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Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat
European Journal of Pharmacology, 237 (1993) 73-81
73
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53120
Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat Christine Gouard~res, Maaja Sutak
a
Jean-Marie Zajac and Khem Jhamandas a
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse C~dex, France and a Department of Pharmacology, Queen's Uniuersity, Kingston, Ontario K7L3N6, Canada Received 12 November 1992, revised MS received 17 February 1993, accepted 23 March 1993
The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRFamide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5/zmol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmoi) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions. F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2); FMRFamide (Phe-Met-Arg-Phe-NH2); Naloxone; Morphine; Spinal antinociception
1. Introduction
The octapeptide F8Famide ( F L F Q P Q R F a m i d e , neuropeptide FF) was isolated from bovine brain by Yang et al. (1985) using antisera against the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH 2) (Dockray et al., 1981). Since this discovery, a number of authors have reported that F8Famide-like immunoreactivity is widely distributed in brain and spinal cord areas, especially those involved in nociceptive signal transmission (Yang et al., 1985; Majane et al., 1989; Allard et al., 1991). The spinal F8Famide-like immunoreactivity appears to be derived from two neuronal sources: descending spinal fibres originating from brain regions, and local interneurons (Ferrarese et al., 1986; Panula et al., 1987; Majane et al., 1989; Kivipelto et al., 1989; Allard et al., 1991; Kivipelto and Panula, 1991). Allard et al. (1989) have demonstrated F8Famide binding sites, distinct from opioid receptor sites, which may
Correspondence to: C. GouardBres, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse C~dex, France. Tel. (33) 61 17 59 73, fax (33) 61 17 59 94.
represent specific receptors for F8Famide and related neuropeptides. High levels of these sites have been found in the dorsal spinal cord (Allard et al., 1992), a region that plays a critical role in pain transmission. However, the functional significance of F8Famide binding sites is unknown. In pharmacological experiments, intracerebroventricular (i.c.v.) administration of F8Famide and FMRFamide was reported to attenuate spontaneous and opiate-induced analgesia in rats (Tang et al., 1984; Yang et al., 1985) and mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). Several studies in which agents were injected i.c.v, have reported a potential role of F8Famide in opioid tolerance and dependence (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991). In addition to anti-opioid actions, some studies have shown pro-opioid effects of F8Famide. F8Famide and A18Famide decreased colonic bead expulsion time in mice (Raffa and Jacoby, 1989), and recently synthetic analogues of F8Famide were reported to inhibit morphine analgesia while other analogues produced analgesic effects after i.c.v, injection (Gicquel et al., 1992). Thus, these peptides have the potential to both inhibit and mimic the action of opioids. However, fewer studies have focused
74 on the action of F8Famide or F M R F a m i d e in the spinal cord. In rats, intrathecally administered F M R Famide inhibited the antinociceptive effect of spinal morphine in the tail flick test, but was inactive by its own (Tang et al., 1984). In electrophysiological studies, F8Famide was reported to attenuate the inhibitory effect of /x-opioid agonists and a2-adrenoceptor agonists on the C-fibre evoked firing of rat spinal neurons (Magnuson et al., 1990; Sullivan et al., 1991). Guzman et al. (1989) described a transient hyperpolarization and subsequent depolarization of cultured mouse spinal neurons exposed to F8Famide. These studies suggest a role for F8Famide in the modulation of nociception at the spinal level. However, at present, little is known about the actions of F8Famide in different models of nociception and about the influence of opioids and nonopioids in these actions. To gain insight into the role of F8Famide in spinal nociception, the present study evaluated the comparative effects of intrathecal F8Famide and the molluscan peptide F M R F a m i d e in two different nociceptive tests in the rat, the tail flick and paw pressure tests. The interaction of these peptides with morphine and naloxone was also examined. This communication describes for the first time a long-lasting antinociceptive spinal action of F8Famide and FMRFamide and their differential effect on morphine-induced antinociception.
2. Materials and methods
peptides. Control animals received an intrathecal injection of an equal volume of saline vehicle.
2.2. Nociceptive assays The nociceptive responses were assessed using the response evoked by thermal (tail flick; D ' A m o u r and Smith, 1941) and mechanical (paw pressure; Randall and Selitto, 1957) stimuli. Baseline tail flick latency was 2 - 3 s. To minimize tissue damage, a 10-s cut-off was imposed (Loomis et al., 1985). The responsiveness to paw pressure was tested using a modification of the procedure of Loomis et al. (1987). Briefly, mechanical pressure was applied to the upper surface of the hind paw until a withdrawal response was observed. The control and cut-off values were 90-110 and 300 m m Hg, respectively. Rats were handled for both tests. They were tested in groups of six. Two determinations were made with each animal, and each animal was used only once. Tests were applied in tandem, with the tail flick determination preceding the paw pressure test. The effects of peptides were evaluated at 10-min intervals for the first 60 min and every 30 min thereafter up to 180 rain. The tail flick and paw pressure responses were assessed 24 and 48 h or longer after the intrathecal injection. All experiments were performed in the morning. At the end of nociceptive tests, the correct placement of catheters in lumbar space was confirmed by intrathecal dye injection. Animals in which catheters were not located in the lumbar region were excluded from experiments reported in this study.
2.1. Intrathecal cannulations and drug injections
2.3. Data analysis
Male Sprague-Dawley rats (250-300 g, Charles River Inc., St. Constant, Que., Canada) were anaesthetized with halothane, and chronic lumbar intrathecal polyethylene catheters (PE 10) were inserted through the cisterna magna to the rostral edge of the lumbar enlargement (7.5 to 8 cm) (Yaksh and Rudy, 1976). The catheters were then flushed with 10 ~1 of physiological NaCI solution. The animals were placed in individual cages for a 4-day recovery period before being used in experiments. Animals showing neurological deficits (flexion of hindlimbs, rigidity or paralysis) were discarded. All drugs for spinal administration were p r e p a r e d in a final volume of saline such that the total dose was delivered in 10 /xl through the indwelling spinal catheter, using a 50-/zl Hamilton syringe. Drug injection was followed by an additional injection of 10/zl of saline to flush the catheter. Naloxone was given i.p. (0.1 ml per 100 g rat weight) 10 rain before intrathecal
All data for response latency (s) or threshold response pressure (mm Hg) were converted to maximum percent effect (MPE) calculated as: M P E = 100 x [(post-drug r e s p o n s e - baseline r e s p o n s e ) / ( c u t - o f f response - baseline response)]. The area under the M P E vs. time curve (AUC) was computed by trapezoidal approximation of the A U C for each rat over two periods: 10-60 and 60-180 min. The A U C is expressed as a percentage of the maxim u m possible A U C that could be obtained if the animal displayed a 100% M P E at each testing time over the experimental period. Statistical significance (to analyse the data with groups, times, doses) was calculated by using a oneor two-way analysis of variance ( A N O V A ) with multiple comparisons followed by appropriate tests ( S T A T V I E W program, Macintosh). Results are presented as the entire time course or as the area under the curve approximation. The differences were consid-
75 FMRFamide
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Fig. 1. T i m e course of the effects o f intrathecally administered F 8 F a m i d e ( A , B ) and F M R F a m i d e ( C , D ) in the tail flick and paw pressure tests. D a t a are m e a n s + S . E . M . o f M P E f r o m 6 t o 11 animals. Saline ( o ) . F 8 F a m i d e (A, B): 0.44 nmol (o), 4.4 n m o l ( [ ] ) , 17.5 n m o l ( • ) . F M R F a m i d e ( C , D ) : 0.05 n m o l (o), 8.8 n m o l ( [] ), 25 nmol ( • ).
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from E n d o Laboratories ( U . S . A . ) and m o r p h i n e sulphate from B D H Pharmaceuticals (Canada).
