Effects of neuropeptide FF system on CB1 and CB2 receptors mediated antinociception in mice

Neuropharmacology 62 (2012) 855e864

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Effects of neuropeptide FF system on CB1 and CB2 receptors mediated antinociception in mice Quan Fanga, Zheng-Lan Hana, Ning Lia, Zi-Long Wanga, Ning Hea, Rui Wanga, b, * a

Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Physiology & Psychology, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou 730000, PR China b State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2011 Received in revised form 4 September 2011 Accepted 13 September 2011

It has been demonstrated that opioid and cannabinoid receptor systems can produce similar signal transduction and behavioural effects. Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in control of pain and analgesia through interactions with the opioid system. We were interested in whether the central and peripheral antinociception of cannabinoids could be influenced by supraspinal NPFF system. The present study examined the effects of NPFF and related peptides on the antinociceptive activities induced by the non-selective cannabinoid receptors agonist WIN55,212-2, given by supraspinal and intraplantar routes. In mice, the central and peripheral antinociception of WIN55,212-2 are mediated by cannabinoid CB1 and CB2 receptors, respectively. Interestingly, central administration of NPFF significantly reduced central and peripheral analgesia of cannabinoids in dose-dependent manners. In contrast, dNPA and NPVF (i.c.v.), two highly selective agonists for NPFF2 and NPFF1 receptors, dose-dependently augmented the antinociception caused by intracerebroventricular and intraplantar injection of WIN55,212-2. Additionally, pretreatment with the NPFF receptors selective antagonist RF9 (i.c.v.) markedly reduced the cannabinoid-modulating activities of NPFF and related peptides in nociceptive assays. These data provide the first evidence for a functional interaction between NPFF and cannabinoid systems, indicating that activation of central NPFF receptors interferes with cannabinoidmediated central and peripheral antinociception. Intriguingly, the present work may pave the way for a new strategy of using combination treatment of cannabinoid and NPFF agonists for pain management. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Neuropeptide FF (NPFF) Cannabinoid WIN55,212-2 Antinociception Mice

1. Introduction Neuropeptide FF (NPFF, FLFQPQRFamide) was originally isolated from bovine brain through its cross-reaction with antibodies to the molluscan cardioexcitory peptide FMRF-NH2, which possessed the similar C-terminal sequence (Yang et al., 1985). Recent reports have

Abbreviations: AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, 6-iodo-2-methyl-1-[2-(4morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone; BIBP3226, (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide; CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor type 2; dNPA, D.NP(N-Me) AFLFQPQRFamide; NPA-NPFF, NPAFLFQPQRFamide; NPVF, VPNLPQRFamide; WIN55,212-2, (R)-(þ)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone. * Corresponding author. Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou 730000, PR China. Tel./fax: þ86 931 8912567. E-mail address: [email protected] (R. Wang). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.09.013

shown that NPFF belongs to a neuropeptide family including two precursors (pro-NPFFA and pro-NPFFB) and two G-protein coupled receptors (NPFF1 and NPFF2) (Bonini et al., 2000; Elshourbagy et al., 2000; Hinuma et al., 2000; Liu et al., 2001; Perry et al., 1997; Vilim et al., 1999). NPFF1 and NPFF2 receptors have about 50% identical structure (Bonini et al., 2000; Elshourbagy et al., 2000; Liu et al., 2001). However, several studies suggested that the pro-NPFFA peptides (such as NPFF and NPA-NPFF) and pro-NPFFB peptides (such as NPVF) were the preferred ligands for NPFF2 and NPFF1 receptors, respectively (Liu et al., 2001; Mollereau et al., 2002). In addition, the structure-activities studies and pharmacological assays demonstrated that NPVF and dNPA (a stable analogue of NPA-NPFF) were the most selective towards NPFF1 and NPFF2 receptors, respectively (Gouarderes et al., 2007; Liu et al., 2001; Mollereau et al., 2002; Roussin et al., 2005). NPFF was isolated and considered as an anti-opioid peptide in 1985 (Yang et al., 1985). It was also shown to exhibit pharmacological opioid-like activities in previous studies (Harrison et al.,

