Involvement of opioid receptors in the antinociception produced by intracerebroventricularly administered spantide in mice

Neuropeptides (1995) 29, 293-299 © Pearson Professional Ltd 1995

Involvement of Opioid Receptors in the Antinociception Produced by Intracerebroventricularly Administered Spantide in Mice K. TAN-NO*, T. SAKURADAI, T. YAMADA*, S. SAKURADA* AND K. KISARA*

*Department of Pharmacology, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendai 981, Japan and fDepartment of Biochemistry, Daiichi College of Pharmaceutical Science, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815, Japan (Reprint requests to KT)

Abstract--The antinociceptive effect of intracerebroventricularly (i.c.v.) administered [D-Arg 1, D-Trp 7'9, Leu11]-substance P (spantide), a non-selective tachykinin antagonist, was examined using the mouse formalin test. Licking behaviour induced by 2% formalin solution in the hindpaw of mice had two peaks, 0-5 min (first phase) and 10-30 min (second phase). I.c.v. spantide produced a dose-dependent antinociception during the first and second phases. The IDs0 values were 2.95 (1.59-5.46) nmol for the first phase and 2.87 (1.49-5.52) nmol for the second phase. The antinociceptive effect in the first phase, but not in the second phase produced by spantide was antagonized by pretreatment with naloxone (1.0 mg/kg, i.p.), an opioid receptor antagonist. An opioid binding study using [3H]naloxone revealed that spantide was able to inhibit [3H]naloxone binding to mouse brain membrane preparations. These results suggest that opioid receptor systems in the mouse brain are involved in spantide-induced antinociception during the first phase, but not during the second phase of the formalin-induced nociceptive behaviour.

Introduction

The undecapeptide substance P (SP) has been proposed as a neurotransmitter and/or neuromodulator in the transmission of nociceptive

Date received 21 March 1995 Date accepted 16 July 1995 Correspondence to: Koichi Tan-No, Department of Pharmacology, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendal 981, Japan, Tel: (0)22-234-4181, Fax: (0)22275-2013.

information from the peripheral to the spinal cord, 1 SP belongs to a family of peptides named tachykinins, which are characterized by a common Cterminal amino acid sequence, Phe-X-Gly-LeuMet-NH2. Apart from SP, two other tachykinins have been discovered in mammalian tissues: neurokinin A (NK A) and neurokinin B (NK B). 2,3 Currently, three distinct types of binding sites for tachykinins have been identified in mammalian tissues. The three mammalian tachykinins interact preferentially with at least three different receptors

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294 called neurokinin (NK)~ for SP, NK2 for NK A and NK3 for NK B. 4 Behavioural studies using mammalian tachykinin agonists have suggested that scratching, biting and licking behaviour is largely evoked by SP, an NKI preferring agonist. 5 Iontophoretic studies using receptor preferring agonists have also revealed that dorsal horn excitation of nociceptive neurons is mediated at least in part by NK1 receptors. 6 The formalin test for the identification of antinociceptive agents, originally described in rats and cats by Dubuisson and Dennis, 7 is now used widely as a model of chemogenic nociception in both rats and mice. s-14 Subcutaneous (s.c.) injection of dilute formalin into the hindpaw produces two distinct periods of high licking and biting behaviours which are considered to be nociceptive. The levels of SPlike immunoreactivity in the dorsal horn of the spinal cord are increased by the injection of formalin, as assayed by the immunohistochemical method.IS ~7 Several SP analogues with D-amino acid substitutions have been shown to be NK receptor antagonists. Of these SP analogues, [D-Arg ~, DTrp 7,9, LeuI~]-SP (spantide), has been widely used as a pharmacological tool for elucidating the roles of endogenous NKs in physiological processes. 18'19 Intrathecal (i.t.) administration of spantide caused a naloxone-reversible reduction in scratching, biting and licking behaviours which were produced by the injection of NK2 and NK3 receptor agonists. 2° In the present study, we investigated the antinociceptive effect of intracerebroventricularly (i.c.v.) administered spantide in the mouse formalin test. Moreover, we sought to examine whether spantide-induced antinociception is mediated through an opioid receptor by the in vivo and in vitro assays.

Materials and methods

Animals Male Std-ddY mice, weighing 20-24 g, were housed in colony cages at room temperature of 23 + 2°C and humidity 55 + 5% in a room with a 12 h light/ dark cycle. They had free access to water and food.