2.4. Drugs 3. Results Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2 (F8Fa m i d e ) was s y n t h e s i z e d by solid-phase m e t h o d s , as previously described ( G i c q u e l et al., 1992). F M R F a m i d e ( P h e - M e t - A r g - P h e - N H 2) was p u r c h a s e d from P e n i n s u l a Laboratories. N a l o x o n e hydrochloride was
3.1. Behavioural effects T h e intrathecal injection of F 8 F a m i d e (0.05 to 17.5 n m o l ) or F M R F a m i d e (0.002 to 25 n m o l ) had no apFMRFamide
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Fig. 2. Dose-response curves for the initial (10-60 rain, o) and the delayed (60-180 min, o) spinal antinociceptive effect of FSFamide (A, B) and FMRFamide (C, D) in the tail flick and paw pressure tests. Data are means+ S.E.M. of % AUC from 6 to 21 animals. For each curve, the values marked * and those obtained with higher doses are considered to be statistically different from corresponding control saline value (P < 0.05).
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DOSES (nmol) Fig. 3. Antinociceptive responses 24 ( [ ] ) and 48 ( [ ] ) h after intrathecally administered F8Famide (A, B) and FMRFamide (C, D) in the tail flick and paw pressure tests. Data are means_+ S.E.M. of MPE from 6 to 21 animals. * Indicates significant differences (P < 0.05) from control saline (dose 0).
parent effect on the general behaviour of the rats or on normal motor function. Very high intrathecal doses of peptides tended to produce an impairment of hindlimb function in some animals, and therefore such doses were not included in our study.
(fig. 1). In fact, examination of the individual time-response curves revealed a biphasic action of F8Famide in both tests at all doses. The first response (increase in tail flick latency or elevation of paw pressure threshold) peaked at 30-40 min and the second response peaked between 150-180 min (fig. 1A, B). The effect of FMRFamide in both tests was characterized by a rapid onset (10-20 min), followed by a gradual increase in response over time, the peak effect occurring 120-180 min after injection (fig. 1C, D). Figure 2 shows the dose-response curves for the initial and the delayed effects of F8Famide (fig. 2A, B) and FMRFamide (fig. 2C, D) in the two tests. As
3.2. Antinociceptive effects The effects of various doses of intrathecally administered F8Famide and FMRFamide in the tail flick and paw pressure tests are illustrated in figs. 1-3. In both tests, these peptides showed dose-related and long-lasting antinociceptive effects but with different patterns
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Fig. 4. Effects of naloxone on the time course of spinal F8Famide (A)- or FMRFamide (B)-induced antinociception in the tail flick test. Rats were injected intrathecally with the peptides (8.8 nmol) 10 min after i.p. saline (©) or naloxone (5.5 /zmol/kg, o). Effects of i.p. naloxone (5.5 /zmol/kg) alone (12]). Data are means_+ S.E.M. of MPE from six animals. * Indicates significant differences from the action of peptide alone
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77 min before 8.8 nmol intrathecal peptides) significantly attenuated the initial phase of the antinociceptive response produced by F8Famide (fig. 4A) and FMRFamide (fig. 4B). In contrast, naloxone significantly enhanced the second phase of the response to F8Famide (fig. 4A) but did not influence the late response produced by FMRFamide (fig. 4B). In naloxone-treated animals, the antinociceptive effects of F8Famide were still apparent 24 h after the peptide injection (fig. 4A). The actions of systemic naloxone were similar in the paw pressure test (data not shown).
shown, at all doses used, the magnitude of the delayed response was higher than the initial response. Over the range of doses used, dose-response relationships for the two peptides were found only in the tail flick test. In this test, the EDs0 values for F8Famide were 12 and 7 nmol for the initial and delayed effects, respectively. Corresponding values for FMRFamide were 15 and 3 nmol. In the paw pressure test, the magnitude of the effect was lower, as evidenced by the flat dose-response curves. The EDs0 values were higher than 20 nmol for both peptides. In both analgesic tests, the effect of F8Famide and FMRFamide was apparent 24 and 48 h after injection (fig. 3). In the case of F8Famide (fig. 3A, B), this delayed antinociception was seen in the dose range 2.5-17.5 nmol, and in the case of FMRFamide (fig. 3C, D), it was apparent in the dose range 0.05-25 nmol. Although not examined systematically, the sustained antinociception was not a manifestation of motor dysfunction. All animals showed normal ambulatory behaviour and recovery from the effect of the peptide. Depending on the dose used, the elevated latencies or thresholds typically returned to baseline levels between 3 or 4 days.
3.4. Effects of F8Famide or FMRFamide on spinal morphine antinociception The effects of a subeffective intrathecal dose of F8Famide or FMRFamide on spinal morphine analgesia in the tail flick and paw pressure tests are represented in fig. 5. Morphine (6.6 nmol) produced a monophasic antinociceptive effect which reached a peak around 30 rain and returned to baseline by 60-90 min in both tests. Intrathecal co-injection of F8Famide (0.05 nmol) increased the magnitude of the effect of morphine from 60.0 + 2.3 (MPE, peak effect) to 71.4 _+ 5.5 in the tail flick test (fig. 5A). This co-injection also produced a marked prolongation of the effect of morphine. The tail flick response returned to the pre-drug level 90 rain after injection of morphine alone. However, after co-injection of F8Famide and morphine, the tail flick response remained elevated at this time (MPE = 81.5 _+ 7.1) and this effect still persisted 180 rain after injection (MPE = 61.2 + 7.4) (fig. 5A). Twentyfour hours after the co-injection, the response was still
3.3. Effects of naloxone on spinal F8Famide FMRFamide antinociception Figure 4 shows the results of experiments in which the effect of i.p. naloxone on peptide-induced responses was tested. As shown (fig. 4A), naloxone (5.5 p~mol/kg) by itself had no effect on the tail flick response. However, this dose of naloxone (given i.p. 10 FSFamide
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T I M E AFTER I N J E C T I O N Fig. 5. E f f e c t s o f F 8 F a m i d e ( A , B) a n d F M R F a m i d e (C, D ) o n t h e t i m e c o u r s e o f t h e a n t i n o c i c e p t i o n i n d u c e d by s p i n a l m o r p h i n e (6.6 n m o l , o ) in t h e tail flick a n d p a w p r e s s u r e tests. F 8 F a m i d e (0.05 n m o l , ~2 ) o r F M R F a m i d e (0.002 n m o l , [ ] ) w a s c o - i n j e c t e d i n t r a t h e c a l l y w i t h m o r p h i n e ( • ) .
Data are means+ S.E.M. of MPE from 6 to 17 animals. * Indicates significant differencesfrom the action of morphine alone (P < 0.05).
78
significantly above control values (MPE = 19.7 _+ 4.8). Full recovery from the antinociceptive effects was seen 48 h after injection. In the paw pressure test (fig. 5B), a similar augmentation of morphine-induced effect was observed. In contrast with the action of F8Famide, 0.002 nmol FMRFamide injected with morphine significantly attenuated the antinociceptive action of the opiate in both the tail flick and paw pressure tests during the initial 50- and 30-min periods, respectively (fig. 5C, D). However, after this, a sustained antinociceptive action of the co-injected agents was apparent in both tests. Full recovery from the antinociceptive effect occurred 24-48 h after the injection. T h u s combination e x p e r i m e n t s showed that F8Famide enhanced the action of morphine and that this action was attenuated by FMRFamide during the 10- to 60-min period after injection. Both peptides prolonged the effect of morphine in the 60- to 180-min period. The enhancement of opioid antinociception occurred at doses of the peptides that had an insignificant effect when injected alone.