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1998; Mollereau et al., 2005b; Mouledous et al., 2010; Panula et al., 1999; Yang and Iadarola, 2006). A considerable number of biological studies suggested that NPFF system was involved in pain modulation, food intake, gastrointestinal and hormonal modulation, modulation of opiate tolerance and abstinence and cardiovascular action (Mouledous et al., 2010; Panula et al., 1996; Roumy and Zajac, 1998). Similar to other opioid-modulating peptides, the link between NPFF and opioid systems has been widely investigated in in vitro and in vivo studies (Mollereau et al., 2005b; Mouledous et al., 2010). At the cellular level, many pharmacological data suggested that NPFF and related peptides exhibited anti-opioid effects via NPFF1 and NPFF2 receptors (Kersante et al., 2006; Mollereau et al., 2005a; Rebeyrolles et al., 1996; Roumy et al., 2003, 2007). Moreover, at the whole animal level, NPFF exhibited complex opioidmodulating activities in different pharmacological studies (Mollereau et al., 2005b; Panula et al., 1996). Intracerebroventricular administration of NPFF exhibited opioid-like inhibition of the mouse colonic bead propulsion time and inhibited small intestinal transit (Gicquel et al., 1993; Raffa and Jacoby, 1989). In contrast, the previous reports suggested that central administration of NPFF acted as an anti-opioid peptide in nociceptive modulation, locomotor activity and feeding behaviour (Mollereau et al., 2005b; Mouledous et al., 2010; Panula et al., 1999; Roumy and Zajac, 1998). Recently, NPFF-related peptides exhibiting different selectivities towards NPFF1 and NPFF2 receptors could evoke different effects on morphine-induced analgesia after i.c.v. co-administration in mice (Quelven et al., 2005; Roussin et al., 2005). So, it demonstrated that opioid-modulating actions of NPFF-related peptides were not strictly related to the selectivity towards NPFF1 and NPFF2 receptors. It is well known that opioid and cannabinoid are two different receptor systems. Activation of opioid or cannabinoid receptors can induce similar pharmacological activities, including analgesia, sedation, hypothermia, inhibition of intestinal motility and motor activity. Both receptor types also were found to share similar signal transduction mechanisms (Bushlin et al., 2010; Pacher et al., 2006). Moreover, the functional interaction between opioid and cannabinoid systems has been extensively explored, especially in the production of antinociception (Bushlin et al., 2010; Cichewicz, 2004). Interestingly, the participation of opioids in antinociception of cannabinoid receptors agonists had been supported by a number of studies, while the participation of cannabinoids in the peripheral and central antinociception induced by opioids had also been observed in recent years (Bushlin et al., 2010; Pacheco et al., 2009, 2008; Reche et al., 1998). Additionally, systemic or intraplantar injection of cannabinoids had shown to result in the release of endogenous opioid peptides (Corchero et al., 1997; Ibrahim et al., 2005; Manzanares et al., 1998). There is considerable evidence describing the common feature of opioid and cannabinoid receptor systems (Bushlin et al., 2010; Pacher et al., 2006). Opioid-modulating peptides had been widely proved as the modulators of opioid functions (Mollereau et al., 2005b). However, a few studies investigated the interactions between opioid-modulating peptides and cannabinoids. To date, several studies had shown that the nociceptin/orphanin FQ (N/OFQ)induced hyperphagia was inhibited by cannabinoid CB1 receptor antagonist SR141716, whereas WIN55,212-2-evoked hypothermia was blocked by N/OFQ receptor antagonist JTC-801 (Pietras and Rowland, 2002; Rawls et al., 2007). In addition, recent reports have shown that the interaction between cholecystokinin and endocannabinoids may underlie the processes of stress-induced analgesia and extinction learning (Chhatwal et al., 2009; Kurrikoff et al., 2008). To our knowledge, no attention has been directed to the functional interaction between NPFF and cannabinoid systems. Therefore, in the present study, the modulating roles of NPFF and related peptides

