected DADELT II was completely abolished by naltrindole, the highly selective delta antagonist. These results confirm the involvement of delta receptors in spinal analgesicactivity in the rat. Deltorphin
Delta receptor stimulation
Spinal analgesia
ISOLATED from skin extracts of frogs belonging to the genus
Phyllomedusa, deltorphins are linear heptapeptides that have a higher affinity and selectivity for delta-opioid binding sites than any other natural compound known (11). Recently, two deltorphins, one with the sequence Tyr-t>-Ala-Phe-Asp-Val-Val-GlyNH2 ([D-Ala2]deltorphin I) and the other with the sequence TyrD-AIa-Phe-GIu-Val-VaI-Gly-NH2 ([D-Ala2]deltorphin II), have been isolated from skin extracts of Phyllomedusa bicolor (7). Pharmacological assays in isolated tissues and binding assays have shown that these peptides possess a higher affinity for delta receptors than the previously characterized deltorphin, which contains D-Met as the second amino acid. These new deltorphins contain the N-terminal sequence Tyr-D-Ala-Phe. This sequence is present in another constituent of Phyllomedusa skin, dermorphin (1), which is highly selective for mu-opioid receptors. Pharmacological evidence has been accumulated strongly implicating both mu- and delta-receptor agonists in spinal antinociception in the rat (6,15,17,18). The superficial dorsal horn of the spinal cord is rich in mu receptors. Although less abundant, delta sites are present in the substantia gelatinosa of the spinal cord (9,16). Processing of nociceptive transmission in the dorsal horn of the spinal cord can be modulated by the opiate receptor system. Yet the role of different subtypes of opioid receptors remains controversial, principally owing to the lack of highly Requests for reprints should to be addressed to Prof. G. Improta.
1123
selective agonists and antagonists. Because the recently available peptide [D-Ala2]deltorphin II (DADELT II) is the most selective delta agonist known to date, it should help to provide further insight into the pharmacological and physiological role of the delta-opioid receptor system. Thus, in the present study we have evaluated and compared DADELT II with dermorphin (DER), after direct intrathecal administration, in an antinociceptive assay in vivo in the rat, to increase our understanding of delta-opioid receptor involvement in mediating spinal analgesia. METHOD
Animal Model Male Sprague-Dawley rats (300-350 g) were chronically implanted with a lumbar intrathecal catheter (PE-10, 0.75 mm in diameter, 12 cm in length) according to Yaksh and Rudy (19). Briefly, in animals lightly anesthetized with ether, a slit was made in the atlanto-occipital membrane and the catheter was inserted to the level of the lumbar cord (8.5 cm). The other end of the catheter was externalized between the eyes and plugged with a steel wire stylet. After 7 days recovery, the animals were used to determine the effect of peptide injection or infusion on a thermal test (tail-flick test).
1124
IMPROTA AND BROCCARDO
100"
50-
60 IJJ e,
40 O"
20 n
0
0
30
-5O
•
60 90 120 POSTINJECTION TIME (rain)
150
FIG. 1. Time course of the analgesia produced by graded IT doses of injected DADELT II (l pg/rat, A; 10 pg/rat, O; 25 pg/rat, m) and DER (10 ng/rat, [~). Each point represents the mean _+ SEM of the MPE determined in seven rats per group.
J
0
1'0
i
,
u
,
i
.
20 30 40 50 POSTINJECTION TIME (mln)
i
60
•
u
70
FIG. 2. Effect ofSC naloxone (1 mg/kg, • and 10 mg/kg, O) and SC naltrindole (l mg/kg, • and l0 mg/kg, El) on the inhibition of tail flick by IT-injected DADELT II (10 ug/rat, A). Each point represents the mean + SEM of the MPE determined in five rats per group. Asterisks indicate significantdifference from DADELT II group at each time point (p < 0.001, Dunnett's t-test).
