- Email: [email protected]
Antinociceptive effects of intrathecal endomorphin-1 and -2 in rats
PI1 SOO24-3205(99)00532-9
ANTINOCICEPTIVE
EFFECTS OF INTRATHECAL
Gyongyi Horvatp,
Life Sciences, Vol. 65, No. 24, pp. 26352641, 199 Copyight 0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/993/$-see hat matter
ENDOMORPHIN-
1 AND -2 IN RATS
Margit SzikszayX, Csaba TGmbSly*, Gyorgy Benedek‘
“Department of Physiology, “Faculty of Health Science, Albert Szent-Gyorgyi Medical University, P.O. Box 427, Szeged, H-6701, Hungary; *Isotope Laboratory, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
(Received in final form August 5, 1999) Summary
Endomorphin-l and endomorphinwere recently postulated to be endogenous uopioid receptor agonists. We have investigated the antinociceptive and antihyperalgesic effects of intrathecally administered endomorphins in cumulative doses (0. l-l 00 pg) on acute and inflammatory pain sensations in awake rats. In the tail-flick test, both peptides caused a dose-dependent short-lasting antinociception. except at the highest dose, which caused motor impairment also. The dose-response curves revealed the development of acute tolerance (tachyphylaxis) to endomorphin. Similarly in the carrageenan-injected paw, the endomorphins (10 pg) exerted transient antinociceptive effects. These are the first data to demonstrate decreased responsivity in models of both acute and inflammatory pain after intrathecal administration of endomorphin- 1 and -2 in awake rats.
Key Words: antinociception, carrageenan, endogenous opioid peptides, endomorphin, hyperalgesia, opioid, spinal administration
Morphine and agonists exerting activity at the u-opioid receptor are well-known analgesics, and at least a proportion of their analgesic efficacy derives from activity at the spinal cord level (1). The existence of endogenous, selective neuropeptide agonists for both the 6- and ic-opioid receptors has repeatedly raised the question of the existence of selective, endogenous agonists for the u-opioid receptor. However, searches for endogenous p-opioid peptides based on the conserved aminoterminal sequence shared by the other endogenous opioid peptides have failed. The recently isolated p-opioid receptor-selective endogenous peptides endomorphin-l (Tyr-Pro-Trp-Phe-NH*) and endomorphin(Tyr-Pro-Phe-Phe-NH2) (2) from the bovine frontal cortex do not share this conserved sequence and for this reason may have been missed in previous screens. Correspondence to: Gyongyi Horvath Dept. Physiol. Albert Szent-Gyorgyi Medical P.O. Box: 427. H-6701 Szeged, Hungary Tel: 36-62-455101, Fax: 36-62-455842. email: [email protected]
University
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Intratliecal Effects of Endomorphin- and -2
Vol. 65, No. 24, 1999
Both of these peptides were reported to display high affinity and selectivity for the u-opioid receptor in vivo and in vitro (?;3) and may therefore be endogenous ligands for the u-opioid receptor. Endomorphin-l is 4000 and 15000 times as selective for the p-opioid receptor as compared to the 6- and ic-opioid receptors, respectively, whereas endomorphinis >13000 and > 7500 times as selective, respectively (2). Both peptides mediate naloxone-sensitive analgesia after intracerebroventricular or intrathecal injection, with similar potency to morphine in mice (2-S). Furthermore in u-receptor knockout mice the endomorphins do not cause any antinociceptive effect (4). After intravenous administration to anesthetized rabbits and rodents these peptides have also been demonstrated to exhibit hypotensive activity mediated by the release of nitric oxide from the endothelium of vascular bed (6-8), and they decrease the heart rate in rodents (7;8). Shane et al. observed the antinociceptive effect of endomorphinafter intracerebroventricular administration using the tailflick test in rats (9). The only data relating to the analgesic properties of spinal endomorphins originated from an electrophysiological study in anesthetized rats (10). Since the species differences in the concentrations and distributions of opioid binding sites is well-known, it is important to investigate the spinal antinociceptive effect of endomorphins in awake unrestrained rats (11). Furthermore little if anything is known about the effects of endomorphins on inflammatory pain sensation. The objective of the present investigation was to assess the antinociceptive effects of endomorphin-l and -2 after intrathecal administration in acute and inflammatory pain tests in awake rats. Methods Intrathecal catheterization After approval had been obtained from the Animal Care Committee of Albert Szent-Gyorgyi Medical University, 64 male Wistar rats weighing 200-300 g were studied. The rats were surgically prepared under ketamine-xylazine anesthesia (87 and 13 mg/kg intraperitoneally, respectively). An intrathecal catheter (PE-10 tubing) was inserted through a small opening in the cisterna magna and passed 8.5 