(i.c.v.) in the central and peripheral antinocieption of cannabinoids were investigated in mice. 2. Materials and methods 2.1. Animals The experiments were performed on male Kunming strain mice from the Experimental Animal Center of Lanzhou University. The mice were housed in a temperaturecontrolled room (22  1  C). Food and water were freely available until the onset of the nociceptive test. All animals were cared for and experiments were carried out in accordance with the European Community guidelines for the use of experimental animals (86/609/EEC). All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China. 2.2. Chemicals NPFF, dNPA, NPVF and RF9 were prepared by manual solid-phase synthesis using standard Fmoc chemistry, as described earlier (Fang et al., 2011). The cannabinoid agonist WIN55,212-2 and naloxone were obtained from Sigma-Aldrich. The selective CB1 and CB2 receptors antagonists AM251 and AM630 were purchased from Tocris. Morphine hydrochloride was the product of Shenyang First Pharmaceutical Factory, China. WIN55,212-2, AM251, AM630 and naloxone were dissolved in the vehicle (a 1:1:18 ratio of cremophor:dimethyl sulfoxide:saline solution) before injection. All other drugs were dissolved in sterilized distilled saline and stored at 20  C. Drugs were i.c.v. administered at a fixed volume of 5 ml (at a constant rate of 10 ml/min), which was followed by 1 ml saline to flush in the drug by using a 25-ml microsyringe. In addition, WIN55,212-2, AM251, AM630 and naloxone were injected intraplantarly at a volume of 10 ml in the paw-withdrawal assay. 2.3. Implantation of cannula into lateral ventricle Surgical implantation of cannula was conducted in an aseptic environment, as described earlier (Li et al., 2009). Mice were anesthetized with pentobarbital sodium (80 mg/kg, intraperitoneally), and placed in a stereotaxic apparatus. The incision area of the scalp was shaved, and a sagittal incision was made in the midline exposing the surface of the skull. A single hole was drilled through the skull targeted above the left or right lateral ventricle, 3.0 mm posterior and 1.0 mm lateral to the bregma. A stainless steel guide cannula was implanted 3.0 mm ventrally from the surface of skull. To prevent occlusion, a dummy cannula was inserted into the guide cannula. The dummy cannula protruded 0.5 mm from the guide cannula. Dental cement was used to fix the guide cannula to the skull. After surgery, the animals were allowed to recover for at least 5 days, and during this time mice were gently handled daily to minimize the stress associated with manipulation of the animals throughout the experiments. 2.4. Tail-flick test The nociceptive response was assessed by the radiant heat tail-flick test. Briefly, male Kunming mice weighing 20e22 g were used. The animals were gently restrained by hand, and a light beam was focused onto the tail. At the beginning of the study, the lamp intensity was adjusted to elicit a response in control animals within 3e5 s. A cut-off time was set at 10 s to minimize tissue damage. Tail-flick time was determined before injection and then at 5, 10, 15, 20, 30, 40, 50 and 60 min after injection. Every male mouse was used only once. 2.5. Paw-withdrawal test (lamp-foot-flick assay) Experiments were performed according to an adaptation of the previous studies (Chen et al., 2006; Ibrahim et al., 2006). Male Kunming mice weighing 24e26 g were used. The paw-withdrawal responses were examined by the same method as the tail-flick using radiant heat. The male mice were gently handled and held the body without restraining the head or legs. A radiant heat source was focused onto the plantar surface of the hindpaw. The time that elapsed before the mouse withdrew its paw was recorded as the paw-withdrawal latency. At the beginning of the study, the lamp intensity was adjusted to elicit a response in control animals within 3e5 s. A cut-off time was set at 10 s to minimize tissue damage. Paw-flick time was determined before injection and then at 5, 10, 15, 20, 30, 40, 50 and 60 min after injection. Every male mouse was used only once. 2.6. Experimental protocol In the mouse tail-flick test, experiment was designed to examine the effect of the NPFF system on the central antinociception induced by cannabinoids. The nonselective cannabinoid agonist WIN55,212-2 was administered into the lateral ventricle and produced the central antinociception of cannabinoids. A series of antagonists AM251, AM630 and naloxone were injected i.c.v. 5 min prior to WIN55,212-2 to investigate whether cannabinoid and opioid receptors were