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Fig. 6. Modulation of spinal morphine antinociception by F8Famide and F M R F a m i d e in the tail flick and paw pressure tests. Rats were treated intrathecally with saline (S), morphine (M, 6.6 nmol). F8Famide (F8, 2.5 nmol), F M R F a m i d e (FM, 2.5 nmol), or a combination of morphine with each peptide ( F 8 + M , F M + M ) . The antinociceptive effect is expressed as % A U C from 10 to 60 rain (A, B) and 60 to 180 min (C, D) after drug administration. Data are m e a n s + S.E.M. of % A U C from 6 to 17 animals. Values significantly different (P < 0.05) from control saline (+), peptide alone (°) and morphine alone (*).
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Fig. 7. Modulation of spinal morphine antinociception by F8Famide and F M R F a m i d e in the tail flick test. Rats were treated intrathecally with saline (S), morphine (M, 6.6 nmol), F8Famide (F8, 2.5 nmol), F M R F a m i d e (FM, 2.5 nmol), or a combination of morphine with each peptide ( F 8 + M , F M + M ) . The antinociceptive response is expressed as M P E observed at 24 h (A) and 48 h (B) after drug administration. Data are m e a n s _+S.E.M. of M P E values from 6 to 17 animals. Values significantly different (P < 0.05) from control saline (+), peptide alone (°) and morphine alone (*).
To investigate the effect of antinociceptive doses of F8Famide or FMRFamide on spinal morphine analgesia, 6.6 nmol morphine and 2.5 nmol of each peptide were co-injected intrathecally. The individual and combined antinociceptive effects are expressed as % AUC (as described under Methods) from 10 to 60 rain and 60 to 180 min after injection (fig. 6) and as MPE obtained at 24 and 48 h (fig. 7). The antinociceptive effect of morphine (6.6 nmol) in both tests was reduced 60 min after injection (fig. 6) and was not significantly different from that of control saline-treated animals 24 and 48 h later (fig. 7). Intrathecal administration of F8Famide or FMRFamide (2.5 nmol) produced similar long-lasting antinociceptive responses that were different from that of morphine (figs. 6, 7). The response was elevated 60 min after injection (fig. 6) and declined thereafter to return to control saline values by 48 h (fig. 7). In the paw pressure test, in the initial as well as in the delayed period, each peptide co-injected with morphine produced an analgesic effect which corresponded to the sum of their individual effects (fig. 6B, D). This additivity of the analgesic effects of peptides co-injected with morphine was also observed in the tail flick test, but only during the 60- to 180-min period (fig. 6C). In this test, F8Famide did not modify the effect of morphine in the first hour (10-60 min), whereas FMRFamide decreased this effect (fig. 6A) as did a subeffective dose (fig. 5C). Measurement of nociceptive responses at 24 h and 48 h showed that the tail flick latency of the F8Famide and morphine co-injected group of animals at 24 h was elevated well above that observed in the morphine- or peptide-injected groups (fig. 7A). This elevation was not observed in the FMRFamide- and morphine-
79
treated animals. A similar profile of activity was observed for the combination 48 h after injection (fig. 7B) but the magnitude of response was lower than that seen at 24 h. The combination of morphine with each peptide did not change the paw pressure threshold observed in the corresponding peptide-injected group (data not shown). None of the animals in this part of the study showed a visible impairment of motor function.
4. Discussion
The present study demonstrates that spinal administration of F8Famide, an endogenous mammalian opioid modulatory peptide (Yang et al., 1985), and FMRFamide, a molluscan peptide (Dockray et al., 1981), produces dose-related and sustained antinociception against thermal and mechanical stimuli. Previous studies, largely involving i.c.v, injections and the tail flick test, have not indicated that these peptides have antinociceptive effects but have described their antiopioid properties in rats (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991) and mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). Tang et al. (1984) have shown the same results after intrathecal administration of FMRFamide in the rat. However, one study has reported that i.c.v. F M R F a m i d e exerted an antinociceptive effect in the mouse hot plate test (Hill et al., 1987). The reason for this discrepancy between the results of i.c.v. and intrathecally administered peptides is not clear. However, the dissociation seen here is not without precedence. Norepinephrine and az-adrenoceptor agonists produce potent antinociception after spinal but not i.c.v, injection (Reddy et al., 1980; Ossipov and Gebhart, 1983). It is possible that sites targeted by intrathecal F8Famide are a part of an endogenous mechanism modulating nociceptive transmission at a spinal level. The presence of a high density of F8Famide immunoreactivity (Yang et al., 1985; Majane et al., 1989; Allard et al., 1991) as well as binding sites (Allard et al., 1989, 1992) in the dorsal cord tends to support this notion. However, the possibility that the action of F8Famide at the spinal level is modulated by supraspinal mechanisms cannot be excluded. An unusual characteristic of the action of F8Famide in both tests was its biphasic action, a rapid initial response followed by an extended response that was prominent in the 60- to 180-min observation period but which persisted in the 24- to 48-h period after injection. FMRFamide also produced sustained antinociception. This extended response is reminiscent of the sustained spinal action of dynorphin in the tail flick test (Jhamandas et al., 1986) that has been associated with motor impairment (Faden and Jacobs, 1984; Her-
man and Goldstein, 1985). However, unlike dynorphin injection, administration of antinociceptive doses of the peptides in this study produced no visible motor impairment and all animals showed normal ambulation and full recovery from antinociception. The two phases of the antinociceptive response evoked by thermal stimuli in the present work showed a differential sensitivity to systemic naloxone. Both the dose and route of naloxone administration did not affect the baseline responses. Naloxone attenuated the early but not late response, suggesting involvement of opioid mechanisms in this phase. In the rat spinal cord, F8Famide receptors (Allard et al., 1992) and opioid receptors (Gouard6res et al., 1985, 1991, 1993) are similarly distributed. However, spinal F8Famide binding sites exhibit a very low affinity for opioid agonists (Allard et al., 1989) and F8Famide does not bind to opioid receptors in vitro (data not shown). Therefore, it is unlikely that the naloxone-sensitive response is due to direct opioid receptor stimulation by F8Famide. This response probably involves an indirect action by release of an endogenous opioid peptide. Surprisingly, naloxone did not antagonize the action of F8Famide in the paw pressure test. This suggests that the dose of naloxone may have been inadequate or that the response involves an interaction of the peptide with a non-opioid mechanism. The use of higher antagonist doses was avoided because of the development of hyperalgesia or analgesia (Sawynok et al., 1979) due to the action of naloxone itself. The possibility of a differential action of F8Famide in the tail flick and paw pressure tests is suggested by previous results showing that different neuromodulatory systems participate in thermal and mechanical nociception (Kuraishi et al., 1983, 1985a,b). In contrast, mechanical antinociception induced by FMRFamide during the first hour after injection was reversed by naloxone. This suggests the existence of different mechanisms mediating the activity of F8Famide and FMRFamide in the paw pressure test. Opioid mechanisms appear to be involved in the sustained response evoked by both peptides. Although the basis for its appearance is unknown, the possibility that this response is caused by metabolites of the peptides or their rostral redistribution to the brain cannot be discounted. It would be of interest to determine if inhibition of metabolism or use of stable peptide analogues (Gicquel et al., 1992) also yields a similar activity profile. The sustained response to F8Famide and FMRFamide could also involve recruitment of non-opioid mechanisms, such as desensitization to the action of a nociceptive substance released from primary afferents or reinforcement of descending inhibitory controls. Previous reports have described the antagonism of morphine or other opioids by F M R F a m i d e or