cm caudally into the intrathecal space (12). After surgery, the rats were housed individually with free access to food and water and allowed to recover for at least 3 days before use. Rats showing postoperative neurologic deficits were not used. All experiments were performed in freely-moving animals during the same period of the day (8:00-13:00 h) in order to exclude diurnal variations in pharmacological effects. The animals were randomly assigned to treatment groups, and the observer was blind to the treatment administered. Drugs and their administration The following drugs were administered in this study: Ketamine (Ketalar, Parke-Davis, Austria), xylazine (Rompun, Bayer, FRG.), carrageenan (Sigma-Aldrich Kfi. Budapest, Hungary), and endomorphin-l and -2 synthesized using solid-state method and purified by means of HPLC in the Isotope Laboratory of Biological Research Center of the Hungarian Academy of Sciences. The peptides were dissolved in sterile physiological saline, freshly prepared on the day of the experiment, and were injected intrathecally over 30 s in a volume of 5 ~1, followed by a 10 ul flush of physiological saline. The drugs were administered in cumulative doses. Acute heat pain test (tail-flick test) The acute nociceptive sensitivity was assessed by using the tail-flick technique described by Janssen et al. (13). The reaction time in the tail-flick test was determined by immersing the lower 5 cm portion of the tail in the hot water until the typical tail-withdrawal response was observed (5 1.5
Vol. 65, No. 24, 1999
Intrathecal Effects of Endomorphin- and -2
2637
oC water, cut-off time: 20 s). Baseline latencies were obtained immediately before and then 10 and 30 min after the drug injections. Three series of experiments were performed with different cumulative doses of endomorphins, e.g. 0.1-0.3-l ug or l-3-10 ug or 10-30-100 ug (n=6131group). Inflammatory pain test (paw withdrawal test) The rats were placed on a glass surface in a plastic chamber and allowed to acclimatize to their environment for 15-30 min before testing, and baseline hindpaw withdrawal latencies (precarrageenan values at -180 min) were obtained. The heat stimulus was directed onto the plantar surface of each hindpaw. A detailed description of this method has been published elsewhere (14). Unilateral inflammation was induced by intraplantar injection of 2 mg carrageenan in 0.1 ml physiological saline into the right hindpaw. The paw withdrawal latencies were obtained again 3 h after carrageenan (post-carrageenan baseline values at 0 min), and then 10 and 30 min after the drug injections. The experiments were performed with different cumulative doses of endomorphins, e.g. l-3-10 ug (n=5-9/group). Data analysis Analgesic latencies in the acute pain test were converted to percentage maximum possible effects (% MPE) by using the formula % MPE = [(observed latency - baseline latency)/(cut-off time - baseline latency)]* 100 Data are presented as means f SEM. Analysis of variance (ANOVA) of data for repeated measures was used for overall effects, with the Newman-Keuls test for post-hoc comparison of means. A level of PcO.05 was considered significant. Results Effects of intrathecal endomorphins in acute pain test There was no significant difference in tail-flick latency between the groups before the drug administration. The overall mean tail-flick latency was 7.4 + 0.24 s (n=44). Both endomorphin-l and -2 caused a dose-dependent antinociceptive effect at 10 min with similar potency (Fig. 1.). The latency had decreased to below the baseline value at 30 min following the injections. This decrease was significant in some cases, suggesting hyperalgesia. The highest dose of endomorphin(100 ug) caused motor impairment too. The half-maximal effective dose (EDSo) for endomorphinat 10 min was 2.51 ug (4.05 nmol) [95% confidence interval = 1.122 - 5.618 ug = 1,82 - 9,lO nmol]. The dose-response curve revealed that 10 ug had a higher efficacy when it was applied in the first injection than following the third administration, suggesting the development of tachyphylaxis to endomorphin(Fig. 2.). Effects of intrathecal endomorphins on normal and inflamed paws There was no significant difference in the paw withdrawal responses to noxious thermal stimuli between the right and left hindpaws prior to the intraplantar injection of carrageenan. The overall mean paw withdrawal times for the ipsilateral and contralateral paws were 9.77 k 0.29 and 10.37 f 0.45 s (n=20), respectively. Carrageenan injection induced inflammation of the injected hindpaw, as evidenced by edema and erythema. The paw withdrawal latencies of the carrageenaninjected paw were significantly reduced to 2.1 * 0.05 s (P
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IntrathecalEffects of Endomorphin-l and -2
had a significant short-lasting antinociceptive effect at the highest dose (10 ug), although they did not relieve the hyperalgesia (Fig. 3., right side).
