B 1996 Stockton Press All rights reserved 0007-1188/96 $12.00 S

Role of opioid receptors in the spinal antinociceptive effects ofneuropeptide FF analogues

'Christine Gouarderes, *Khem Jhamandas, *Maaja Sutak & Jean-Marie Zajac

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, Franceand *Department of Pharmacology, Queen's University, Kingston, Ontario, Canada K7L 3N6

1 Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphine-induced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFFanalogues, [D-Tyr',(NMe)Phe']NPFF (IDMe) and [D-Tyr',D-Leu2,D-Phe']NPFF (3D), which areresistant to degradation and exhibit a high affinity for NPFF binding sites, were examined in tests ofthermal and mechanical nociception.2 IDMe and 3D produced potent dose-dependent spinal antinociception in the tail-flick test. On amolar basis, IDMe was 20 and 50 times more potent than 3D and morphine, respectively, and highdoses of lDMe and 3D produced a sustained antinociceptive effect without visible signs of motorimpairment.3 Spinal antinociceptive effects produced by IDMe (0.86 nmol) or 3D (8.6 nmol) were significantlyreduced by i.t. co-administration of naloxone (11 nmol) or i.t. pre-administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 9.25 nmol) or ,B-funaltrexamine (fl-FNA, 2 nmol) or naltrindole(2.2 nmol). The doses of the ,-antagonists (CTOP and f,-FNA) or the 5-antagonist (naltrindole) used inIDMe and 3D experiments blocked the antinociceptive effects of i-or 3-receptor-selective agonists.4 When administered in combination with antinociceptive doses of the p-receptor agonist, morphine(13.2 nmol) or the (-receptor agonist, [D-Ala2]deltorphin I (20 nmol), sub-effective dose of IDMe or 3D(0.009 nmol) enhanced and prolonged the spinal effects of these opioid agonists.5 The results of this study show that spinal ,-and 6-opioid receptors play a role in antinociceptionproduced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptivesignals at the spinal level.

Keywords: Neuropeptide FF; ,u- and 6-opioids; nociception; intrathecal; rat spinal cord

Introduction

Neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2,NPFF) was first isolated from bovine brain and shown toantagonize morphine-induced supraspinal antinociception(Yang et al., 1985). NPFF is widely distributed in the ratcentral nervous system (Majane et al., 1989; Kivipelto et al.,1989; 1991; Kivipelto & Panula, 1991a, b; Allard et al., 1991)and in porcine spinal cord (Wasowicz & Panula, 1994). Thisneuropeptide binds with high affinity to specific sites (Allard etal., 1989; Payza et al., 1993; Devillers et al., 1994) that arelocalized in the superficial laminae of the rat (Allard et al.,1992), mouse (Desprat & Zajac, 1994) and human spinal cord(Allard et al., 1994).

In rodents, NPFF exerts a modulatory action on a varietyof behavioural and physiological responses (Roth et al., 1987;Guzman et al., 1989; Magnuson et al., 1990; Sullivan et al.,1991; Kavaliers & Colwell, 1993; Demichel et al., 1993).Pharmacological studies have shown that intracere-broventricular (i.c.v.) administration of NPFF attenuatesmorphine-induced analgesia in rats (Yang et al., 1985; Millionet al., 1993; Oberling et al., 1993) and mice (Kavaliers, 1990;Gicquel et al., 1992; Desprat & Zajac, 1994). Injection of IgGdirected against NPFF potentiates analgesia induced by mor-phine or stress (Kavaliers & Yang, 1989). Furthermore, NPFFhas been implicated in opioid tolerance and dependence (Malinet al., 1990). These findings have led to the suggestion thatNPFF may function as an endogenous anti-opioid agent(Yang et al., 1985; Malin et al., 1991; 1993; Lake et al., 1992;Rothman, 1992).

However, in addition to its potential anti-opioid actions,

1 Author for correspondence.

NPFF has also been shown to possess pro-opioid effects. Likemorphine, NPFF decreased colonic bead expulsion in mice(Raffa & Jacoby, 1989; 1990). In rats, intrathecal NPFF in-jection elicited a long-lasting spinal antinociception that waspartly attenuated by naloxone (Gouarderes et al., 1993c).