80 F8Famide given i.c.v, in rats (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991) or mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). In the present study, which focused on the rat spinal cord and examined the action of both sub-antinociceptive and antinociceptive doses of peptide, F8Famide did not antagonize the effect of morphine. In contrast, the low dose of peptide, which was inactive on its own, produced a remarkable enhancement of morphine-induced antinociception in both tests. The action of intrathecal morphine, which normally lasted for 60-90 min, was significantly extended to 24 h after injection of a sub-analgesic dose of F8Famide. An antinociceptive dose of the peptide produced the same effect. In contrast, in both tests, a sub-antinociceptive dose of FMRFamide attenuated the action of morphine in the early phase (10-60 min), as was previously reported (Tang et al., 1984), but extended this effect later. An analgesic dose of FMRFamide decreased the initial action of morphine only in the tail flick test, but prolonged its action in the later period. Thus, the antagonist action of FMRFamide against the action of morphine in the early phase was not seen with F8Famide. The reasons for this difference between the action of the two peptides are not known, but the results suggest that the antiopioid component of F M R F a m i d e was not apparent in the action of FSFamide. The fact that F8Famide increased morphine analgesia, an agonist that preferentially activates ~-type opioid receptors in the spinal cord (Tung and Yaksh, 1982), suggests that endogenous F8Famide in the spinal cord may function to amplify the action of endogenous opioids that normally interact with mu receptors. The mechanism of interaction of F M R F a m i d e with the opioid system could be different in regard to the results mentioned above. In addition, FMRFamide exhibits a weak affinity for opioid receptors (Zhu and Raffa, 1986) and displays, in contrast to F8Famide, a low affinity for F8Famide binding sites in the rat spinal cord (Allard et al., 1989). These findings could explain the differential modulation of the effects of morphine by these peptides after intrathecal injection. In fact, F8Famide and F M R F a m i d e may have a role in modulating sensory transmission via different neurochemical systems (Kuraishi et al., 1983, 1985a,b; Satoh et al., 1991) in the dorsal horn of the rat spinal cord. The enhancement of the effect of morphine seen in this behavioural study contrasts with the antagonism of the effect of D A G O (Tyr-D-Ala-Gly-(N-Me)Phe-Glyol, a/~-opioid agonist) by F8Famide in an electrophysiological study of C-fibre-driven spinal nociceptive neurons in anaesthetized rats (Magnuson et al., 1990). The reason for this discrepancy is not clear but factors such as anaesthesia, nociceptive stimulation modality, peptide dose and observation period could contribute to
the differences between behavioural and electrophysiological experiments. Moreover, these authors have also reported that the activity of D S T B U L E T (Tyr-DSer(OtBu)-Gly-Phe-Leu-Thr), a a-opioid agonist, is not modified by F8Famide. It remains to be determined if the augmentation of antinociception seen here is specific to morphine or extends to other antinociceptive agents. Although the sustained response induced by intrathecal administration of F8Famide may seem unusual, it has been reported that intrathecal injection of substance P can produce a very long-lasting change in the response to intrathecal norepinephrine (Nance and Sawynok, 1987). The presence of F8Famide-like immunoreactivity in the dorsal horn of the rat spinal cord (Majane et al., 1989; Allard et al., 1991), the presence of its own receptors in the same region (Allard et al., 1989, 1992), together with its effects on dorsal horn neurons (Magnuson et al., 1990; Sullivan et al., 1991), suggest that F8Famide may have a dual action, acting as a transmitter of nociceptive information as well as producing antinociception, these effects being elicited at different sites. An alternative possibility is that F8Famide in the spinal cord may be involved in modulating functions other than nociception.
Acknowledgements This work was supported in part by grants from the Medical Research Council (MRC) of Canada (MRC-MI5690 to K.J.) and funds from INSERM (CRE 910815) and CNRS, France. C.G. was sponsored by a Visiting Scientist award from the MRC and the CNRS.
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81 Ferrarese, C., M.J. ladorola, H.-Y.T. Yang and E. Costa, 1986, Peripheral and central origin of Phe-Met-Arg-Phe-amide immunoreactivity in rat spinal cord, Reg. Pept. 13, 245. Gicquel, S., H. Mazarguil, M. Allard, G. Simonnet and J.-M. Zajac, 1992, Analogues of F8Famide resistant to degradation, possessing high affinity and in vivo effects, Eur. J. Pharmacol. 222, 61. Gouard6res, C., J. Cros and R. Quirion, 1985, Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea-pig spinal cord, Neuropeptides 6, 331. Gouard6res, C., A. Beaudet, J.-M. Zajac, J. Cros and R. Quirion, 1991, High resolution radioautographic localization of [12SI]FK33824-labelled mu opioid receptors in the spinal cord of normal and deafferented rats, Neuroscience 43, 197. Gouard~res, C., S. Tellez, J.A.M. Tafani and J.-M. Zajac, 1993, Quantitative autoradiographic mapping of &opioid receptors in the rat central nervous system using [~2Sl] [D.AlaZ]deltorphin-I, Synapse 13, 231. Guzman, A., P. Legendre, M. Allard, S. Geoffre, J.D. Vincent and G. Simonnet, 1989, Electrophysiological effects of FLFQPQRFamide, an endogenous brain morphine modulating peptide, on cultured mouse spinal cord neurons, Neuropeptides 14, 253. Herman, B.H. and A. Goldstein, 1985, Antinociception and paralysis induced by intrathecal dynorphin A, J. Pharmacol. Exp. Ther. 232, 27. Hill, R.G., J. Hughes and K.M. Pittaway, 1987, Antinociceptive action of cholecystokinin octapeptide (CCKS) and related peptides in rats and mice: effects of naloxone and peptidase inhibitors, Neuropharmacology 26, 289. Jhamandas, K., M. Sutak and S. Lemaire, 1986, Comparative spinal analgesic action of dynorphin 1-8, dynorphin 1-13, and a kappareceptor agonist U50,488, Can. J. Physiol. Pharmacol. 64, 263. Kavaliers, M., 1990, Inhibitory influences of mammalian FMRFamide (Phe-Met-Arg-Phe-amide)-related peptides on nociception and morphine and stress-induced analgesia in mice, Neurosci. Lett. 115,307. Kavaliers, M. and H.-Y.T. Yang, 1989, IgG from antiserum against endogenous mammalian FMRF-NH2-related peptides augments morphine and stress-induced analgesia in mice, Peptides 10, 741. Kivipelto, L. and P. Panula, 1991, Central neuronal pathways containing FLFQPQRFamide-like (morphine-modulating) peptides in the rat brain, Neuroscience 41, 137. Kivipelto, L., E.A. Majane, H.-Y.T. Yang and P. Panula, 1989, Immunohistochemical distribution and partial characterization of FLFQPQRFamide-like peptides in the central nervous system of rats, J. Comp. Neurol. 286, 269. Kuraishi, Y., Y. Harada, S. Aratani, M. Satoh and H. Takagi, 1983, Separate involvement of the spinal noradrenergic and serotoninergic systems in morphine analgesia: the differences in mechanical and thermal analgesic tests, Brain Res. 273, 245. Kuraishi, Y., N. Hirota, Y. Sato, Y. Hino, M. Satoh and H. Takagi, 1985a, Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn, Brain Res. 325, 294. Kuraishi, Y., N. Hirota, M. Satoh and H. Takagi, 1985b, Antinociceptive effects of intrathecal opioids, noradrenaline and serotonin in the rats: mechanical and thermal analgesic tests, Brain Res. 326, 168. Lake, J.R., M.V. Hammond, R.C. Shaddox, L.M. Hunsicker, H.-Y.T. Yang and D.H. Malin, 1991, IgG from neuropeptide FF antiserum reverses morphine tolerance in the rat, Neurosci. Lett. 132, 29. Loomis, C.W., F.W. Cervenko, K. Jhamandas, M. Sutak and B. Milne, 1985, Analgesia and autonomic function following intratbecal administration of morphine and norepinephrine to the rat, Can. J. Physiol. Pharmacol. 63, 656. Loomis, C.W., B. Milne and F.W. Cervenko, 1987, Determination of