Endoatozphin-2
Endomorphin-l 100 80 60
30
60
90
0
Time (minutes)
Inhibition of endomorphin10 ug: filled administration antinociception The results are control group. administrations.
30
60
Time (minutes)
Fig. 1 acute thermal nociceptive responses by endomorphin-l and treatments (control: open squares, 0.1-0.3-l ug: open circles, 1-3circles, 10-30-100 ug: filled squares). Cumulative intrathecal of endomorphin-l and -2 produced short-lasting, dose-dependent in the hot-water tail-flick test, with maximum efficacy at 10 min. expressed as means f S.E.M. *P
1
10
100
hes (Icg) Fig. 2 The dose-dependent effects of cumulative treatments with endomorphinat 10 min after injection (0.1-0.3-l ug: circles, l-3-10 ug: triangles, 10-30-100 ug: squares) on tail-flick latencies. The results are expressed as means z!zS.E.M.
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Discussion This is the first study to examine the antinociceptive potency of intrathecally administered endomorphins on carrageenan-induced thermal hyperalgesia in rats. We have shown that the intrathecal endomorphin-l and -2 caused a short-lasting antinociception on tail-flick and carrageenan-induced thermal hyperalgesia, with similar potency. The dose-response curve revealed the development of tachyphylaxis to endomorphins.
Right Paw
Left Paw 12.
12,
10 1
8
s
6.
;
4. 2
carrageenan
Time (min)
Fig. 3 Attenuation of the inflammatory pain sensation by endomorphin-l and -2 (l-3-10 up). None of the treatments influenced the heat-pain latency of the non-injected paws (left side). Endomorphin-l (filled circles) and endomorphin(tilled triangles) at the highest dose significantly attenuated the carrageenan-induced thermal hyperalgesia at 10 min after endomorphin injection (right side), saline treatment: open squares. The results are expressed as means f S.E.M. xP<0.05 compared to post-carrageenan baseline values. *P
Radioimmunoassay studies have localized the endomorphin-l immunoreactivity within the bovine central nervous system (2). Antisera were raised against endomorphinto localize this peptide in the spinal cords of rats and the rhesus macaque (15-17). Dense endomorphinimmunoreactivity was found in the marginal zone, the substantia gelatinosa and ventral to the central canal. Additionally, endomorphinimmunoreactivity was dramatically reduced following dorsal rhizotomy. These data suggest that endomorphinoccurs in the small diameter primary afferent fibers in rodents and primates. It appears possible that the release of neurotransmitters from nociceptive primary afferents might be regulated by the release of endomorphinfrom primary afferent terminals. Recent studies examined the ability of these peptides to stimulate G-protein activation via the uopioid receptor (18-20). These findings demonstrate that the endomorphins exert activity similar to that of morphine at the u-opioid receptor and suggest that these peptides have the potential to modulate neuronal activity in viva We observed that the antinociceptive min after intrathecal administration.