Recently, two analogues of NPFF, [D-Tyr', (NMe) Phe3]NPFF (IDMe) and [D-Tyr', D-Leu2,D-Phe3]NPFF (3D) whichare partially protected against enzymatic breakdown, espe-cially against aminopeptidase activity (Gicquel et al., 1992),were reported both to inhibit as well as mimic the action ofopioids after i.c.v. injection in mice (Gicquel et al., 1992; 1993;Desprat & Zajac, 1994) or in rats (Million et al., 1993). BothIDMe and 3D were shown to bind with a high affinity to ratspinal cord NPFF binding sites (Gicquel et al., 1992; Devillerset al., 1994). Therefore, to gain additional insight into the roleofNPFF in spinal nociception, we have investigated the effectsof intrathecally administered NPFF analogues, IDMe and 3D,using the tail-flick and paw-pressure tests. The interaction ofthese peptides with p-(morphine) and 6-([D-Ala2]deltorphin Ior DPDPE) opioid agonists, and with y-(D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or fl-funaltrexamine (fi-FNA)) and 6-(naltrindole) opioid antagonists have been ex-amined to determine the relative role of spinal opioid receptortypes in the antinociception elicited by spinally administeredpeptides.

Methods

Intrathecal cannulation and drug injectionMale Sprague-Dawley rats (250 - 300 g, Charles River Inc., St.Constant, Que., Canada) were anaesthetized with halothane

British Journal of Pharmacology (1996) 117, 493-501

C. Gouarderes et a! Spinal antinociception of NPFF and opioid receptors

and intrathecal polyethylene catheters (PE 10, 7.5-8 cmlength) were inserted through the cisterna magna to lie at therostral edge of the lumbar enlargement (Yaksh & Rudy, 1976).The catheters were then flushed with 10 M1 of physiologicalsaline (0.9% NaCl). Following surgery, the animals were al-lowed to recover and housed in individual cages for a four-dayperiod. Animals showing neurological deficits (flexion of hin-dlimbs, rigidity or paralysis) were excluded from experiments.

Agents injected intrathecally alone or in combination withanother agent were dissolved in saline to deliver the dose understudy in a 10 M1 volume. The injections were made through theindwelling intrathecal catheter via a 50 pl Hamilton syringe.Each drug injection was followed by an additional injection of10 yl of saline to flush the catheter. Control animals received10 MI intrathecal saline followed by a flush of 10pl saline. Inthe antagonism experiments, the antagonist under study wasadministered intrathecally in 8 pl volume followed by 8 plsaline flush 15 min or 24 h (fl-FNA) prior to the agonist. Thelatter was delivered in an 8 MI volume followed by a flush with8 pl saline. Other animals received intrathecal injections by thesame procedure: 8 MI saline +8 M1 saline flush 15 min or 24 hprior to 8 pl saline followed by 8 pl saline flush to determinethe effects of multiple injections on the nociceptive responses.Saline injections did not influence these responses.

Nociceptive assays

The nociceptive responses were assessed by the tail-flick(D'Amour & Smith, 1941) and paw-pressure (Loomis et al.,1987) tests. In the tail-flick test, the baseline latency was setat 2-3 s and the cut-off time was set at 10 s to minimizetissue damage (Loomis et al., 1985). In the paw-pressure test,mechanical pressure was applied with a pressurized syringe tothe upper surface of the hind paw until a withdrawal re-sponse was elicited. The pressure producing a withdrawalresponse was 90-110 mmHg, and the maximum pressureapplied (cut-off) was 300 mmHg. The rats were handled andtested in groups of six in both tests and each animal was usedonce only. The nociception tests were used in tandem, withthe tail-flick test preceding the paw-pressure test. The effectsof peptides were evaluated at 10 min intervals for the first60 min and every 30 min thereafter up to 180 min. Ad-ditionally, the tail-flick and paw-pressure responses were as-sessed 24 and 48 h or longer after the intrathecal injection todetermine the reversibility of the observed effects. All ex-periments were performed in the morning between 09 h00 min-12 h 00 min.

Following the end of nociceptive tests, the placement ofcatheters in the lumbar space was confirmed by intrathecal dyeinjection. Animals in which catheters were not located in thelumbar region were excluded from the study.

Data analysis

All data of response latency(s) or threshold response pressure(mmHg) were converted to maximum percentage effect (MPE)calculated as:

MPE = 100 x [(post-drug response- baseline response)/(cut-off response- baseline response)].

The results of experiments are presented as the entire time-course of the MPE.

To determine the dose-response curves for the intrathecaleffects of drugs, the area under the curve (AUC), depictingtotal MPE vs. time, was computed by trapezoidal approx-imation over the period 10-180 min and was expressed as apercentage of the maximum possible AUC that could be ob-tained if the animal displayed a 100% MPE at each time pointin the experimental period.

Data were analyzed by one-way analysis of variance(ANOVA). Post-hoc comparisons were made with Dunnett's ttest or Newman-Keuls test for multiple comparisons. The levelof significance was set at P<0.05.