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involved in the central antinociception of cannabinoids. Moreover, to further test the effects of NPFF system on cannabinoid-induced central antinociception, NPFF, dNPA and NPVF were injected alone or co-injected with the antagonist RF9 by the i.c.v. route 20 min prior to the WIN55,212-2. In the paw-withdrawal assay, experiment was designed to determine whether the peripheral antinociception of cannabinoids could be influenced by central NPFF system. Injection WIN55,212-2 into the plantar region of the hindpaw induced peripheral antinociception to a thermal stimulus applied to the same paw. To investigate the analgesic mechanisms, AM251, AM630 and naloxone were injected into the hindpaw ipsilateral or contralateral to the side of nociceptive testing 5 min prior to WIN55,212-2, respectively. Furthermore, to further test the effects of NPFF receptor agonists on the peripheral antinociception of WIN55,212-2, NPFF, dNPA and NPVF were injected i.c.v. alone or co-injected with RF9 (i.c.v.) 10 min before the i.paw administration of WIN55,212-2. 2.7. Histology and statistical analysis After completion of behavioural testing, mice were injected with methylene blue dye (3 ml), which was allowed to diffuse for 10 min. Then mice were decapitated, and their brains were removed and frozen. Gross dissection of the brains was used to verify the placement of the cannula. Only the data from those animals with dispersion of the dye throughout the ventricles were used in the study. Data were given as means  S.E.M. Data are expressed as the percent maximum possible effect (MPE) calculated as: MPE (%) ¼ 100  [(post-drug response  baseline response)/(cut-off response  baseline response)]. The raw data from each animal were converted to area under the curve (AUC). We calculated the AUC data over the period 0e30 min. The area under the curves depicting total %MPE versus time was computed by trapezoidal approximation over the period 0e30 min. Data were statistically compared by means of one-way ANOVA followed by the Dunnett’s post-hoc test or Bonferroni’s post-hoc test. Two-way ANOVA with repeated measurements followed by Dunnett’s test were with PRISM 5.0 (GraphPad, San Diego). Probabilities of less than 5% (P < 0.05) were considered statistically significant.

3. Results 3.1. Effects of i.c.v. administration of AM251, AM630 and naloxone on the central antinociception induced by WIN55,212-2 In the mouse tail-flick test, central administration of vehicle did not significantly alter the nociceptive threshold. Compared with vehicle treated animals, intracerebroventricular (i.c.v.) injection of WIN55,212-2 (1.3, 2.6 and 3.9 mg) dose-dependently produced significant increases in tail withdrawal latencies, reaching maximal antinociceptive response at 10 min after the injection in conscious mice (Fig. 1). The two-way ANOVA of these data revealed significant effects for both dose and time, F3, 288 ¼ 131.7, P < 0.0001 and F8, 288 ¼ 126.6, P < 0.0001, respectively, as well as, a statistically significant dose  time interaction, F24, 288 ¼ 27.35, P < 0.0001. To characterize the central antinociception of WIN55,212-2, antagonists of cannabinoid and opioid receptors were further used in the present study. As shown in Fig. 2, co-injection of the CB1 receptor antagonist AM251 (10 mg, i.c.v.) significantly blocked the WIN55,212-2-induced central antinociception. However, neither the CB2 receptor antagonist AM630 (10 mg, i.c.v.) nor the opioid receptors antagonist naloxone (5 nmol, i.c.v.) alter the antinociceptive effect induced by i.c.v. administration of WIN55,212-2 (3.9 mg, i.c.v.). In addition, at the same doses, these three antagonists did not modify the tail-flick latency in mice (P > 0.05, data not shown).

Fig. 1. Dose- and time-related analgesic effects of i.c.v. administration of WIN55,212-2 (1.3, 2.6 and 3.9 mg) in the mouse tail-flick assays. Data points represent means  S.E.M. from experiments conducted on 9 mice. AUC calculated during 0e30 min from these data are statistically analyzed and are presented in the insert. Significant differences from the action of vehicle control alone with one-way ANOVA followed by the Dunnett’s post-hoc test (**P < 0.01 and ***P < 0.001).