cross tolerance in rat spinal cord using intrathecal infusion via sequential mini-osmotic pumps, Pharmacol. Biochem. Behav. 26, 131. Magnuson, D.S.K., A.F. Sullivan, G. Simonnet, B.P. Roques and A.H. Dickenson, 1990, Differential interactions of cholecystokin and FLFQPQRF-NH 2 with /~ and 6 opioid antinociception in the rat spinal cord, Neuropeptides 16, 213. Majane, E.A., P. Panula and H.-Y.T. Yang, 1989, Rat brain regional distribution and spinal cord neuronal pathway of FLFQPQRFNH 2, a mammalian FMRF-NH~-Iike peptide, Brain Res. 494, 1. Malin, D.H., L. Harter, P.D. Jenkins, R.D. Monfort, P.D. Bruce, P.A. Farley, R. Ferebee, K.L. Thrasher and D.S. Marullo, 1987, Cerebrospinal fluid from morphine abstinent rats precipitates opiate abstinence syndrome, Life Sci. 41,377. Malin, D.H., J.R. Lake, D.E. Fowler, M.V. Hammond, S.L. Brown, J.E. Leyva, P.E. Prasco and T.M. Dougherty, 1990a, FMRFNHz-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat, Peptides 11,277. Malin, D.H., J.R. Lake, M.V. Hammond, D.E. Fowler, R.B. Rogillio, S.L. Brown, J.L. Sims, B.M. Leecraft and H.-Y.T. Yang, 1990b, FMRF-NH2-1ike mammalian octapeptide: possible role in opiate dependence and abstinence, Peptides 11,969. Nance, P.W. and J. Sawynok, 1987, Substance P-induced long term blockade of spinal adrenergic analgesia: reversal by morphine and naloxone, J. Pharmacol. Exp. Ther. 240, 972. Ossipov, M.H. and G.F. Gebhart, 1983, Absence of anti-nociceptive effect of alpha2-agonists microinjected in the periaqueducta[ gray of the rat, Brain Res. 289, 349. Panula, P., L. Kivipelto, O. Nieminen, E.A. Majane and H.-Y.T. Yang, 1987, Neuroanatomy of morphine-modulating peptides, Med. Biol. 65, 127. Raffa, R.B. and H.I. Jacoby, 1989, A-18-Famide and F-8-Famide, endogenous mammalian equivalents of the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), inhibit colonic bead expulsion time in mice, Peptides 10, 873. Randall, L.O. and J.J. Selitto, 1957, A method for the measurement of analgesic activity on inflammed tissue, Arch. Int. Pharmacodyn. Ther. 111,409. Reddy, S.V.R., J.L. Maderdrut and T.L. Yaksh, 1980, Spinal cord pharmacology of adrenergic agonist-mediated antinociception, J. Pharmacol. Exp. Ther. 213, 525. Satoh, M. and Y. Kuraishi, 1991, Opioid interaction with other neuropeptides in the spinal cord: relevance to nociception, in: Neurobiology of Opioids, eds. O.F.X. Almeida and T.S. Shippenberg (Springer-Verlag, Berlin) p. 261. Sawynok, J., C. Pinsky and F.S. LaBella, 1979, On the specificity of naloxone as an opiate antagonist, Life Sci. 25, 1621. Sullivan, A.F., E.A. Kalso, H.J. McQuay and A.H. Dickenson, 1991, FLFQPQRFamide modulates a2-adrenergic antinociception in the rat dorsal horn in vivo, Brain Res. 562, 327. Tang, J., H.-Y.T. Yang and E. Costa, 1984, Inhibition of spontaneous and opiate-modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-NH2-1ike immunoreactivity, Proc. Natl. Acad. Sci. U.S.A. 81, 5002. Tung, A.S. and T.L. Yaksh, 1982, In vivo evidence for multiple opiate receptors mediating analgesia in rat spinal cord, Brain Res. 247, 75. Yaksh, T.L. and T.A. Rudy, 1976, Analgesia mediated by a direct spinal action of narcotics, Science 192, 1357. Yang, H.-Y.T., W. Fratta, E.A. Majane and E. Costa, 1985, Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine, Proc. Natl. Acad. Sci. U.S.A. 82, 7757. Zhu, X.Z. and R.B. Raffa, 1986, Low affinity inhibition of opioid receptor binding by FMRFamide, Neuropeptides 8, 55.
73
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53120
Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat Christine Gouard~res, Maaja Sutak
a
Jean-Marie Zajac and Khem Jhamandas a
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse C~dex, France and a Department of Pharmacology, Queen's Uniuersity, Kingston, Ontario K7L3N6, Canada Received 12 November 1992, revised MS received 17 February 1993, accepted 23 March 1993
The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRFamide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5/zmol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmoi) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions. F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2); FMRFamide (Phe-Met-Arg-Phe-NH2); Naloxone; Morphine; Spinal antinociception
1. Introduction
The octapeptide F8Famide ( F L F Q P Q R F a m i d e , neuropeptide FF) was isolated from bovine brain by Yang et al. (1985) using antisera against the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH 2) (Dockray et al., 1981). Since this discovery, a number of authors have reported that F8Famide-like immunoreactivity is widely distributed in brain and spinal cord areas, especially those involved in nociceptive signal transmission (Yang et al., 1985; Majane et al., 1989; Allard et al., 1991). The spinal F8Famide-like immunoreactivity appears to be derived from two neuronal sources: descending spinal fibres originating from brain regions, and local interneurons (Ferrarese et al., 1986; Panula et al., 1987; Majane et al., 1989; Kivipelto et al., 1989; Allard et al., 1991; Kivipelto and Panula, 1991). Allard et al. (1989) have demonstrated F8Famide binding sites, distinct from opioid receptor sites, which may
Correspondence to: C. GouardBres, Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse C~dex, France. Tel. (33) 61 17 59 73, fax (33) 61 17 59 94.
represent specific receptors for F8Famide and related neuropeptides. High levels of these sites have been found in the dorsal spinal cord (Allard et al., 1992), a region that plays a critical role in pain transmission. However, the functional significance of F8Famide binding sites is unknown. In pharmacological experiments, intracerebroventricular (i.c.v.) administration of F8Famide and FMRFamide was reported to attenuate spontaneous and opiate-induced analgesia in rats (Tang et al., 1984; Yang et al., 1985) and mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). Several studies in which agents were injected i.c.v, have reported a potential role of F8Famide in opioid tolerance and dependence (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991). In addition to anti-opioid actions, some studies have shown pro-opioid effects of F8Famide. F8Famide and A18Famide decreased colonic bead expulsion time in mice (Raffa and Jacoby, 1989), and recently synthetic analogues of F8Famide were reported to inhibit morphine analgesia while other analogues produced analgesic effects after i.c.v, injection (Gicquel et al., 1992). Thus, these peptides have the potential to both inhibit and mimic the action of opioids. However, fewer studies have focused
74 on the action of F8Famide or F M R F a m i d e in the spinal cord. In rats, intrathecally administered F M R Famide inhibited the antinociceptive effect of spinal morphine in the tail flick test, but was inactive by its own (Tang et al., 1984). In electrophysiological studies, F8Famide was reported to attenuate the inhibitory effect of /x-opioid agonists and a2-adrenoceptor agonists on the C-fibre evoked firing of rat spinal neurons (Magnuson et al., 1990; Sullivan et al., 1991). Guzman et al. (1989) described a transient hyperpolarization and subsequent depolarization of cultured mouse spinal neurons exposed to F8Famide. These studies suggest a role for F8Famide in the modulation of nociception at the spinal level. However, at present, little is known about the actions of F8Famide in different models of nociception and about the influence of opioids and nonopioids in these actions. To gain insight into the role of F8Famide in spinal nociception, the present study evaluated the comparative effects of intrathecal F8Famide and the molluscan peptide F M R F a m i d e in two different nociceptive tests in the rat, the tail flick and paw pressure tests. The interaction of these peptides with morphine and naloxone was also examined. This communication describes for the first time a long-lasting antinociceptive spinal action of F8Famide and FMRFamide and their differential effect on morphine-induced antinociception.