Drug Administration Injections. Drugs for intrathecal (IT) injections, DADELT II (1, 10, and 25 pg/rat), and DER (10 ng/rat) were administered in a 10 #1 volume followed by t0 pl sterile and distilled water to flush the catheter. Subcutaneous (SC) naloxone (NLX), the mu-preferring antagonist, or naitrindole (NLD), the delta-selective antagonist (13), were given 5 rain before the peptide injections. Infusions. Continuous IT infusions of DADELT II at different doses (0.1, 1, and 3 pg/pl/min) were administered for a period of 120 rain, with a Hamilton microliter syringe connected to a constant infusion Harvard pump. During this experimental session, animals were restrained in Plexiglas tubes from which their tail extended; they were never disconnected from the apparatus. In rats treated with opioid antagonists, DADELT II (0.5 #g/pl/ min) was infused continuously for 210 rain and NLD (1 mg/ kg) and NLX (1 and 10 mg/kg) were injected SC 90 rain after the start of the peptide infusion. Analgesia was tested at 30-rain intervals throughout DADELT II infusion. Analgesic Assay The analgesic effect of IT drugs was examined on the tailflick test (TF) (5). The TF response was evoked by placing the rat's tail under an infrared source (100 W bulb), whose radiant energy is focused onto a photo cell, by an aluminized parabolic mirror (Socrel Tail-flick Unit, BRA, Comerio, Varese, Italy). The response latency was taken as the time required for the rat to vigorously flick its tail and was determined, via a suitable electronic circuit, with a second counter connected to the TF unit. Animals not flicking their tails within 5 s were discarded. To prevent tissue damage, the cutofflatency for the TF response was 10 s. The TF latency thus obtained was expressed as maximum percent effect (MPE), calculated as follows:
RESULTS
Injections Figure l shows the dose- and time-related inhibition of the TF response induced by IT injections ofDADELT II compared with the TF response induced by DER. The maximal antinociceptive response occurred 5-l 0 min after DADELT II administration and 30-60 min after DER injection. The DADELT II analgesia lasted 10-60 min, depending on the dose, and appeared to be of shorter duration than the analgesia produced in rats after IT injection of DER (20-120 min). The effect of l0 pg DADELT II on the TF response was partially and completely abolished by pretreatment with SC NLD at l and l0 mg/kg, respectively (Fig. 2). When given subcutaneously at a dose of 1 mg/kg, 5 min before the administration ofDADELT II, NLX failed to modify the analgesic response. At a tenfold higher dose (t0 mg/kg), although unable to antagonize DADELT II-induced analgesia completely, NLX reduced the intensity and duration of the effect.
Infusions Figure 3 shows the antinociceptive time-response curves of graded IT doses (0.1,0.5, and 3 pg/pl/min) ofinfused DADELT
INFUISlON
_|.
10080 'e"t
60
:E
MPE =
posttreatment latency - pretreatment latency × 100 cutoff latency - pretreatment latency
40 20
Chemicals 0
The DADELT I1 was synthesized as previously described (7), dissolved in sterile distilled water plus acetic acid, frozen in aliquots, and redissolved immediately before use. Dermorphin (Peninsula Labs), NLX (1-10 mg/kg) (Narcan, Endo Laboratories, Inc.), and NLD (1-10 mg/kg) (Research Biochemicals Inc.) were dissolved in sterile distilled water just before use.
•
0
i
1
•
i
2
•
,
3 TIME (hours)
u
4
,
v
5
6
FIG. 3. Time course of the analgesia produced by graded IT doses (0.1 pg//A/min, A; 0.5 pe,/#l/min, O; 3 t~g/pl/min,B) of infused DADELT II. Each point represents the mean +_SEM of the MPE determined in six rats per group.
DELTORPHIN MEDIATION OF SPINAL ANALGESIA I1 in the rat TF test. At the smallest dose used (0.1/~g/#l/min), the peptide induced a transitory analgesia that disappeared before the end of the infusion. At the doses of 0.5 and 3 #g/#l/min an analgesic effect appeared and lasted for 90 or 180 min, respectively, after the end of the infusion. The analgesic action induced by an infusion of 0.5/~g/#l/min of DADELT II was completely antagonized by SC administration of NLD (1 mg/kg). Naloxone also antagonized DADELT II-induced analgesia. After a SC dose of 1 mg/kg the reversal lasted 90 min; after a SC dose of 10 mg/ kg the antagonistic effect lasted throughout the infusion period (Fig. 4).