effect was significant at 10 min and it had disappeared at 30 Therefore putative endogenous u-opioid receptor agonists
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endomorphin-l and -2 are potent but short-acting antinociceptive agents in the spinal cord of rats in agreement with the earlier results (3;5;7;9). The behavioral analgesic effects of intrathecally administered endomorphins are comparable to the effects of spinally applied endomorphins on the C-fiber evoked neuronal response (10). Chapman et al. have shown that intrathecal endomorphin-l and -2 (0.25-50 pg) reduced all components of the electrically evoked C-fiber responses of spinal neurons in anesthetized rats, in a dose-related manner. The peak inhibitory effects of the endomorphins were observed at 15-20 min post-administration. We found that the two compounds have similar potencies and time courses, and they are also effective antihyperalgesic agents in the model of carrageenan-induced inflammatory pain. It is well established that the antinociceptive potency of spinal opioids is enhanced in inflammatory states (21). In agreement with this, 10 ug endomorphins significantly decreased the unilateral thermalhyperalgesia, but did not influence the normal paw latencies. There is some dicrepancy between the tail-flick and paw withdrawal results, i.e. same dose of endomorphins (10 pg) caused about 40 %MPE in the tail-flick test, but we could not observe any antinociceptive effect on the normal paw on the paw withdrawal test. There are several possibilities to explain these observation. First, there are some data suggesting that carrageenan-induced inflammation causes a slight increase in the pain sensitivity at the contralateral paw (22) which might be due to the bilateral release of neuropeptides (e.g. substance-P and calcitonin gene related peptides) (23) and activation of c-FOS (24). This fact might explain the lower efficacy of endomorphins at paw withdrawal test. Second, there are some differences between either the two methods (contact heat vs radiant heat) or the sensitivity of the tail and paw. This would agree with preliminary experiments when we determined both the paw- and the tail-withdrawal radiant heat pain latencies in normal rats and observed that endomorphin was more effective on the tail than on the hindpaws (data are not shown). Endomorphinand -2 significantly attenuated the thermal hyperalgesia with a short time course similar to the observed in the tail flick assay. The transient action observed in both tests suggests tachyphylaxis or fast degradation of the peptides. This latter option seems likely in view of the longer duration of action of 100 ug endomorphin-2, and the longer duration of synthesized analogs @X,9,25).The continuous infusion of these compounds or more stable, longer-lasting analogs may prove to be useful therapeutically against acute or inflammatory pain. Some promising substances are the recently synthesized analogs of endomorphin-2, D-[Ala’lendomorphin-2 and D-proline*endomorphin(8;9;25). The magnitude of responses (cardiovascular or antinociceptive) were not different, but the response duration was longer with the analogs. This finding may be interpreted as suggesting that the D-amino acid substitution inhibits the degradation of the peptide by proteolytic processing and increases the duration of action. Similarly to the results of Stone et al. in mice (3), we observed the rapid development of tachyphylaxis. These results suggests that these compounds may serve as useful tools to study of the mechanism of action of compounds modulating either the development or the expression of tachyphylaxis. Following the appropriate administered endomorphins studies.
preclinical toxicological studies, the clinical potential of spinally would appear to have gained theoretical support from the present