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Drugs and chemicals The following drugs and chemicals were purchased from commercial sources: spantide (Peptide Institute, Osaka, Japan), naloxone hydrochloride (Sigma Chemical Co., St. Louis, Mo., USA), [3H]naloxone (New England Nuclear, Boston, Mass., USA) and formalin (Nacalai Tesque, Osaka, Japan). All other chemicals were of the purest grade commercially available. Administration procedure Spantide was dissolved in artificial cerebrospinal fluid (CSF), containing NaC1 7.4 g, KC1 0.19 g, MgC12 0.19 g, CaC12 0.14 g/1000 ml of sterilized and distilled water. Spantide or artificial CSF (5 #1 volume) was injected free hand at a rate of 10-15 s into the right-side lateral cerebroventricle of a mouse using a 50 #1 Hamilton microsyringe according to the method of Haley and McCormick. 2~ Naloxone was dissolved in physiological saline and was administered intraperitoneally (i.p.) in a volume of 0.1 ml/10 g/mouse. Naloxone (1.0 mg/kg) was administered i.p. 15 min before spantide for the study of the first phase in the formalin test, and naloxone (1.0-4.0 mg/kg) was administered simultaneously with spantide for the study of the second phase. Procedure of formalin test Approximately 1 h before testing, the animals were placed individually in transparent cages (22.0 x 15.0 x 12.5 cm), which also served as the observation chamber. A mirror was placed behind the chamber to allow clear observation of the hindpaws. After this period of adaptation, the mouse was taken out of the cage and 20 pl of 2% formalin solution (0.74% formaldehyde in saline) was injected s.c. into the dorsal surface of the right hindpaw. Each animal was immediately put back into the observation cage. The amount of time the animals spent licking the injected paw, the toe or the leg was timed with a stopwatch. The animals were observed individually for 30 min, immediately after the s.c. injection of formalin. On the basis of the response pattern described elsewhere, 2z two distinct periods of intensive licking activity were identified. Recording of the first behavioural response (first phase) started immediately after the

INTRACEREBROVENTRICULARLY ADMINISTERED SPANTIDE IN MICE

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injection of 2% formalin solution and lasted for 5 rain (0-5 rain). The recording of the second behavioral response (second phase started 10 rain after 2% formalin solution injection and lasted for 20 rain (10-30 min). The injection timing of spantide was determined by the previously reported data that spantide had a peak effect at 5-10 rain after i.t. injection. 2°'23'24Consequently, spantide (1.0-8.0 nmol) was administered 5 rain before or 10 min after 2% formalin solution in order to evaluate the peak effect of spantide in the two phases independently.

Analyses of data Statistical analysis of the results included determination of IDs0 values by the method of Litchfield and Wilcoxon 28 and Dunnett's test for multiple comparisons, after analysis of variance (aNOVA). The criterion of significance was set at P < 0.05. All results are given as mean + SEM.

Procedure of opioid receptor binding assay Brain homegenates extracted from the whole brains of mice without cerebellum were prepared as previously described by Chang and Cuatrecasas 25 and stored frozen at -90°C. The binding of [3H]naloxone to brain homogenates was performed for 40 rain at 25°C in a total volume of 250 #1:50 mM Tris-HCl buffer (pH 7.4) containing crude membranes (300 #g of protein), 10 mM MgCl2, 0.04% BSA, 10 #M phosphoramidon, 40 #g/ml bacitracin, 4 #g/ml leupeptin, 2 #g/ml chymostatin, [3H] naloxone (0.1-5 nM) and either 10 #M unlabelled naloxone (non-specific binding) or absence (total binding). In competition studies, 2 nM [3H] naloxone was incubated with varying concentrations of spantide or unlabelled naloxone as reference for comparison. At the end of the incubation period, bound [3H]naloxone was separated from free [3H]naloxone by rapid filtration under vacuum through Whatman (GF/B) glass filters presoaked overnight at 4°C in 0.1% polyethyleneimine using a receptor-binding harvester. The filter was washed twice with the same ice-cold Tris-HC1 buffer (5 ml) and transferred to vials. A 3 ml volume of liquid scintillation fluid was added and radioactivity was determined by liquid scintillation spectrometry (Beckman LS7800). Specific binding to the opioid receptor was calculated by subtracting non-specific binding from total binding. Equilibrium dissociation constants (KD) and binding capacities (Bin,x) were determined from Scatchard transformation. 26 The Ki values were converted from ICs0 values. Protein content was determined by the method of Bradford. 27 Binding assays were performed in duplicate, and each titration was repeated three times at least.