Chemicals

IDMe ([D-Tyr',(NMe)Phe3]NPFF) and 3D ([D-Tyr', D-Leu2,D-Phe3]NPFF) were synthesized by solid phase meth-ods as described by Gicquel et al. (1992). [D-Ala2]deltorphinI (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) was synthesized asdescribed previously (Dupin et al., 1991). CTOP hydro-chloride (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) waspurchased from Peninsula Laboratories. Naloxone hydro-chloride was obtained from Endo Laboratories (U.S.A.) andmorphine sulphate from BDH Pharmaceuticals (Canada). fl-FNA (fi-funaltrexamine), DPDPE ([D-Pen2,D-Pen5]enkepha-lin) and naltrindole were obtained from RBI (France).

Results

Behavioural effects of peptides

The intrathecal (i.t.) injection oflDMe (0.009-6.4 nmol) or3D (0.009-25.8 nmol) had no apparent effects on the generalbehaviour of the rat or on normal motor functions as assessedby the methods of Faden & Jacobs (1984) and Rivlin & Tator(1977) (data not shown). Intrathecal doses of IDMe higherthan 6.4 nmol tended to produce an impairment of hindlimbfunction in some animals, and therefore such doses were notincluded in this study. The i.t. injection of morphine (6.6-52.8 nmol) and [D-Ala2]deltorphin I (5-20 nmol) did not in-duce any detectable effect on righting reflexes or hind-pawplacing and stepping reflexes. However, the highest dose of[D-Ala2]deltorphin I (40 nmol) produced suppression ofspontaneous activity in animals and was excluded from thestudy.

Antinociceptive effects of peptides

The comparative effects of different doses of intrathecally ad-ministered IDMe and 3D in the tail-flick and paw-pressuretests are illustrated in Figure 1. Spinal antinociceptive effects ofmorphine, a p-agonist, and [D-Ala2]deltorphin I, a 6-agonist,are shown in Figure 2. Both NPFF analogues produced longlasting antinociceptive effects (Figure 1). The effect of IDMe inboth nociception tests was characterized by a rapid onset (10-20 min) and was long lasting, 30-90% effects in the tail-flicktest being still observable at 180 min post-injection (Figure la).The increase of tail-flick latency peaked at 30-40 min for 3Dand declined progressively thereafter (Figure Ic). Both p- andc-opioid agonists produced similar effects (Figure 2). However,the 25.8 nmol dose of 3D produced significant antinociception24 h after the i.t. injection, while the maximal doses of the twoopioid agonists were ineffective at this time period (Table 1). Inboth nociception tests, the effect of the highest dose of IDMe(6.4 nmol) was still apparent 24 and 48 h post-injection (Table1). The elevated latencies or thresholds induced by both NPFFanalogues returned to baseline levels between 24 and 72 h andnone of the animals showed visible motor deficits.

Both IDMe and 3D produced dose-dependent increases inthe tail-flick antinociceptive effect expressed as % area underthe curve (% AUC) during the 10 to 180 min period followingthe i.t. injection with ED50 values approximating to 1 and20 nmol, respectively (data not shown). In contrast to themarked effects seen in the tail-flick test, the magnitude of ef-fects of the two NPFF analogues was lower in the paw-pres-sure test, as evinced by shallow dose-response curves.Morphine was equipotent in both tests with a value for ED:50 ofapproximately 50 nmol. [D-Ala2]deltorphin I displayed anti-nociceptive activity in both tests, but as the highest dose of the6-agonist was limited to 20 nmol, since higher doses tended tosuppress motor activity, the value of apparent ED50 was notderived.

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C. Gouarderes et al Spinal antinociception of NPFF and opiold receptors

Effects of naloxone on the spinal antinociceptive actionof NPFF analogues and morphine

The effect of intrathecal naloxone (11 nmol) on the spinal ef-fects of IDMe, 3D and morphine, at doses producing com-parable antinociceptive responses in the two nociceptive tests,are shown in Figure 3. Intrathecal naloxone alone had no effecton tail-flick latency or paw-withdrawal response (Figure 3aand b). However, naloxone administration significantly atte-nuated the effect of lDMe in both tests during the time period10-60 min (Figure 3a and b). Naloxone also attenuated theantinociceptive response to 3D but this was evident only in thetail-flick test (Figure 3c). The dose of intrathecal naloxone usedhere significantly reduced the antinociceptive action of mor-phine in both tests (Figure 3e and f).