Acknowledgements This work was supported by ETT T-03 590/96 and OTKA 029817. Ketamine was generously provided by Parke-Davis Austria. The authors are grateful to Ildiko Dobos for her excellent technical assistance.
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and -2
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References 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
T.L. YAKSH and T.A. RUDY, J.Pharmacol.Exp.Ther. 202 411-413 (1977). J.E. ZADINA, L. HACKLER, L.-J. GE and A.J. KASTIN, Nature 386 499-502 (1997). L.S. STONE, CA. FAIRBANKS, T.M. LAUGHLIN, H.O. NGUYEN, T.M. BUSHY, M. W. WESSENDORF and G.L. WILCOX, NeuroReport 8 3 13 l-3 135 (1997). H.H. LOH, H.-C. LIU, A. CAVALLI, W. YANG, Y.-F. CHEN and L.-N. WEI, Mol.Brain Res. 54 321-326 (1999). I.E. GOLDBERG, G.C. ROSSI, S.R. LETCHWORTH, J.P. MATHIS, J. RYAN-MORO. L. LEVENTHAL, W. SU, D. EMMEL, E.A. BOLAN and G.W. PASTERNAK, J.Pharmacol.Exp.Ther. 286 1007-l 0 13 (1999). H.C. CHAMPION, J.E. ZADINA, A.J. KASTIN, L. HACKLER, L.-J. GE and P.J. KADOWITZ, Biochem.Biophys.Res.Commun. 235 567-570 (1997). H.C. CHAMPION, J.E. ZADINA, A.J. KASTIN and P.J. KADOWITZ, Peptides 19 925929 (1998). H.C. CHAMPION and P.J. KADOWITZ, Am.J.Physiol. 274(S) H1690-H1697(1998). R. SHANE, S. WILK and R.J. BODNAR, Brain Res. 815 278-286 (1999). V. CHAPMAN, A. DIAZ and A.H. DICKENSON, Br.J.Pharmacol. 122 1537-l 539 (1997). L.E. ROBSON, M.G.C. GILLAN AND H.W. KOSTERLITZ, Eur. J. Pharmacol. 112 65-71 (1985). T.L. YAKSH and T.A. RUDY, PhysiolBehav. 17 1031-1036 (1976). P.A.J. JANSSEN, C.J.E. NIEMEGEERS and J.C.G. DONY, Arzneim Forsch./Drug Res. 13 502-507 (1963). K. HARGREAVES, R. DUBNER, F. BROWN, C. FLORES and J. JORIS, Pain 32 77-88 (1988). T.L. PIERCE, M.D. GRAHEK and M.W. WESSENDORF, NeuroReport 9 385-389 (1998). S. MARTIN-SCHILD, J.E. ZADINA, A.A. GERALL, S. VIGH and A.J. KASTIN, Peptides 18 1645-1649 (1999). M. SCHERFF, S. SCHULZ, D. WIBORNY and V. HGLLT, NeuroReport 9 103 l-1034 1998). K. HOSOHATA and T.H. BURKEY, Eur.J.Pharmacol. 346 11 l-l 14 (1998). L.J. SIM, Q. LIU, S.R. CHILDERS and D.E. SELLEY, J.Neurochem. 70 1567-l 576 (1998). M. SPETEA, K. MONORY, Cs. TGMBijLY, G. TGTH, E. TZAVARA, S. BENYHE. J. HANOUNE and A. BORSODI, Biochem.Biophys.Res.Commun. 250 720-725 (1999). J.L.K. HYLDEN, D.A. THOMAS, M.J. IADAROLA, R.L. NAHIN and R. DUBNER, Eur.J.Pharmacol. 194 135-143 (1991). G. HORVATH, M. KOVACS, M.SZIKSZAY AND G. BENEDEK, Eur.J.Pharmacol. 253 61-66 (1994). J.DONNERER, R. SCHULIGOI and C. STEIN, Neuroscience 49 693-698 (1992). KREN and M.A. RUDA, NeuroReport 7 2 186-2190 (1996). M. SPETEA, F. OTVOS, G. TOTH, T.M. NGUYEN, P.W. SCHILLER, Z. VOGEL and A. BORSODI, Peptides 19 1091-1098 (1998).
ANTINOCICEPTIVE
EFFECTS OF INTRATHECAL
Gyongyi Horvatp,
Life Sciences, Vol. 65, No. 24, pp. 26352641, 199 Copyight 0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/993/$-see hat matter
ENDOMORPHIN-
1 AND -2 IN RATS
Margit SzikszayX, Csaba TGmbSly*, Gyorgy Benedek‘
“Department of Physiology, “Faculty of Health Science, Albert Szent-Gyorgyi Medical University, P.O. Box 427, Szeged, H-6701, Hungary; *Isotope Laboratory, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
(Received in final form August 5, 1999) Summary
Endomorphin-l and endomorphinwere recently postulated to be endogenous uopioid receptor agonists. We have investigated the antinociceptive and antihyperalgesic effects of intrathecally administered endomorphins in cumulative doses (0. l-l 00 pg) on acute and inflammatory pain sensations in awake rats. In the tail-flick test, both peptides caused a dose-dependent short-lasting antinociception. except at the highest dose, which caused motor impairment also. The dose-response curves revealed the development of acute tolerance (tachyphylaxis) to endomorphin. Similarly in the carrageenan-injected paw, the endomorphins (10 pg) exerted transient antinociceptive effects. These are the first data to demonstrate decreased responsivity in models of both acute and inflammatory pain after intrathecal administration of endomorphin- 1 and -2 in awake rats.