Effect of spantide on 2% formalin-induced nociceptire behaviour The injection of 2% formalin solution into the dorsal surface ofa hindpaw caused an acute nociceptive behaviour that lasted about 5 rain (first phase). Subsequently, this behaviour completely disappeared for about 5 rain and then reoccurred and lasted about 20 min (second phase). When administered i.c.v. 5 rain before the injection of formalin, spantide (1.0--8.0 nmol) produced a dose-dependent reduction of the first phase of the nociceptive behaviour (Fig. 1A). The same doses of spantide, injected 5 rain post-formalin, produced a dose-dependent reduction of the second phase of the nociceptive behaviour (Fig. 1B). I.c.v. administration of spantide (1.0-8.0 nmol) produced no signs of flaccid paralysis of the hindlimbs, pain or irritation. The mice behaved normally during the first 10 rain after i.c.v, administration of spantide. The IDs0 values were 2.95 (1.59-5.46) nmol for the first phase and 2.87 (1.49-5.52) nmol for the second phase.

Results

Effect of naloxone on the antinociceptive action of span tide As shown in Figure 2A, pretreatment with noloxone (1.0 mg/kg, i.p.) resulted in a significant antagonism against 4.0 nmol of spantide which can induce a significant antinociception in the first phase. In contrast, even a high dose of naloxone (4.0 mg/kg, i.p.) gave no significant antagonism against spantide in the second phase (Fig. 2B). Naloxone was without affecting the nociceptive behaviour evoked by formalin in artificial CSFtreated control mice.

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Fig. 1 Effect of intracerebroventricularly administered spantide on the 2% formalin-induced licking behaviour during the first phase (A) and second phase (B). Spantide was administered 5 min before 2% formalin solution for the study of the first phase and 10 min after 2% formalin solution for the study of the second phase. The data are given as mean + SEM for groups of 10 mice. *P < 0.05, **P < 0.01 when compared to CSFtreated control.

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Binding properties of spantide for opioid receptors

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I N T R A C E R E B R O V E N T R I C U L A R L Y A D M I N I S T E R E D SPANTIDE IN MICE

shown to increase the reaction time when tested in the tail-flick test. 29 Similarly, i.t. administration of spantide into rats elicits antinociception in the hotplate and tail-flick tests, z3 Recently, we have found that antinociceptive effect of i.t. administered spantide is reversed by pretreatment with naloxone, an opioid antagonistJ 3 In addition, competition studies confirmed that the binding of the opioid ligand [3H]naloxone to mouse spinal cord membrane preparations was displaced by spantide with a low but measurable affinity. 2° Spantide is known to be a broad spectrum antagonist of tachykinin receptors in the spinal cord. 5'32-34However, there is no available report concerning the non-selective mechanism of action of spantide in the supraspinal cord. Therefore, in the present study, we investigated the effect of i.c.v, administered spantide on the formalininduced nociceptive behaviour in mice. The results of the present study indicate that spantide (1.0-8.0 nmol) produced a dose-dependent antinociceptive effect during both phases in the mouse formalin test. Spantide-induced antinociception during the first phase was antagonized significantly by pretreatment with naloxone (1.0 mg/kg, i.p.). Moreover, spantide had an affinity for binding sites of [3H]naloxone on mouse brain membrane preparations, though spantide was approximately 3 500-fold less active than unlabelled naloxone as reference for comparison. These findings suggest that spantide may produce an opioiddependent antinociception by a direct effect within the brain. This suggestion for an opioid activity of spantide is supported by the findings that: 1. Spantide, administered i.t. in mice, produced a naloxone-reversible reduction of the spinally mediated scratching, biting and licking behaviours induced by NK2 and NK3 agonists (NK A, D-septide, NK B and eledoisin), 2° 2. Spantide, administered i.c.v., inhibited naloxone-induced morphine withdrawal response in guinea-pigs 35 and 3. [D-Trp 7,9, Leu11]-SP, substituted D-Arg 1with LArg ~ in the sequence of spantide, administered i.t. in mice, produced antinociception in the tailflick and hotplate assays which are reversed by a relatively high dose of naloxone (5.0 mg/kg, i.p.). 29 In addition, we have recently found that the bind-