Effects of CTOP and f3-FNA on the spinalantinociceptive action of NPFF analogues and morphine

As shown in Figure 4 (left panel), CTOP (9.25 nmol) injectedintrathecally 15 min prior to IDMe (Figure 4a) or 3D (Figure4c) or morphine (Figure 4e) significantly attenuated the firstphase (10-60 min) of the thermal antinociceptive responsesproduced by these compounds. The antinociceptive responsesproduced by IDMe or 3D 60- 180 min post-injection, however,were significantly higher than those obtained with the peptidesin the absence of CTOP (Figure 4a and c). This rebound re-sponse was not seen in similar experiments with morphine(Figure 4e). Thus, CTOP treatment enhanced the second phaseof antinociception produced by the two peptides but not mor-phine in the tail-flick test. As illustrated in Figure 4 (right pa-nel), CTOP pretreatment (9.25 nmol) attenuated theantinociceptive effects elicited in the paw-pressure test by IDMe(Figure 4b) during the first 60 min period after injection of thepeptide. The subsequent response to lDMe was enhanced inCTOP-treated animals (Figure 4b). The antagonistic effect ofCTOP was not evident in experiments with 3D (Figure 4d) but itwas apparent in experiments with morphine (Figure 4f).

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As shown in Figure 5 (left panel), fl-FNA (2 nmol), injectedi.t. 24 h prior to the two NPFF analogues, effectively blockedboth the early and delayed antinociceptive responses produced

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Figure 2 Time-course of the antinociceptive responses induced byintrathecally administered saline (A), 52.8nmol morphine (0) and20 nmol [D-Ala2]deltorphin I (0) in the tail-flick test (a) and paw-pressure test (b). The doses used were the maximal doses that did notproduce visible signs of motor disturbance in rats. Data aremeans+s.e.mean of MPE from 6 to 15 rats.

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Figure 1 Time-course of the antinociceptive responses induced by intrathecally administered NPFF analogues (lDMe and 3D) inthe tail-flick test (a, c) and paw-pressure test (b, d). Data are means+s.e.mean of MPE from 6 to 14 rats. Saline (A); lDMe (a, b):0.086 nmol (A), 0.86 nmol (0), 6.4 nmol (0); 3D (c, d): 4.3 nmol (A), 8.6 nmol (0), 25.8 nmol (0). The absence of an error barindicates that the value of the s.e.mean is smaller than the size of the symbol.

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C. Gouarderes et al Spinal antinociception of NPFF and opioid receptors

Table 1 Antinociceptive responses observed 24, 48 and 72 h after a single intrathecal injection of NPFF analogues and opioidagonists in the tail-flick (TF) and paw-pressure (PP) tests in rat

Treatment TF

SalineIDMe (6.4 nmol)3D (25.8 nmol)Morphine (52.8 nmol)[D-Ala2]deltorphin I

(20 nmol)

24 h

0.6 +0.648.4 i 7.2**14.6 + 2.0*1.2± 0.57.5 + 1.5

PP

1.2+1.342.4 + 4.2**10.7 + 3.2*1.1 +0.98.5+ 1.8

TF48 h

0.6+0.623.2 ± 3.3**5.6+ 1.40.4+0.33.9+1.0

PP

1.3+ 1.025.7+ 6.3**3.6+1.91.1+0.65.3+ 1.1

TF

1.2±1.17.5±1.42.6+ 1.70.3 +0.42.9+ 1.9

72 hPP

1.4+0.87.0+ 1.91.8 i 0.80.4+0.33.9+ 3.2

These doses were the highest used which did not induce any motor dysfunction.Data are means+s.e.mean of MPE from at least 6 rats per treatment.Differences are significantly different from saline. *P<0.05; **P<0.01.

by these peptides in the tail-flick test (Figure 5a and c). It alsoabolished the effect of morphine (Figure Se). P-FNA treatmentalone produced a small but significant antinociceptive action(MPE = 20%) (Figure 5a). Interestingly, the dose of 3-FNA(2 nmol) used here did not influence the antinociceptive effect of40 nmol intrathecal DPDPE, a 6-selective opioid agonist (Figure5g). In the paw-pressure test (Figure 5, right panel), fl-FNA de-creased the antinociceptive effects ofboth IDMe (Figure 5b) andmorphine (Figure 5f) during the 10-40 min period, but en-hanced the effects induced by 3D during the same period (Figure5d). In contrast, j3-FNA failed to influence the antinociceptioninduced by the selective 6-agonist DPDPE (Figure 5h).

Effects of naltrindole on the spinal antinociceptive actionof NPFF analogues and DPDPE

The administration of the 6-receptor antagonist, naltrindole(2.2 nmol), injected intrathecally 15 min prior to IDMe or

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Figure 3 Effects of intrathecal naloxone (11 nmol) on the time-course of spinal antinociception induced by lDMe (a, b), 3D (c, d) ormorphine (e, f) in the tail-flick test (a, c, e) and paw-pressure test (b,d, f). lDMe (0.86nmol, 0, 0), 3D (8.6nmol, 1, *) or morphine(13.2nmol, A, A) were injected intrathecally alone (open symbols)or co-injected with naloxone (solid symbols). Data are means +s.e.mean of MPE from at least 6 rats. *Significant differences fromthe action of agonist alone (P <0.05). At these doses, noantinociceptive effect was observed 24h after the spinal administra-tion of agonists used alone or in combination with naloxone.Intrathecal naloxone alone (V) did not influence baseline nociceptiveresponses. The absence of an error bar indicates that the value of thes.e.mean is smaller than the size of the symbol.