NPVF (30 nmol, i.c.v.), two highly selective agonists for NPFF2 and NPFF1 receptors, markedly increased the central antinociception of WIN55,212-2 compared to that induced by injection at the same dose of WIN55,212-2 alone (Fig. 3 and Table 1). Injected into the lateral ventricle alone, dNPA (30 nmol) and NPVF (30 nmol) did not change the tail-flick latency (Fig. 4B and C). To investigate the modulatory effects of NPFF system on WIN55,212-2-induced central antinociception, the dose-effect relationships of NPFF-related peptides were further studied. When WIN55,212-2 (3.9 mg, i.c.v.) induced 73% of MPE, i.c.v. injection of NPFF (3, 10 and 30 nmol) inhibited the cannabinoid-induced central antinociception in a dose-dependent manner (Fig. 4A and Table 1). The two-way ANOVA revealed that the dose  time interaction was

3.2. Effects of i.c.v. administration of NPFF-related peptides on the central antinociception induced by WIN55,212-2 A dose of 2.6 mg WIN55,212-2 into the lateral ventricle was used to induce a 50% analgesia at the peak effect allowing investigation of both potentiation and reversion of the central antinociception induced by the cannabinoid agonist (Fig. 3). I.c.v. administration of NPFF (30 nmol) alone did not modify nociceptive threshold but significantly reduced WIN55,212-2induced central analgesia. In contrast, dNPA (30 nmol, i.c.v.) and

Fig. 2. Effects of i.c.v. administration of AM251 (10 mg), AM630 (10 mg) and naloxone (5 nmol) on the central antinociception produced by WIN55,212-2 (3.9 mg, i.c.v.) in the mouse tail-flick assays. Data points represent means  S.E.M. from experiments conducted on 8 mice. AUC calculated during 0e30 min from these data are statistically analyzed and are presented in the insert. Significant difference compared to Vehicle þ 3.9 mg WIN55,212-2-injected group (analysis of one-way ANOVA followed by the Bonferroni’s post-hoc test, **P < 0.01 and ***P < 0.001).

858

Q. Fang et al. / Neuropharmacology 62 (2012) 855e864 90

Saline (i.c.v.) + 2.6 µg WIN55,212-2 (i.c.v.) N = 8

Antinociception (MPE%)

80 70

30 nmol NPFF (i.c.v.) + 2.6 µg WIN55,212-2 (i.c.v.) N = 8

60

30 nmol dNPA (i.c.v.)+ 2.6 µg WIN55,212-2 (i.c.v.) N = 7

50

30 nmol NPVF (i.c.v.) + 2.6 µg WIN55,212-2 (i.c.v.) N = 8

3.3. Effects of i.paw administration of AM251, AM630 and naloxone on the peripheral antinociception induced by WIN55,212-2

40 30 20 10 0 -10 0

10

20

30

40

50

tail-flick test, and partially antagonized the strong potentiating effect of NPVF on WIN55,212-2 antinociception at the supraspinal level (Fig. 5).

60

Time(min)

Fig. 3. Effects of i.c.v. administration of NPFF (30 nmol), dNPA (30 nmol) and NPVF (30 nmol) on the central antinociception produced by WIN55,212-2 (2.6 mg, i.c.v.) in the mouse tail-flick assays. Data points represent means  S.E.M. from experiments conducted on 7e8 mice.

statistical significance (F24, 261 ¼ 11.73, P < 0.0001). Whereas, two highly selective agonists dNPA (3, 10 and 30 nmol, i.c.v.) and NPVF (3, 10 and 30 nmol, i.c.v.) dose-dependently enhanced the central antinociception induced by a low dose of WIN55,212-2 (1.3 mg, i.c.v.; Fig. 4 and Table 1). The two-way ANOVA of these data indicated that the dose  time interactions were statistical significant (F24, 252 ¼ 5.233, P < 0.0001 and F24, 225 ¼ 17.3, P < 0.0001, respectively). Based on the doseeresponse relationships for the morphinepotentiating effects, the order of the potency of these two selective agonists to increase WIN55,212-2-induced central analgesia was: NPVF > dNPA. Moreover, effects of the antagonist RF9 on the cannabinoidmodulating activities of NPFF and related peptides were further investigated in the tail-flick test. RF9 (30 nmol, i.c.v.) itself had no significant effect on nociceptive threshold and the central antinociception induced by WIN55,212-2 (1.3 and 3.9 mg, i.c.v.; Fig. 5). However, pretreatment with 30 nmol of RF9 (i.c.v.) fully blocked the WIN55,212-2-modulating actions of NPFF and dNPA in the mouse