2. Materials and methods
peptides. Control animals received an intrathecal injection of an equal volume of saline vehicle.
2.2. Nociceptive assays The nociceptive responses were assessed using the response evoked by thermal (tail flick; D ' A m o u r and Smith, 1941) and mechanical (paw pressure; Randall and Selitto, 1957) stimuli. Baseline tail flick latency was 2 - 3 s. To minimize tissue damage, a 10-s cut-off was imposed (Loomis et al., 1985). The responsiveness to paw pressure was tested using a modification of the procedure of Loomis et al. (1987). Briefly, mechanical pressure was applied to the upper surface of the hind paw until a withdrawal response was observed. The control and cut-off values were 90-110 and 300 m m Hg, respectively. Rats were handled for both tests. They were tested in groups of six. Two determinations were made with each animal, and each animal was used only once. Tests were applied in tandem, with the tail flick determination preceding the paw pressure test. The effects of peptides were evaluated at 10-min intervals for the first 60 min and every 30 min thereafter up to 180 rain. The tail flick and paw pressure responses were assessed 24 and 48 h or longer after the intrathecal injection. All experiments were performed in the morning. At the end of nociceptive tests, the correct placement of catheters in lumbar space was confirmed by intrathecal dye injection. Animals in which catheters were not located in the lumbar region were excluded from experiments reported in this study.
2.1. Intrathecal cannulations and drug injections
2.3. Data analysis
Male Sprague-Dawley rats (250-300 g, Charles River Inc., St. Constant, Que., Canada) were anaesthetized with halothane, and chronic lumbar intrathecal polyethylene catheters (PE 10) were inserted through the cisterna magna to the rostral edge of the lumbar enlargement (7.5 to 8 cm) (Yaksh and Rudy, 1976). The catheters were then flushed with 10 ~1 of physiological NaCI solution. The animals were placed in individual cages for a 4-day recovery period before being used in experiments. Animals showing neurological deficits (flexion of hindlimbs, rigidity or paralysis) were discarded. All drugs for spinal administration were p r e p a r e d in a final volume of saline such that the total dose was delivered in 10 /xl through the indwelling spinal catheter, using a 50-/zl Hamilton syringe. Drug injection was followed by an additional injection of 10/zl of saline to flush the catheter. Naloxone was given i.p. (0.1 ml per 100 g rat weight) 10 rain before intrathecal
All data for response latency (s) or threshold response pressure (mm Hg) were converted to maximum percent effect (MPE) calculated as: M P E = 100 x [(post-drug r e s p o n s e - baseline r e s p o n s e ) / ( c u t - o f f response - baseline response)]. The area under the M P E vs. time curve (AUC) was computed by trapezoidal approximation of the A U C for each rat over two periods: 10-60 and 60-180 min. The A U C is expressed as a percentage of the maxim u m possible A U C that could be obtained if the animal displayed a 100% M P E at each testing time over the experimental period. Statistical significance (to analyse the data with groups, times, doses) was calculated by using a oneor two-way analysis of variance ( A N O V A ) with multiple comparisons followed by appropriate tests ( S T A T V I E W program, Macintosh). Results are presented as the entire time course or as the area under the curve approximation. The differences were consid-
75 FMRFamide
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from E n d o Laboratories ( U . S . A . ) and m o r p h i n e sulphate from B D H Pharmaceuticals (Canada).
2.4. Drugs 3. Results Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2 (F8Fa m i d e ) was s y n t h e s i z e d by solid-phase m e t h o d s , as previously described ( G i c q u e l et al., 1992). F M R F a m i d e ( P h e - M e t - A r g - P h e - N H 2) was p u r c h a s e d from P e n i n s u l a Laboratories. N a l o x o n e hydrochloride was
3.1. Behavioural effects T h e intrathecal injection of F 8 F a m i d e (0.05 to 17.5 n m o l ) or F M R F a m i d e (0.002 to 25 n m o l ) had no apFMRFamide
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Fig. 2. Dose-response curves for the initial (10-60 rain, o) and the delayed (60-180 min, o) spinal antinociceptive effect of FSFamide (A, B) and FMRFamide (C, D) in the tail flick and paw pressure tests. Data are means+ S.E.M. of % AUC from 6 to 21 animals. For each curve, the values marked * and those obtained with higher doses are considered to be statistically different from corresponding control saline value (P < 0.05).
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DOSES (nmol) Fig. 3. Antinociceptive responses 24 ( [ ] ) and 48 ( [ ] ) h after intrathecally administered F8Famide (A, B) and FMRFamide (C, D) in the tail flick and paw pressure tests. Data are means_+ S.E.M. of MPE from 6 to 21 animals. * Indicates significant differences (P < 0.05) from control saline (dose 0).
parent effect on the general behaviour of the rats or on normal motor function. Very high intrathecal doses of peptides tended to produce an impairment of hindlimb function in some animals, and therefore such doses were not included in our study.
(fig. 1). In fact, examination of the individual time-response curves revealed a biphasic action of F8Famide in both tests at all doses. The first response (increase in tail flick latency or elevation of paw pressure threshold) peaked at 30-40 min and the second response peaked between 150-180 min (fig. 1A, B). The effect of FMRFamide in both tests was characterized by a rapid onset (10-20 min), followed by a gradual increase in response over time, the peak effect occurring 120-180 min after injection (fig. 1C, D). Figure 2 shows the dose-response curves for the initial and the delayed effects of F8Famide (fig. 2A, B) and FMRFamide (fig. 2C, D) in the two tests. As
3.2. Antinociceptive effects The effects of various doses of intrathecally administered F8Famide and FMRFamide in the tail flick and paw pressure tests are illustrated in figs. 1-3. In both tests, these peptides showed dose-related and long-lasting antinociceptive effects but with different patterns
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Fig. 4. Effects of naloxone on the time course of spinal F8Famide (A)- or FMRFamide (B)-induced antinociception in the tail flick test. Rats were injected intrathecally with the peptides (8.8 nmol) 10 min after i.p. saline (©) or naloxone (5.5 /zmol/kg, o). Effects of i.p. naloxone (5.5 /zmol/kg) alone (12]). Data are means_+ S.E.M. of MPE from six animals. * Indicates significant differences from the action of peptide alone
(P < 0.05).
77 min before 8.8 nmol intrathecal peptides) significantly attenuated the initial phase of the antinociceptive response produced by F8Famide (fig. 4A) and FMRFamide (fig. 4B). In contrast, naloxone significantly enhanced the second phase of the response to F8Famide (fig. 4A) but did not influence the late response produced by FMRFamide (fig. 4B). In naloxone-treated animals, the antinociceptive effects of F8Famide were still apparent 24 h after the peptide injection (fig. 4A). The actions of systemic naloxone were similar in the paw pressure test (data not shown).
shown, at all doses used, the magnitude of the delayed response was higher than the initial response. Over the range of doses used, dose-response relationships for the two peptides were found only in the tail flick test. In this test, the EDs0 values for F8Famide were 12 and 7 nmol for the initial and delayed effects, respectively. Corresponding values for FMRFamide were 15 and 3 nmol. In the paw pressure test, the magnitude of the effect was lower, as evidenced by the flat dose-response curves. The EDs0 values were higher than 20 nmol for both peptides. In both analgesic tests, the effect of F8Famide and FMRFamide was apparent 24 and 48 h after injection (fig. 3). In the case of F8Famide (fig. 3A, B), this delayed antinociception was seen in the dose range 2.5-17.5 nmol, and in the case of FMRFamide (fig. 3C, D), it was apparent in the dose range 0.05-25 nmol. Although not examined systematically, the sustained antinociception was not a manifestation of motor dysfunction. All animals showed normal ambulatory behaviour and recovery from the effect of the peptide. Depending on the dose used, the elevated latencies or thresholds typically returned to baseline levels between 3 or 4 days.