3D, significantly attenuated the antinociceptive responsesproduced by either peptide during the initial 60 min periodin the tail-flick and paw-pressure tests (Figure 6a, b, c andd). However, in the following period, the combination ofnaltrindole with 1DMe or 3D produced greater anti-nociceptive responses than those produced by the peptidesalone in either test (Figure 6a, b, c and d). The dose ofnaltrindole used in these experiments was effective inblocking the action of DPDPE (40 nmol) in both nocicep-tion tests (Figure 6e and f).

Action of IDMe or 3D on morphine-inducedantinociception

The effects of a sub-effective intrathecal dose of lDMe or 3Don the spinal antinociceptive action of morphine in the tail-flick and paw-pressure tests are shown in Figure 7. In bothtests, morphine (13.2 nmol) produced a monophasic responsethat peaked between 30-40 min and returned to baseline by60-90 min. When morphine was injected along with a sub-effective dose of lDMe (0.009 nmol), the action of morphine

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Figure 4 Effects of the p-opioid receptor-selective antagonist CTOPon the time-course of spinal antinociception induced by lDMe (a, b),3D (c, d) or morphine (e, f) in the tail-flick test (a, c, e) and paw-pressure test (b, d, f). CTOP (9.25nmol) was injected intrathecallyalone (V) or 15min prior to lDMe (0.86 nmol, 0), 3D (8.6 nmol, *)or morphine (13.2 nmol, A). Separate animals were injectedintrathecally with lDMe (0), 3D (E) or morphine (A\) alone. Dataare means + s.e.mean of MPE from at least 6 rats. *Significantdifferences from the action of the agonist alone (P<0.05).

496

C. Gouard~~~~~res et a! Spinal antinociception - W---of PFFan opoi receptors49

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Figure 5 Effects of the i-opioid receptor-selective antagonist fl-FNAon the time-course of spinal antinociception induced by 1DMe (a, b),3D (c, d), morphine (e, f) or DPDPE (g, h) in the tail-ffick test (a, c,e, g) and paw-pressure test (b, d, f, h). fl-FNA (2nmol) was injectedintrathecally alone (x) or 24h prior to lDMe (0.86nmol, 0), 3D(8.6nmol, *), morphine (13.2nmol, A) or DPDPE (40nmol, V).Separate animals were injected intrathecally with IDMe (0), 3D([]), morphine (A) or DPDPE (V) alone. Data are means+ s.e.meanof MPE from at least 6 rats *Significant differences from the actionof the agonist alone (P<0.05). The absence of an error bar indicatesthat the value of the s.e.mean is smaller than the size of the symbol.

was attenuated during the first 20 min period but markedlyenhanced and prolonged during the subsequent time period(40- 180 min) (Figure 7a and b). Co-injection ofmorphine anda sub-effective dose of 3D (0.009 nmol) also resulted in a

prolongation and enhancement of the effect of morphine inboth tests in the same time period (Figure 7c and d). To de-termine if the actions of the combination of peptide andmorphine were reversible, the antinociceptive actions wereevaluated in both tests at 24 h post injection. The anti-nociceptive responses (MPE) of the IDMe or 3D and mor-

phine combination 24 h after injection was MPE = 0.1 + 0.4(n = 11) and 3.6 + 1.8 (n = 12) (tail-flick test) and 3.3 + 1.2(n = 11) and 5.2 + 3.1 (n = 12) (paw-pressure test), respectively.These responses were not significantly different from thebaseline values in either test. Thus, both lDMe and 3D re-versibly enhanced the antinociceptive actions of morphine inthe two tests.

Effects of JDMe or 3D on [D-Ala2]deltorphin I-inducedantinociception

[D-Ala2]deltorphin I (20 nmol, i.t.) produced a prolongedmonophasic antinociceptive effect that reached a peak at30 min and slowly returned to baseline in both tests (Figure8). Intrathecal co-injection of a sub-effective dose(0.009 nmol) of IDMe or 3D produced a marked and longlasting significant increase in the effect of [D-Ala2]deltorphinI in the tail-flick test over the 10 min to 48 h period forIDMe (Figure 8a) and over the 40 min to 24 h period for

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Figure 6 Effects of the b-opioid receptor-selective antagonist,naltrindole, on the time-course of spinal antinociception induced byIDMe (a, b), 3D (c, d) or DPDPE (e, f) in the tail-flick test (a, c, e)and paw-pressure test (b, d, f). Naltrindole (2.2nmol) was injectedintrathecally alone (V) or 15 min prior to lDMe (0.86 nmol, *), 3D(8.6 nmol, *) or DPDPE (40 nmol, A). Separate animals wereinjected intrathecally with lDMe (0), 3D (EQ) or DPDPE (A\) alone.Data are means+s.e.mean of MPE from at least 6 rats. *Significantdifferences from the action of the agonist alone (P<0.05).