The non-selective cannabinoid agonist, WIN55,212-2 (15, 25 and 35 mg, i.paw) produced the dose- and time-related antinociception to a thermal stimulus applied to the hindpaw, when administered into the hindpaw on the side of testing (Fig. 6). The two-way ANOVA of these data revealed significant effects for both dose and time, F3, 252 ¼ 100.3, P < 0.0001 and F8, 252 ¼ 94.96, P < 0.0001, respectively, as well as, a statistically significant dose  time interaction, F24, 252 ¼ 14.93, P < 0.0001. To explore whether the cannabinoid receptors or the opioid receptors are involved in WIN55,212-2-induced peripheral antinociception, the CB1 receptor antagonist AM251, the CB2 receptor antagonist AM630 and the classical opioid receptors antagonist naloxone were co-injected into the hindpaw ipsilateral or contralateral to the side of nociceptive testing, respectively. In Fig. 7A, the CB2 receptor antagonist AM630 (2.5 mg), administered into the ipsilateral paw, completely prevented the peripheral antinociception induced by WIN55,212-2. Interestingly, ipsilateral intrapaw the classical opioid receptors antagonist naloxone (25 mg) markedly blocked the antinociceptive effect of i.paw the cannabinoid agonist. In contrast, an equivalent dose of AM630 or naloxone administered into the paw contralateral to the side of testing did not antagonize the peripheral antinociception of WIN55,212-2 (Fig. 7A and C). However, the CB1 receptor selective antagonist AM251 (7.5 mg) had no effect on WIN55,212-2-induced peripheral antinociception, when given into either hindpaw (Fig. 7B). In addition, these three antagonists did not alter the nociceptive threshold in control mice (P > 0.05, versus Vehicle þ Vehicle-injected group with Bonferroni’s post-hoc test). 3.4. Effects of i.c.v. administration of NPFF-related peptides on the peripheral antinociception induced by WIN55,212-2 In order to investigate the modulating effects of NPFF-related peptides on the peripheral antinociception of cannabinoid, a dose of 25 mg WIN55,212-2 into the hindpaw was used to induce a 57%

Table 1 Effects of i.c.v. administration of NPFF, dNPA and NPVF on WIN55,212-2-induced peripheral and central antinociception in mice. Peptides (i.c.v.)

Tail-flick assay n

WIN55,212-2

Saline 30 nmol NPFF 30 nmol dNPA 30 nmol NPVF

2.6 2.6 2.6 2.6

mg (i.c.v.) mg (i.c.v.) mg (i.c.v.) mg (i.c.v.)

703 359 1172 1584

   

45 84** 71*** 36***

8 8 7 8

25 25 25 25

mg (i.paw) mg (i.paw) mg (i.paw) mg (i.paw)

933 383 1656 1569

   

56 61*** 106*** 65***

8 7 7 8

Saline 3 nmol NPFF 10 nmol NPFF 30 nmol NPFF

3.9 3.9 3.9 3.9

mg (i.c.v.) mg (i.c.v.) mg (i.c.v.) mg (i.c.v.)

1140 834 607 396

   

65 53** 35*** 46***

8 8 8 9

35 35 35 35

mg (i.paw) mg (i.paw) mg (i.paw) mg (i.paw)

1675 1294 910 499

   

64 53*** 45*** 39***

7 7 7 7

Saline 3 nmol dNPA 10 nmol dNPA 30 nmol dNPA

1.3 1.3 1.3 1.3

mg (i.c.v.) mg (i.c.v.) mg (i.c.v.) mg (i.c.v.)

227 316 553 792

   

59 35 73** 81***

8 9 7 8

15 15 15 15

mg (i.paw) mg (i.paw) mg (i.paw) mg (i.paw)

328 597 787 1218

   

17 32** 56*** 80***

8 7 7 7

Saline 3 nmol NPVF 10 nmol NPVF 30 nmol NPVF

1.3 1.3 1.3 1.3

mg (i.c.v.) mg (i.c.v.) mg (i.c.v.) mg (i.c.v.)