3.4. Effects of F8Famide or FMRFamide on spinal morphine antinociception The effects of a subeffective intrathecal dose of F8Famide or FMRFamide on spinal morphine analgesia in the tail flick and paw pressure tests are represented in fig. 5. Morphine (6.6 nmol) produced a monophasic antinociceptive effect which reached a peak around 30 rain and returned to baseline by 60-90 min in both tests. Intrathecal co-injection of F8Famide (0.05 nmol) increased the magnitude of the effect of morphine from 60.0 + 2.3 (MPE, peak effect) to 71.4 _+ 5.5 in the tail flick test (fig. 5A). This co-injection also produced a marked prolongation of the effect of morphine. The tail flick response returned to the pre-drug level 90 rain after injection of morphine alone. However, after co-injection of F8Famide and morphine, the tail flick response remained elevated at this time (MPE = 81.5 _+ 7.1) and this effect still persisted 180 rain after injection (MPE = 61.2 + 7.4) (fig. 5A). Twentyfour hours after the co-injection, the response was still
3.3. Effects of naloxone on spinal F8Famide FMRFamide antinociception Figure 4 shows the results of experiments in which the effect of i.p. naloxone on peptide-induced responses was tested. As shown (fig. 4A), naloxone (5.5 p~mol/kg) by itself had no effect on the tail flick response. However, this dose of naloxone (given i.p. 10 FSFamide
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T I M E AFTER I N J E C T I O N Fig. 5. E f f e c t s o f F 8 F a m i d e ( A , B) a n d F M R F a m i d e (C, D ) o n t h e t i m e c o u r s e o f t h e a n t i n o c i c e p t i o n i n d u c e d by s p i n a l m o r p h i n e (6.6 n m o l , o ) in t h e tail flick a n d p a w p r e s s u r e tests. F 8 F a m i d e (0.05 n m o l , ~2 ) o r F M R F a m i d e (0.002 n m o l , [ ] ) w a s c o - i n j e c t e d i n t r a t h e c a l l y w i t h m o r p h i n e ( • ) .
Data are means+ S.E.M. of MPE from 6 to 17 animals. * Indicates significant differencesfrom the action of morphine alone (P < 0.05).
78
significantly above control values (MPE = 19.7 _+ 4.8). Full recovery from the antinociceptive effects was seen 48 h after injection. In the paw pressure test (fig. 5B), a similar augmentation of morphine-induced effect was observed. In contrast with the action of F8Famide, 0.002 nmol FMRFamide injected with morphine significantly attenuated the antinociceptive action of the opiate in both the tail flick and paw pressure tests during the initial 50- and 30-min periods, respectively (fig. 5C, D). However, after this, a sustained antinociceptive action of the co-injected agents was apparent in both tests. Full recovery from the antinociceptive effect occurred 24-48 h after the injection. T h u s combination e x p e r i m e n t s showed that F8Famide enhanced the action of morphine and that this action was attenuated by FMRFamide during the 10- to 60-min period after injection. Both peptides prolonged the effect of morphine in the 60- to 180-min period. The enhancement of opioid antinociception occurred at doses of the peptides that had an insignificant effect when injected alone.
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Fig. 6. Modulation of spinal morphine antinociception by F8Famide and F M R F a m i d e in the tail flick and paw pressure tests. Rats were treated intrathecally with saline (S), morphine (M, 6.6 nmol). F8Famide (F8, 2.5 nmol), F M R F a m i d e (FM, 2.5 nmol), or a combination of morphine with each peptide ( F 8 + M , F M + M ) . The antinociceptive effect is expressed as % A U C from 10 to 60 rain (A, B) and 60 to 180 min (C, D) after drug administration. Data are m e a n s + S.E.M. of % A U C from 6 to 17 animals. Values significantly different (P < 0.05) from control saline (+), peptide alone (°) and morphine alone (*).
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Fig. 7. Modulation of spinal morphine antinociception by F8Famide and F M R F a m i d e in the tail flick test. Rats were treated intrathecally with saline (S), morphine (M, 6.6 nmol), F8Famide (F8, 2.5 nmol), F M R F a m i d e (FM, 2.5 nmol), or a combination of morphine with each peptide ( F 8 + M , F M + M ) . The antinociceptive response is expressed as M P E observed at 24 h (A) and 48 h (B) after drug administration. Data are m e a n s _+S.E.M. of M P E values from 6 to 17 animals. Values significantly different (P < 0.05) from control saline (+), peptide alone (°) and morphine alone (*).
To investigate the effect of antinociceptive doses of F8Famide or FMRFamide on spinal morphine analgesia, 6.6 nmol morphine and 2.5 nmol of each peptide were co-injected intrathecally. The individual and combined antinociceptive effects are expressed as % AUC (as described under Methods) from 10 to 60 rain and 60 to 180 min after injection (fig. 6) and as MPE obtained at 24 and 48 h (fig. 7). The antinociceptive effect of morphine (6.6 nmol) in both tests was reduced 60 min after injection (fig. 6) and was not significantly different from that of control saline-treated animals 24 and 48 h later (fig. 7). Intrathecal administration of F8Famide or FMRFamide (2.5 nmol) produced similar long-lasting antinociceptive responses that were different from that of morphine (figs. 6, 7). The response was elevated 60 min after injection (fig. 6) and declined thereafter to return to control saline values by 48 h (fig. 7). In the paw pressure test, in the initial as well as in the delayed period, each peptide co-injected with morphine produced an analgesic effect which corresponded to the sum of their individual effects (fig. 6B, D). This additivity of the analgesic effects of peptides co-injected with morphine was also observed in the tail flick test, but only during the 60- to 180-min period (fig. 6C). In this test, F8Famide did not modify the effect of morphine in the first hour (10-60 min), whereas FMRFamide decreased this effect (fig. 6A) as did a subeffective dose (fig. 5C). Measurement of nociceptive responses at 24 h and 48 h showed that the tail flick latency of the F8Famide and morphine co-injected group of animals at 24 h was elevated well above that observed in the morphine- or peptide-injected groups (fig. 7A). This elevation was not observed in the FMRFamide- and morphine-
79
treated animals. A similar profile of activity was observed for the combination 48 h after injection (fig. 7B) but the magnitude of response was lower than that seen at 24 h. The combination of morphine with each peptide did not change the paw pressure threshold observed in the corresponding peptide-injected group (data not shown). None of the animals in this part of the study showed a visible impairment of motor function.