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ing of [3H]-[D-Ala 2, MePhe 4, Gly(ol) 5] enkephalin (DAMaO) to mouse spinal cord membranes was displaced by spantide at large concentrations (10 -410 -6 M), while spantide had no measurable affinity for [3H]-(D-Pen2, D-Pen s] enkephalin (DPDPE) to delta sites and [3H]U69,593 to kappa sites (unpublished data). On the other hand, receptor mechanisms underlying opioid antinociception have been discussed in the formalin test. 36 39 Calcagnetti et aP 7 tested the selective receptor agonists, DAMGO, Dt'DeE and U50488H for their ability to produce antinociception on the formalin test after i.c.v. administration. On the basis of dose-response data, the rank order of antinociceptive efficacy was DAMGO> DPDPE > U5048814. The other authors also suggested that mu receptor may be important in opioid-induced antinociception in the formalin test, while involvement of delta or kappa receptor are contradictory. Collectively, it seems likely that spantide may act at least as a mu receptor agonist in the brain and elicit naloxone-reversible antinociception in the first phase. However, spantide-induced antinociception during the second phase was not antagonized by even a high dose of naloxone (4.0 mg/kg, i.p.). This finding in the second phase response cannot simply be explained by the opioid mechanism in the brain. Recently, we have found a difference of antagonistic activity of naloxone on morphine-induced antinociception in the first and second phases of the nociceptive behaviour; antinociception induced by i.c.v, administration of morphine during the first phase was antagonized readily by 0.5 mg/kg, i.p. of naloxone, while a higher dose (4.0 mg/kg, i.p.) of naloxone was required to antagonize the antinociceptive activity induced by morphine during the second phase. 4° On the other hand, using a similar approach to study the involvement of opioid systems in the spinal cord, we have shown that the antinociceptive effect of spantide was reversed significantly in the first phase of the formalininduced nociceptive behaviour by 1.0 and 2.0 mg/kg of naloxone and the second phase by 2.0 and 4.0 mg/kg of naloxone. 13 Thus, the difference of i.c.v. and i.t. morphine is that i.c.v, morphine had a naloxone-resistant antinociceptive effect during the second phase. These results suggest that the brain opioid mechanism may not be involved in mediating the antinociceptive effect of spantide in the

298 second phase o f the formalin-induced nociceptive behaviour. In reviewing the literature, the study o f the dose-dependent relationships between naloxone and m o r p h i n e indicated that naloxone attenuated m o r p h i n e antinociception at the lower dose (0.1 and 0.3 mg/kg) and potentiated m o r p h i n e antinociception at the largest dose (10 mg/kg) in the formalin test, while naloxone did not affect m o r phine antinociception at the intermediate dose (1 mg/kg) 41. In the tail flick test, 0.3 m g / k g naloxone completely reversed m o r p h i n e antiociception. These results led us to speculate that naloxone m a y have similar effect on spantide-induced antinociception. In the present study, therefore, naloxone ( 1 . 0 4 . 0 mg/kg) pretreatment failed to antagonize spantide-induced antinociception in the second phase response. Verification o f this concept, however, requires m u c h m o r e extensive studies o f the dose-dependent relationships between naloxone and spantide. These studies are currently in progress. O n the other hand, the opioid-independent mechanism o f spantide-induced antinociception should also be considered in the further study. The m e c h a n i s m o f spantide-induced antinociception in the central nervous system m a y be explained in part by the i m m u n e system. M o r p h i n e induced antinociception during the second phase was reversed significantly by pretreatment with hydrocortisone acetate, an i m m u n o s u p p r e s s a n t in mice. 4° Moreover, there is an interesting report that r e c o m b i n a n t h u m a n t u m o r necrosis factor and r e c o m b i n a n t h u m a n interleukin-l~ had a potent antinociceptive effect when given intravenously in the mouse phenylquinone writhing test and that their effect is due to a central mechanism but is n o t naloxone-reversible. 42 Spantide has been widely used in tackykinin studies as a putative antagonist, and appears to have relatively high selectivity for SP in the guinea-pig ileum. 43 A t the m o u s e spinal level, however, spantide significantly inhibited the nociceptive behavioral response elicited by N K A, eledoisin, somatostatin, 5,33 N-methyl-D-aspartate, 32 serotonin 44 and pilocarpine 4s as well as NK1 agonists. The N K A-induced depolarization on m o t o n e urones of the isolated spinal cord o f the n e w b o r n rat was also depressed by spantide. 34 Moreover, spantide, administered i.t. in the rat, p r o d u c e d bilateral m o t o r blockade o f the hindlegs, lasting

NEUROPEPTIDES for 3 d a y s Y These previous studies indicate that spantide is a non-selective tachykinin antagonist. In summary, the present results indicate that spantide has a naloxone-reversible antinociceptive action on the formalin-induced nociceptive behaviour during the first phase and an affinity for binding sites o f [3H]naloxone on mouse brain m e m b r a n e preparations.

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