3D (Figure 8c). The responses returned to baseline valuesby 72 h post-injection for IDMe (data not shown) and by48 h for 3D (Figure 8c). In the paw-pressure test, IDMealso enhanced the action of [D-Ala2]deltorphin I but theincrease was not statistically significant (Figure 8b). Simi-larly, 3D also tended to enhance the antinociceptive re-

sponses produced by the 6-opioid agonist but this effect was

significant only at 90 and 120 min after coinjection (Figure8d).

Discussion

The results of the present study demonstrate that the in-trathecal injection of IDMe and 3D, two relatively peptidase-resistant NPFF analogues (Gicquel et al., 1992), producesantinociception in the tail-flick and paw-pressure tests in rats.This confirms our previous observations showing that NPFFexerts an antinociceptive effect at the spinal level (Gouardereset al., 1993c). Like NPFF, the spinal antinociceptive action ofIDMe and 3D was dose-related in the tail-flick but not the inpaw-pressure test. This difference may be consistent withfindings showing that different neuromodulatory systemsparticipate in thermal and mechanical nociception (Kuraishi etal., 1983; 1985a,b). Thus, neural mechanisms involved inthermal nociception may be more sensitive to the action ofthese peptides. Interestingly, on a molar basis, IDMe appearsto be 10 to 20 times more potent than both NPFF and 3D inthe thermal antinociception test.

Like NPFF (Gouarderes et al., 1993c), its analogues pro-duced sustained antinociception. This prolonged effect is un-likely to result from the action of intact peptide since NPFFanalogues are degraded by mouse brain homogenates within20 min in the absence of peptidase inhibitors (Gicquel et al.,1992). Thus, their prolonged effect could be due to formationof active metabolites or other factors such as sustained releaseof endogenous opioids.NPFF analogues, pu- and 6-opioids or their combination, at

doses tested in the current study did not produce any visiblesigns of motor disturbances. The fact that NPFF analogues in

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C. Gouarderes et al 497Spinal antinociception of NPFF and oploid receptors

I

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60

40

20

b

0 30 60 90 120 150 180 0 30 60 90 120 150 180

Time (min) after intrathecal injection

Figure 7 Effects of sub-effective doses of DMe (a, b) and 3D (c, d) on the time-course of the antinociception induced by spinal morphine inthe tail-flick test (a, c) and paw-pressure test (b, d). A dose of 0.009nmol DMe or 3D was co-injected intrathecally with 13.2nmol morphine(0). Separate animals were injected intrathecally with the peptides (L) or the morphine (0) alone. Data are means + s.e.mean of MPE from 11

to 21 animals. *Significant differences from the action of morphine alone (P<0.05). The responses from the combination returned to baselinevalues by 24h post-injection without any signs of motor impairment.

spinal cord may be involved more in sensory rather than inmotor function seems to be in accord with the finding that thecontent of endogenous NPFF in the dorsal spinal cord is muchhigher than that in the ventral cord (Majane et al., 1989; Allardet al., 1991). In addition, the dorsal cord contains a largerportion of the spinal NPFF binding sites (Allard et al., 1992).A spinal site for the observed antinociceptive effects is sug-gested by results of previous studies in which i.c.v. adminis-tration of a similar concentration of peptides failed to inducean antinociceptive effect in mice or rats (Gicquel et al., 1992;Million et al., 1993; Desprat & Zajac, 1994; Dupouy & Zajac,1995). Thus, the ability of IDMe and 3D to produce anti-nociception following their intrathecal injection is not due toan effect at a supraspinal site following diffusion to that site.Additionally, diffusion of peptides from the spinal sub-arachnoid space to supraspinal sites is minimized by use of asmall injection volume and by focusing drug application tocaudal (lumbosacral) areas of the spinal cord. These con-siderations strongly suggest that the observed antinociceptiveeffect of intrathecal NPFF analogues results from a directaction at the spinal level. It is possible that sites targeted byintrathecal NPFF analogues form part of an endogenousmechanism modulating nociceptive transmission at the spinallevel. The presence of a high density of NPFF im-munoreactivity (Majane et al., 1989; Allard et al., 1991; Kivi-pelto et al., 1991; Kivipelto & Panula, 1991a, b) as well asbinding sites (Allard et al., 1992; 1994; Devillers et al., 1994),that are partly localized on terminals of the primary afferents(Gouarderes et al., 1993b), supports this notion. Furthermore,in cultured spinal neurones, NPFF has been found to produceboth depolarizing and hyperpolarizing responses (Guzman etal., 1989). NPFF was also reported to decrease the inhibitoryaction of a p-opioid agonist (Magnuson et al., 1990) or an a2adrenoceptor agonist (Sullivan et al., 1991) on evoked C-fibrefiring in the rat spinal cord. In addition, Zhu et al. (1992) have

shown a release of NPFF from rat spinal cord following sti-mulation by potassium or substance P. These findings suggestthat NPFF may be involved in modulation of nociception atthe spinal level.