227 647 1059 1599

   

59 67*** 54*** 52***

8 7 7 7

15 15 15 15

mg (i.paw) mg (i.paw) mg (i.paw) mg (i.paw)

328 637 1048 1526

   

17 46*** 64*** 67***

8 7 7 7

WIN55,212-2

Paw-withdrawal test AUC (0e30 min)

AUC (0e30 min)

n

Data are means  S.E.M. The values for antinociceptive response, expressed as area under the time-effect curve (AUC) during 0e30 min **P < 0.01 and ***P < 0.001 indicating significant differences from antinociception of WIN55,212-2 in the absence of NPFF-related peptides (Saline þ WIN55,212-2-injected group) with one-way ANOVA followed by the Bonferroni’s post-hoc test.

Q. Fang et al. / Neuropharmacology 62 (2012) 855e864

A 80 Saline (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N=8 3 nmol NPFF (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N = 8 10 nmol NPFF (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N = 8 30 nmol NPFF (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N = 9 30 nmol NPFF (i.c.v.) + Vehicle (i.c.v.) N = 8

70 60

Antinociception (MPE%)

50 40 30 20

Saline (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N=8 30 nmol RF9 (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N=8 30 nmol NPFF (i.c.v.)+ 3.9 µg WIN55,212-2 (i.c.v.) N=9 30 nmol (NPFF+RF9) (i.c.v.) + 3.9 µg WIN55,212-2 (i.c.v.) N=9 1200 ###

1000 AUC(0-30 min)

A

859

10

800 600 ***

400

0

200 -10 0

10

20

40

50

60

0

B

Saline (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N = 8

Saline (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N=8 30 nmol RF9 (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N=8 30 nmol dNPA (i.c.v.)+ 1.3 µg WIN55,212-2 (i.c.v.) N=8 30 nmol (dNPA+RF9) (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N=8 30 nmol NPVF (i.c.v.)+ 1.3 µg WIN55,212-2 (i.c.v.) N=7 30 nmol (NPVF+RF9) (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N=7 1800

50

3 nmol dNPA (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N = 9

40

10 nmol dNPA (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N = 7

30

30 nmol dNPA (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N = 8

1600

20

30 nmol dNPA (i.c.v.) + Vehicle (i.c.v.) N=8

1400

10

0

AUC(0-30 min)

Antinociception (MPE%)

B 60

30 Time(min)

***

1200 1000 ***

###

800 600

-10 0

C

10

20

90

30 Time(min)

40

50

60

3 nmol NPVF (i.c.v.) + 1.3 µg WIN55,212-2 (i.c.v.) N = 7

Antinociception (MPE%)

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Fig. 4. Dose-related effects of i.c.v. administration of NPFF-related peptides on the central antinociception produced by WIN55,212-2 (i.c.v.) in the mouse tail-flick assays. (A) Reduction of 3.9 mg WIN55,212-2-induced central antinociception by NPFF (3, 10 and 30 nmol). (B) and (C) Potentiation of 1.3 mg WIN55,212-2-induced central antinociception by dNPA (3, 10 and 30 nmol) and NPVF (3, 10 and 30 nmol). Data points represent means  S.E.M. from experiments conducted on 7e9 mice.

0 Fig. 5. The effects of NPFF-related peptides (30 nmol, i.c.v.) on WIN55,212-2-induced central antinociception were antagonized by co-administration of RF9 (30 nmol, i.c.v.) in mouse tail-flick test. (A) Data are expressed as differences in AUC between WIN55,212-2 (3.9 mg) and WIN55,212-2 co-injected with NPFF, RF9 or NPFF plus RF9, S.E.M. during 0e30 min. (B) Data are expressed as differences in AUC between WIN55,212-2 (1.3 mg) and WIN55,212-2 co-injected with NPFF receptors selective agonists (dNPA and NPVF), RF9 or agonists plus RF9, S.E.M. during 0e30 min. Data points represent means  S.E.M. from experiments conducted on 7e9 mice. ***P < 0.001, versus Saline þ WIN55,212-2-injected group; # # #P < 0.001 indicating significant differences from the modulatory effects of NPFF-related peptides in the absence of RF9 with one-way ANOVA followed by the Bonferroni’s post-hoc test.