Key Words: antinociception, carrageenan, endogenous opioid peptides, endomorphin, hyperalgesia, opioid, spinal administration
Morphine and agonists exerting activity at the u-opioid receptor are well-known analgesics, and at least a proportion of their analgesic efficacy derives from activity at the spinal cord level (1). The existence of endogenous, selective neuropeptide agonists for both the 6- and ic-opioid receptors has repeatedly raised the question of the existence of selective, endogenous agonists for the u-opioid receptor. However, searches for endogenous p-opioid peptides based on the conserved aminoterminal sequence shared by the other endogenous opioid peptides have failed. The recently isolated p-opioid receptor-selective endogenous peptides endomorphin-l (Tyr-Pro-Trp-Phe-NH*) and endomorphin(Tyr-Pro-Phe-Phe-NH2) (2) from the bovine frontal cortex do not share this conserved sequence and for this reason may have been missed in previous screens. Correspondence to: Gyongyi Horvath Dept. Physiol. Albert Szent-Gyorgyi Medical P.O. Box: 427. H-6701 Szeged, Hungary Tel: 36-62-455101, Fax: 36-62-455842. email: [email protected]
University
2636
Intratliecal Effects of Endomorphin- and -2
Vol. 65, No. 24, 1999
Both of these peptides were reported to display high affinity and selectivity for the u-opioid receptor in vivo and in vitro (?;3) and may therefore be endogenous ligands for the u-opioid receptor. Endomorphin-l is 4000 and 15000 times as selective for the p-opioid receptor as compared to the 6- and ic-opioid receptors, respectively, whereas endomorphinis >13000 and > 7500 times as selective, respectively (2). Both peptides mediate naloxone-sensitive analgesia after intracerebroventricular or intrathecal injection, with similar potency to morphine in mice (2-S). Furthermore in u-receptor knockout mice the endomorphins do not cause any antinociceptive effect (4). After intravenous administration to anesthetized rabbits and rodents these peptides have also been demonstrated to exhibit hypotensive activity mediated by the release of nitric oxide from the endothelium of vascular bed (6-8), and they decrease the heart rate in rodents (7;8). Shane et al. observed the antinociceptive effect of endomorphinafter intracerebroventricular administration using the tailflick test in rats (9). The only data relating to the analgesic properties of spinal endomorphins originated from an electrophysiological study in anesthetized rats (10). Since the species differences in the concentrations and distributions of opioid binding sites is well-known, it is important to investigate the spinal antinociceptive effect of endomorphins in awake unrestrained rats (11). Furthermore little if anything is known about the effects of endomorphins on inflammatory pain sensation. The objective of the present investigation was to assess the antinociceptive effects of endomorphin-l and -2 after intrathecal administration in acute and inflammatory pain tests in awake rats. Methods Intrathecal catheterization After approval had been obtained from the Animal Care Committee of Albert Szent-Gyorgyi Medical University, 64 male Wistar rats weighing 200-300 g were studied. The rats were surgically prepared under ketamine-xylazine anesthesia (87 and 13 mg/kg intraperitoneally, respectively). An intrathecal catheter (PE-10 tubing) was inserted through a small opening in the cisterna magna and passed 8.5 cm caudally into the intrathecal space (12). After surgery, the rats were housed individually with free access to food and water and allowed to recover for at least 3 days before use. Rats showing postoperative neurologic deficits were not used. All experiments were performed in freely-moving animals during the same period of the day (8:00-13:00 h) in order to exclude diurnal variations in pharmacological effects. The animals were randomly assigned to treatment groups, and the observer was blind to the treatment administered. Drugs and their administration The following drugs were administered in this study: Ketamine (Ketalar, Parke-Davis, Austria), xylazine (Rompun, Bayer, FRG.), carrageenan (Sigma-Aldrich Kfi. Budapest, Hungary), and endomorphin-l and -2 synthesized using solid-state method and purified by means of HPLC in the Isotope Laboratory of Biological Research Center of the Hungarian Academy of Sciences. The peptides were dissolved in sterile physiological saline, freshly prepared on the day of the experiment, and were injected intrathecally over 30 s in a volume of 5 ~1, followed by a 10 ul flush of physiological saline. The drugs were administered in cumulative doses. Acute heat pain test (tail-flick test) The acute nociceptive sensitivity was assessed by using the tail-flick technique described by Janssen et al. (13). The reaction time in the tail-flick test was determined by immersing the lower 5 cm portion of the tail in the hot water until the typical tail-withdrawal response was observed (5 1.5