1125
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These results present the first evidence for a spinal antinociceptive action in rats of DADELT lI, a naturally occurring peptide with high affinity and selectivity for delta-opioid receptors. Intrathecal injection or infusion of DADELT II produced a dose-related inhibition of the tail-flick response in rats. Pretreatment with the selective delta antagonist naltrindole completely blocked the analgesic effect of injected or infused DADELT II, indicating that this response is mediated through spinal delta receptors. The possibility that DADELT II spinally infused or injected exerts its analgesic effect through a supraspinal action was excluded by previous studies showing that intracerebroventricular injection of DADELT If, at doses up to 100 ~tg/rat, never induces analgesia (12). Although a great deal of evidence points towards a role for delta spinal receptors in the rat spinal pain processing evoked by thermal stimuli in rat, it is also true that delta agonists are generally less potent than mu agonists (10). Our study has confirmed this by showing that DADELT II is an effective analgesic, albeit considerably less potent than the mu-selective compound, dermorphin. The reason for the differing potencies might be the different rates of tissue diffusion and access to receptor sites. Alternatively, delta agonists may possess less analgesic potency because they operate through different neuronal mechanisms. The limited action of delta agonists has been correlated to the release of other peptides, such as the indirect opioid antagonist, cholecystokinin-8 (8), or substance P, which may enhance nociception (2). Arguing against this explanation are recent reports (3) indicating that IT infusions of two selective agonists ofdeltaopioid receptors significantly reduce the spinal outflow of substance P-like material. Stimulation of delta receptors might therefore exert a tonic inhibitory control of the release of substance P. However, this controversial point requires further study. In modifying the potency of opioid agonists in vitro (4), the receptor number is also an important factor and possibly also determines potency in vivo. Previous studies have shown that in the rat at supraspinal level, the selective mu agonist dermorphin alone is a potent analgesic (1), unlike DADELT II, which never induces analgesia (12). This interesting dichotomy is thought to depend on the scarcity of delta receptors in the rat substantia nigra, thalamus, and hypothalamus, supraspinal areas mediating the analgesic effect of opioids (16). At present, however, we can only say that afferent transmission of nociceptive information in the rat spinal cord could be attenuated with different potencies by a dual action at mu and delta sites. A striking difference also occurred in the temporal profiles of analgesia elicited by DADELT II and DER. This dissimilar time course suggests that different analgesic receptor mechanisms mediate the effect of the two peptides. Our study also provided another intriguing finding. The mu-
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FIG. 4. Effect of SC injection of naloxone (NLX, 1 mg/kg, A; 10 mg/ kg, O) and naltrindole (NLD, 1 mg/kg, l ) on analgesia induced by IT infusion of DADELT I1 (0.5 ~g/ul/min, I-1). Each point represents the mean _+ SEM of the MPE in five rats per group. Asterisks indicate a significant difference from DADELT 1I alone at each time point (p < 0.001, Dunnett's t-test).
preferring antagonist naloxone partially reduced the analgesic action induced by injections of DADELT II and completely blocked analgesia induced by infusions of DADELT II. Naloxone is not selective for mu receptors and in part also occupies delta receptors. This might explain the partial antagonism obtained with high doses of naloxone (l 0 mg/kg) when DADELT II was given by injection; whereas, in contrast, after continuous infusion with DADELT II, naloxone displayed an antagonist potency equal to that of naltrindole. These equal potencies suggest that the spinal analgesic effect of DADELT II does not involve a delta component alone: a mu component also intervenes. Other authors (12) have shown that N-terminal fragments of DADELT II possess a high affinity for mu receptors. One may therefore argue that by shortening brain peptide chains, carboxypeptidases produce mu agonists, which participate in the antinociceptive effect during the infusion period. During such a long treatment period, these mu-agonist fragments might of course exert some of their effects through a direct supraspinal action or through a mu-mediated influence on spinal delta function. On the other hand, recent reports have hypothesized that delta receptors also exist in a functional or physical complex with mu receptors (14). Thus, DADELT II could produce analgesia through a delta binding site of an opioid mu/deita receptor complex. The occupation of a delta site by DADELT II may alter the conformation of the mu site and favor coupling of the mu site to the effector system. If this were so, it would explain why, when the mu site is occupied by naloxone, the message of the delta ligand is not transmitted and the analgesic effect is suppressed. Delta agonists therefore offer significant promise as modulators of the potency and efficacy of mu agonists. In conclusion, the discovery of [D-Ala2]deltorphin II, a peptide with a high degree of selectivity for delta receptors, has allowed us to confirm the involvement of delta receptors in mediating spinal analgesia in the rat and to provide further insights into the pharmacological and physiological significance of this class of opioid receptors. ACKNOWLEDGEMENTS This work was supported by the target project "Chimica Fine II" from the National Research Council. The authors wish to thank Claudio Munari for his kind technical assistance.
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IMPROTA AND BROCCARDO REFERENCES
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