4. Discussion
The present study demonstrates that spinal administration of F8Famide, an endogenous mammalian opioid modulatory peptide (Yang et al., 1985), and FMRFamide, a molluscan peptide (Dockray et al., 1981), produces dose-related and sustained antinociception against thermal and mechanical stimuli. Previous studies, largely involving i.c.v, injections and the tail flick test, have not indicated that these peptides have antinociceptive effects but have described their antiopioid properties in rats (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991) and mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). Tang et al. (1984) have shown the same results after intrathecal administration of FMRFamide in the rat. However, one study has reported that i.c.v. F M R F a m i d e exerted an antinociceptive effect in the mouse hot plate test (Hill et al., 1987). The reason for this discrepancy between the results of i.c.v. and intrathecally administered peptides is not clear. However, the dissociation seen here is not without precedence. Norepinephrine and az-adrenoceptor agonists produce potent antinociception after spinal but not i.c.v, injection (Reddy et al., 1980; Ossipov and Gebhart, 1983). It is possible that sites targeted by intrathecal F8Famide are a part of an endogenous mechanism modulating nociceptive transmission at a spinal level. The presence of a high density of F8Famide immunoreactivity (Yang et al., 1985; Majane et al., 1989; Allard et al., 1991) as well as binding sites (Allard et al., 1989, 1992) in the dorsal cord tends to support this notion. However, the possibility that the action of F8Famide at the spinal level is modulated by supraspinal mechanisms cannot be excluded. An unusual characteristic of the action of F8Famide in both tests was its biphasic action, a rapid initial response followed by an extended response that was prominent in the 60- to 180-min observation period but which persisted in the 24- to 48-h period after injection. FMRFamide also produced sustained antinociception. This extended response is reminiscent of the sustained spinal action of dynorphin in the tail flick test (Jhamandas et al., 1986) that has been associated with motor impairment (Faden and Jacobs, 1984; Her-
man and Goldstein, 1985). However, unlike dynorphin injection, administration of antinociceptive doses of the peptides in this study produced no visible motor impairment and all animals showed normal ambulation and full recovery from antinociception. The two phases of the antinociceptive response evoked by thermal stimuli in the present work showed a differential sensitivity to systemic naloxone. Both the dose and route of naloxone administration did not affect the baseline responses. Naloxone attenuated the early but not late response, suggesting involvement of opioid mechanisms in this phase. In the rat spinal cord, F8Famide receptors (Allard et al., 1992) and opioid receptors (Gouard6res et al., 1985, 1991, 1993) are similarly distributed. However, spinal F8Famide binding sites exhibit a very low affinity for opioid agonists (Allard et al., 1989) and F8Famide does not bind to opioid receptors in vitro (data not shown). Therefore, it is unlikely that the naloxone-sensitive response is due to direct opioid receptor stimulation by F8Famide. This response probably involves an indirect action by release of an endogenous opioid peptide. Surprisingly, naloxone did not antagonize the action of F8Famide in the paw pressure test. This suggests that the dose of naloxone may have been inadequate or that the response involves an interaction of the peptide with a non-opioid mechanism. The use of higher antagonist doses was avoided because of the development of hyperalgesia or analgesia (Sawynok et al., 1979) due to the action of naloxone itself. The possibility of a differential action of F8Famide in the tail flick and paw pressure tests is suggested by previous results showing that different neuromodulatory systems participate in thermal and mechanical nociception (Kuraishi et al., 1983, 1985a,b). In contrast, mechanical antinociception induced by FMRFamide during the first hour after injection was reversed by naloxone. This suggests the existence of different mechanisms mediating the activity of F8Famide and FMRFamide in the paw pressure test. Opioid mechanisms appear to be involved in the sustained response evoked by both peptides. Although the basis for its appearance is unknown, the possibility that this response is caused by metabolites of the peptides or their rostral redistribution to the brain cannot be discounted. It would be of interest to determine if inhibition of metabolism or use of stable peptide analogues (Gicquel et al., 1992) also yields a similar activity profile. The sustained response to F8Famide and FMRFamide could also involve recruitment of non-opioid mechanisms, such as desensitization to the action of a nociceptive substance released from primary afferents or reinforcement of descending inhibitory controls. Previous reports have described the antagonism of morphine or other opioids by F M R F a m i d e or
80 F8Famide given i.c.v, in rats (Tang et al., 1984; Yang et al., 1985; Malin et al., 1987, 1990a,b; Lake et al., 1991) or mice (Kavaliers and Yang, 1989; Kavaliers, 1990; Gicquel et al., 1992). In the present study, which focused on the rat spinal cord and examined the action of both sub-antinociceptive and antinociceptive doses of peptide, F8Famide did not antagonize the effect of morphine. In contrast, the low dose of peptide, which was inactive on its own, produced a remarkable enhancement of morphine-induced antinociception in both tests. The action of intrathecal morphine, which normally lasted for 60-90 min, was significantly extended to 24 h after injection of a sub-analgesic dose of F8Famide. An antinociceptive dose of the peptide produced the same effect. In contrast, in both tests, a sub-antinociceptive dose of FMRFamide attenuated the action of morphine in the early phase (10-60 min), as was previously reported (Tang et al., 1984), but extended this effect later. An analgesic dose of FMRFamide decreased the initial action of morphine only in the tail flick test, but prolonged its action in the later period. Thus, the antagonist action of FMRFamide against the action of morphine in the early phase was not seen with F8Famide. The reasons for this difference between the action of the two peptides are not known, but the results suggest that the antiopioid component of F M R F a m i d e was not apparent in the action of FSFamide. The fact that F8Famide increased morphine analgesia, an agonist that preferentially activates ~-type opioid receptors in the spinal cord (Tung and Yaksh, 1982), suggests that endogenous F8Famide in the spinal cord may function to amplify the action of endogenous opioids that normally interact with mu receptors. The mechanism of interaction of F M R F a m i d e with the opioid system could be different in regard to the results mentioned above. In addition, FMRFamide exhibits a weak affinity for opioid receptors (Zhu and Raffa, 1986) and displays, in contrast to F8Famide, a low affinity for F8Famide binding sites in the rat spinal cord (Allard et al., 1989). These findings could explain the differential modulation of the effects of morphine by these peptides after intrathecal injection. In fact, F8Famide and F M R F a m i d e may have a role in modulating sensory transmission via different neurochemical systems (Kuraishi et al., 1983, 1985a,b; Satoh et al., 1991) in the dorsal horn of the rat spinal cord. The enhancement of the effect of morphine seen in this behavioural study contrasts with the antagonism of the effect of D A G O (Tyr-D-Ala-Gly-(N-Me)Phe-Glyol, a/~-opioid agonist) by F8Famide in an electrophysiological study of C-fibre-driven spinal nociceptive neurons in anaesthetized rats (Magnuson et al., 1990). The reason for this discrepancy is not clear but factors such as anaesthesia, nociceptive stimulation modality, peptide dose and observation period could contribute to
the differences between behavioural and electrophysiological experiments. Moreover, these authors have also reported that the activity of D S T B U L E T (Tyr-DSer(OtBu)-Gly-Phe-Leu-Thr), a a-opioid agonist, is not modified by F8Famide. It remains to be determined if the augmentation of antinociception seen here is specific to morphine or extends to other antinociceptive agents. Although the sustained response induced by intrathecal administration of F8Famide may seem unusual, it has been reported that intrathecal injection of substance P can produce a very long-lasting change in the response to intrathecal norepinephrine (Nance and Sawynok, 1987). The presence of F8Famide-like immunoreactivity in the dorsal horn of the rat spinal cord (Majane et al., 1989; Allard et al., 1991), the presence of its own receptors in the same region (Allard et al., 1989, 1992), together with its effects on dorsal horn neurons (Magnuson et al., 1990; Sullivan et al., 1991), suggest that F8Famide may have a dual action, acting as a transmitter of nociceptive information as well as producing antinociception, these effects being elicited at different sites. An alternative possibility is that F8Famide in the spinal cord may be involved in modulating functions other than nociception.
Acknowledgements This work was supported in part by grants from the Medical Research Council (MRC) of Canada (MRC-MI5690 to K.J.) and funds from INSERM (CRE 910815) and CNRS, France. C.G. was sponsored by a Visiting Scientist award from the MRC and the CNRS.
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