Vol. 65, No. 24, 1999
Intrathecal Effects of Endomorphin- and -2
2637
oC water, cut-off time: 20 s). Baseline latencies were obtained immediately before and then 10 and 30 min after the drug injections. Three series of experiments were performed with different cumulative doses of endomorphins, e.g. 0.1-0.3-l ug or l-3-10 ug or 10-30-100 ug (n=6131group). Inflammatory pain test (paw withdrawal test) The rats were placed on a glass surface in a plastic chamber and allowed to acclimatize to their environment for 15-30 min before testing, and baseline hindpaw withdrawal latencies (precarrageenan values at -180 min) were obtained. The heat stimulus was directed onto the plantar surface of each hindpaw. A detailed description of this method has been published elsewhere (14). Unilateral inflammation was induced by intraplantar injection of 2 mg carrageenan in 0.1 ml physiological saline into the right hindpaw. The paw withdrawal latencies were obtained again 3 h after carrageenan (post-carrageenan baseline values at 0 min), and then 10 and 30 min after the drug injections. The experiments were performed with different cumulative doses of endomorphins, e.g. l-3-10 ug (n=5-9/group). Data analysis Analgesic latencies in the acute pain test were converted to percentage maximum possible effects (% MPE) by using the formula % MPE = [(observed latency - baseline latency)/(cut-off time - baseline latency)]* 100 Data are presented as means f SEM. Analysis of variance (ANOVA) of data for repeated measures was used for overall effects, with the Newman-Keuls test for post-hoc comparison of means. A level of PcO.05 was considered significant. Results Effects of intrathecal endomorphins in acute pain test There was no significant difference in tail-flick latency between the groups before the drug administration. The overall mean tail-flick latency was 7.4 + 0.24 s (n=44). Both endomorphin-l and -2 caused a dose-dependent antinociceptive effect at 10 min with similar potency (Fig. 1.). The latency had decreased to below the baseline value at 30 min following the injections. This decrease was significant in some cases, suggesting hyperalgesia. The highest dose of endomorphin(100 ug) caused motor impairment too. The half-maximal effective dose (EDSo) for endomorphinat 10 min was 2.51 ug (4.05 nmol) [95% confidence interval = 1.122 - 5.618 ug = 1,82 - 9,lO nmol]. The dose-response curve revealed that 10 ug had a higher efficacy when it was applied in the first injection than following the third administration, suggesting the development of tachyphylaxis to endomorphin(Fig. 2.). Effects of intrathecal endomorphins on normal and inflamed paws There was no significant difference in the paw withdrawal responses to noxious thermal stimuli between the right and left hindpaws prior to the intraplantar injection of carrageenan. The overall mean paw withdrawal times for the ipsilateral and contralateral paws were 9.77 k 0.29 and 10.37 f 0.45 s (n=20), respectively. Carrageenan injection induced inflammation of the injected hindpaw, as evidenced by edema and erythema. The paw withdrawal latencies of the carrageenaninjected paw were significantly reduced to 2.1 * 0.05 s (P
2638
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had a significant short-lasting antinociceptive effect at the highest dose (10 ug), although they did not relieve the hyperalgesia (Fig. 3., right side).
Endoatozphin-2
Endomorphin-l 100 80 60
30
60
90
0
Time (minutes)
Inhibition of endomorphin10 ug: filled administration antinociception The results are control group. administrations.
30
60
Time (minutes)
Fig. 1 acute thermal nociceptive responses by endomorphin-l and treatments (control: open squares, 0.1-0.3-l ug: open circles, 1-3circles, 10-30-100 ug: filled squares). Cumulative intrathecal of endomorphin-l and -2 produced short-lasting, dose-dependent in the hot-water tail-flick test, with maximum efficacy at 10 min. expressed as means f S.E.M. *P
1
10
100
hes (Icg) Fig. 2 The dose-dependent effects of cumulative treatments with endomorphinat 10 min after injection (0.1-0.3-l ug: circles, l-3-10 ug: triangles, 10-30-100 ug: squares) on tail-flick latencies. The results are expressed as means z!zS.E.M.
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Intrathecal Effects of Endomorphin- and -2
Vol. 65, No. 24, 1999
2639
Discussion This is the first study to examine the antinociceptive potency of intrathecally administered endomorphins on carrageenan-induced thermal hyperalgesia in rats. We have shown that the intrathecal endomorphin-l and -2 caused a short-lasting antinociception on tail-flick and carrageenan-induced thermal hyperalgesia, with similar potency. The dose-response curve revealed the development of tachyphylaxis to endomorphins.
Right Paw
Left Paw 12.
12,
10 1
8
s
6.