The spinal antinociceptive response produced by the twoNPFF analogues was partially but significantly attenuated byintrathecal naloxone at a dose that was effective against in-trathecal morphine. Naloxone was previously shown to at-tenuate spinal NPFF effects suggesting involvement of opioidsystems in its action (Gouarderes et al., 1993c). In the ratspinal cord, NPFF receptors (Allard et al., 1992; Devillers etal., 1994) and opioid receptors (Gouarderes et al., 1985; 1991;1993d) are similarly distributed. However, spinal NPFFbinding sites exhibited a very low affinity for opioid agonists(Allard et al., 1989; Devillers et al., 1994; Gouarderes et al.,unpublished results) and NPFF and its analogues do not bindto opioid receptors in vitro (Raffa et al., 1994; Gouarderes &Zajac, unpublished data). Therefore, the effect of naloxone onthe antinociceptive effect of NPFF analogues suggests theirindirect action in releasing endogenous opioid peptides in thespinal cord, the mechanism of which is not known.

Since naloxone can interact with y-, 6- and x-opioid re-ceptors that are present in the rat spinal cord (Gouarderes etal., 1985; 1991; 1993d), the action of u-receptor-selective(CTOP, ,B-FNA) and 6-receptor-selective (naltrindole) an-

tagonists on the peptide-induced effects was evaluated. The ji-

selective antagonists, CTOP (Gulya et al., 1988) and ,B-FNA(Zimmerman et al., 1987; Mjanger & Yaksh, 1991) pre-injectedintrathecally partially attenuated the antinociceptive effects ofIDMe and 3D at doses that were ineffective alone but antag-onized the actions of morphine. Indeed, the dose of P-FNAused here did not influence the antinociceptive effects of theopioid 6-agonist DPDPE (as was shown previously by Mjan-ger & Yaksh, 1991) suggesting that the action of,-FNA onantinociception induced by the two peptides involved interac-

E01.°20

8

*0 100 -

o-C 8C 0-.(

498 C. Gouard6res et al Spinal antinociception of NPFF and oplold receptors

C. Gouarderes et al Spinal antinociception of NPFF and opld receptors

b

d

0 30 60 90 120 150min

'24 h 48 h 0 30 60

Time after intrathecal injection

90 120 150 180" 24 h 48 hmin

Figure 8 Effects of sub-effective doses of 1DMe (a, b) and 3D (c, d) on the time-course of the antinociception induced by spinal [D-Ala2]deltorphin I in the tail-flick test (a, c) and paw-pressure test (b, d). A dose of 0.009nmol lDMe or 3D was co-injected intrathecally with20 nmol [D-Ala2]deltorphin I (0). Separate animals were injected intrathecally with the peptides (LI) or the eD-Ala2]deltorphin I (0) alone. Dataare means+s.e.mean of MPE from 5 to 15 animals. *Significant differences from the action of [D-Ala ]deltorphin I alone (P<0.05). Theresponses from the combination returned to baseline values by 72h post-injection without any signs of motor impairment.

tion with spinal ,u-receptors. Similarly, the 6-selective antago-nist, naltrindole (Portoghese et al., 1988; Calcagnetti &Holtzman, 1991; Drower et al., 1991; Tiseo & Yaksh, 1993)attenuated the spinal antinociceptive effects of NPFF analo-gues at a dose that blocked the action of DPDPE. This sug-gests that the naltrindole-sensitive antinociceptive effect ofNPFF analogues involves interaction with 6-receptors via an

indirect mechanism since NPFF-like agonists do not bind to 6-

opioid receptors in rat spinal cord sections (Gouarderes &Zajac, unpublished results). Unlike ,B-FNA, both CTOP andnaltrindole affected only the first phase of the peptide-inducedantinociceptive response. It is likely that these antagonists were

not present in sufficient amounts to influence the second phaseof response sensitive to fl-FNA.