;
4. 2
carrageenan
Time (min)
Fig. 3 Attenuation of the inflammatory pain sensation by endomorphin-l and -2 (l-3-10 up). None of the treatments influenced the heat-pain latency of the non-injected paws (left side). Endomorphin-l (filled circles) and endomorphin(tilled triangles) at the highest dose significantly attenuated the carrageenan-induced thermal hyperalgesia at 10 min after endomorphin injection (right side), saline treatment: open squares. The results are expressed as means f S.E.M. xP<0.05 compared to post-carrageenan baseline values. *P
Radioimmunoassay studies have localized the endomorphin-l immunoreactivity within the bovine central nervous system (2). Antisera were raised against endomorphinto localize this peptide in the spinal cords of rats and the rhesus macaque (15-17). Dense endomorphinimmunoreactivity was found in the marginal zone, the substantia gelatinosa and ventral to the central canal. Additionally, endomorphinimmunoreactivity was dramatically reduced following dorsal rhizotomy. These data suggest that endomorphinoccurs in the small diameter primary afferent fibers in rodents and primates. It appears possible that the release of neurotransmitters from nociceptive primary afferents might be regulated by the release of endomorphinfrom primary afferent terminals. Recent studies examined the ability of these peptides to stimulate G-protein activation via the uopioid receptor (18-20). These findings demonstrate that the endomorphins exert activity similar to that of morphine at the u-opioid receptor and suggest that these peptides have the potential to modulate neuronal activity in viva We observed that the antinociceptive min after intrathecal administration.
effect was significant at 10 min and it had disappeared at 30 Therefore putative endogenous u-opioid receptor agonists
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endomorphin-l and -2 are potent but short-acting antinociceptive agents in the spinal cord of rats in agreement with the earlier results (3;5;7;9). The behavioral analgesic effects of intrathecally administered endomorphins are comparable to the effects of spinally applied endomorphins on the C-fiber evoked neuronal response (10). Chapman et al. have shown that intrathecal endomorphin-l and -2 (0.25-50 pg) reduced all components of the electrically evoked C-fiber responses of spinal neurons in anesthetized rats, in a dose-related manner. The peak inhibitory effects of the endomorphins were observed at 15-20 min post-administration. We found that the two compounds have similar potencies and time courses, and they are also effective antihyperalgesic agents in the model of carrageenan-induced inflammatory pain. It is well established that the antinociceptive potency of spinal opioids is enhanced in inflammatory states (21). In agreement with this, 10 ug endomorphins significantly decreased the unilateral thermalhyperalgesia, but did not influence the normal paw latencies. There is some dicrepancy between the tail-flick and paw withdrawal results, i.e. same dose of endomorphins (10 pg) caused about 40 %MPE in the tail-flick test, but we could not observe any antinociceptive effect on the normal paw on the paw withdrawal test. There are several possibilities to explain these observation. First, there are some data suggesting that carrageenan-induced inflammation causes a slight increase in the pain sensitivity at the contralateral paw (22) which might be due to the bilateral release of neuropeptides (e.g. substance-P and calcitonin gene related peptides) (23) and activation of c-FOS (24). This fact might explain the lower efficacy of endomorphins at paw withdrawal test. Second, there are some differences between either the two methods (contact heat vs radiant heat) or the sensitivity of the tail and paw. This would agree with preliminary experiments when we determined both the paw- and the tail-withdrawal radiant heat pain latencies in normal rats and observed that endomorphin was more effective on the tail than on the hindpaws (data are not shown). Endomorphinand -2 significantly attenuated the thermal hyperalgesia with a short time course similar to the observed in the tail flick assay. The transient action observed in both tests suggests tachyphylaxis or fast degradation of the peptides. This latter option seems likely in view of the longer duration of action of 100 ug endomorphin-2, and the longer duration of synthesized analogs @X,9,25).The continuous infusion of these compounds or more stable, longer-lasting analogs may prove to be useful therapeutically against acute or inflammatory pain. Some promising substances are the recently synthesized analogs of endomorphin-2, D-[Ala’lendomorphin-2 and D-proline*endomorphin(8;9;25). The magnitude of responses (cardiovascular or antinociceptive) were not different, but the response duration was longer with the analogs. This finding may be interpreted as suggesting that the D-amino acid substitution inhibits the degradation of the peptide by proteolytic processing and increases the duration of action. Similarly to the results of Stone et al. in mice (3), we observed the rapid development of tachyphylaxis. These results suggests that these compounds may serve as useful tools to study of the mechanism of action of compounds modulating either the development or the expression of tachyphylaxis. Following the appropriate administered endomorphins studies.
preclinical toxicological studies, the clinical potential of spinally would appear to have gained theoretical support from the present
Acknowledgements This work was supported by ETT T-03 590/96 and OTKA 029817. Ketamine was generously provided by Parke-Davis Austria. The authors are grateful to Ildiko Dobos for her excellent technical assistance.
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