At a low dose (0.009 nmol) inactive on its own in anti-nociception tests, both NPFF analogues produced a remark-able enhancement of morphine antinociception in both tests,as was observed previously with NPFF (Gouarderes et al.,1993c). Similarly, co-injection of this sub-effective dose oflDMe or 3D with [D-Ala2]deltorphin I both enhanced andprolonged the effect of this 6-opioid agonist. The fact thatNPFF-like drugs induced an increase of the spinal anti-nociceptive effects of morphine and [D-Ala2]deltorphin I,

opioid agonists that preferentially activate ,u- and b-opioidreceptors, respectively, in the spinal cord (Tung & Yaksh,1982; Omote et al., 1990; 1991; Mattia et al., 1992) suggeststhat one of the functions of endogenous NPFF in the spinalcord may be to modulate the action of endogenous opioidssuch as enkephalins or ,B-endorphin that normally interact with,u- and 6-receptors. The interactions of these peptides withendogenous opioids are under study.

The enhancement of morphine and [D-Ala2]deltorphin Ieffects observed here in unanaesthetized animals contrasts withthe antagonism by NPFF of the effects of DAMGO (Tyr-D-

Ala-Gly-(NMe)Phe-Gly-ol, a ,u-opioid agonist) in an electro-physiological study of C-fibre driven spinal nociceptive neu-

rones in anaesthetized rats (Magnuson et al., 1990). However,in that study the activity of DSTBULET (Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr), a 6-opioid agonist, was not blocked byNPFF. In contrast, the results of the present study show thatNPFF analogues can enhance the effects of a 6-receptor-se-lective agonist, [D-Ala2]deltorphin I. The reason for this dis-crepancy is not clear but factors such as anaesthesia,nociceptive stimulus modality, peptide dose and period ofobservation may be contributing factors in differences betweenbehavioural and electrophysiological experiments.

The interaction between NPFF analogues and morphine or

[D-Ala2]deltorphin I may have some practical implications.Since only a minimal activation of NPFF receptors and yi- or

6-receptors is required to produce a substantial antinocicep-tion, one would anticipate a reduction in the development oftolerance for analgesia when a combination of an NPFFanalogue and an opioid agonist is administered.

The prolonged enhancement of ,u- and 6-opioid anti-nociceptive action by agents previously thought to have op-

posite roles in the mediation of nociceptive processes isreminescent of the potentiation of morphine analgesia in thespinal cord by 10 to 100 pmol substance P (without any effecton tail-flick thresholds alone) (Kream et al., 1993). Anotherinteresting finding in our study is the sharp contrast betweenbrain and spinal cord for NPFF/opioid interaction. WhileNPFF and NPFF analogues clearly enhanced morphine anti-nociception in the spinal cord (Gouarderes et al., 1993c; thisstudy), they have been reported to inhibit morphine anti-nociception following brain injections (see Introduction).However, the doses used for combination studies in the brainare 250-2500 times higher than the dose of NPFF analoguesused in the spinal study. Indeed, the amount of NPFF or

a100 r

CL 0

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Ca c.0 100-

+1 80-

60

40

20

-20

499

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500 C. Gouarderes et al Spinal antinociception of NPFF and opiold receptors

NPFF analogues required for the negative modulation ofopioid action is relatively large, approximately 2-10 nmol inthe rat (Million et al., 1993; Dupouy & Zajac, 1995) and 9-22 nmol in the mouse (Gicquel et al., 1992; Desprat & Zajac,1994). These supraspinal doses were reported to have no an-tinociceptive effects when used alone. Their actions on noci-ception were seen only in combination with opioid agonists(anti-opioid action). However, in the spinal cord, these dosesof NPFF analogues (this study) and NPFF (Gouarderes et al.,1993c) were clearly effective in producing antinociceptive ef-fects.NPFF analogues, as well NPFF, may influence supraspinal

and spinal nociception by different mechanisms. In the brain, itis suggested that NPFF acts as an endogenous opioid an-tagonist. The evidence for this is largely provided by studies onmorphine-dependent animals in which NPFF has a profile ofactivity that corresponds to that of an antagonist (Malin et al.,1990; 1991; 1993). However, Rothman et al. (1993) have re-

cently shown that chronic i.c.v. infusion of NPFF down-reg-ulates ,u-opioid binding sites in brain, an effect that isinconsistent with that of naloxone which has been shown toupregulate P-receptors (Gouarderes et al., 1993a; Yoburn etal., 1993). In the spinal cord, the nociception tests involvinguse of NPFF and its two analogues have yielded a profile ofactivity that suggests an agonist-like role, although electro-physiological studies have shown an antagonist-like actionagainst y- but not 6-agonists (Magnuson et al., 1990). Thus,the role of NPFF-like peptides in spinal and supraspinalstructures remains to be determined.

This work was supported by grants from the Medical ResearchCouncil (MRC) of Canada (MRC to K.J.) and funds from CNRSand Association pour la Recherche sur le Cancer and Fondationpour la Recherche Medicale, France (to J.-M.Z.).

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(Received May 1, 1995Revised September 14, 1995Accepted October 3, 1995)