analgesia at the peak effect (Fig. 8). Surprisingly, i.c.v. administration of NPFF (30 nmol) markedly evoked a significant decrease of the peripheral antinociception induced by WIN55,212-2, while the selective agonists dNPA (30 nmol, i.c.v.) and NPVF (30 nmol, i.c.v.) markedly augmented the peripheral antinociception of WIN55,212-2 (Fig. 8 and Table 1). Injected into the lateral ventricle alone, these three NPFF-related peptides (30 nmol) did not modify nociceptive threshold (Fig. 9). To further determine the effects of NPFF system on the peripheral antinociception of WIN55,212-2, the dose-effect relationships of NPFF-related peptides were investigated. Central injection of NPFF (3,10 and 30 nmol, i.c.v.) significantly reversed WIN55,212-2-induced antinociception (35 mg, i.paw) in a dose-dependent manner (Fig. 9A

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and Table 1). The two-way ANOVA revealed that the dose  time interaction was statistical significance (F24, 216 ¼ 15.21, P < 0.0001). However, when WIN55,212-2 (15 mg, i.paw) induced 27% of MPE, i.c.v. administration of both dNPA (3, 10 and 30 nmol, i.c.v.) and NPVF (3, 10 and 30 nmol, i.c.v.) dose-dependently potentiated the peripheral antinociceptive effect induced by WIN55,212-2 (15 mg, i.paw; Fig. 9 and Table 1). Additionally, NPVF displayed a much higher potency than dNPA in this assay. The two-way ANOVA of these data indicated that the dose  time interactions were statistical significance (F24, 225 ¼ 10.29, P < 0.0001 and F24, 225 ¼ 14.97, P < 0.0001, respectively). Furthermore, to explore whether the NPFF receptors are involved in the cannabinoid-modulating activities of NPFF-related peptides, the antagonist RF9 was co-injected with NPFF receptors agonists. The results showed that RF9 (30 nmol, i.c.v.) had no significant effect on nociceptive threshold and the peripheral antinociception induced by WIN55,212-2 (15 and 35 mg, i.paw; Fig. 10). While co-administration with 30 nmol RF9 (i.c.v.) completely blocked the modulating activities induced by NPFF, dNPA and NPVF on WIN55,212-2-induced peripheral antinociception in this nociceptive test (Fig. 10).

Vehicle (i.paw) N = 7 15 µg WIN55,212-2 (i.paw) N = 8 25 µg WIN55,212-2 (i.paw) N = 10 35 µg WIN55,212-2 (i.paw) N = 7

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Fig. 6. Dose- and time-related analgesic effects of i.paw administration of WIN55,2122 (15, 25 and 35 mg) in paw-withdrawal test in mice. Data points represent means  S.E.M. from experiments conducted on 7e10 mice. AUC calculated during 0e30 min from these data are statistically analyzed and are presented in the insert. Significant differences from the action of vehicle control alone with one-way ANOVA followed by the Dunnett’s post-hoc test (**P < 0.01 and ***P < 0.001).

A 100

Ipsil Vehicle (i.paw) + 35 µg WIN55,212-2 (i.paw) N = 8 Ipsil 2.5 µg AM630 (i.paw) + Vehicle (i.paw) N=7 Ipsil 2.5 µg AM630 (i.paw) + 35 µg WIN55,212-2(i.paw) N = 7 Contra 2.5 µg AM630 (i.paw) + 35 µg WIN55,212-2 (i.paw) N = 7

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NPFF represents a neurotransmitter system acting as a modulator of opioid functions (Mollereau et al., 2005b; Mouledous et al., 2010). Several publications have suggested that NPFF and related analogues play important roles in nociceptive regulation through interactions with the opioid system (Panula et al., 1999; Roumy and Zajac, 1998; Yang and Iadarola, 2006; Yang et al., 2008). Very recently, growing

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Ipsil Vehicle (i.paw) + 35 µg WIN55,212-2 (i.paw) N = 8 Ipsil 25 µg Naloxone (i.paw) + Vehicle (i.paw) N=7 Ipsil 25 µg Naloxone (i.paw) + 35 µg WIN55,212-2(i.paw) N = 7 Contra 25 µg Naloxone(i.paw) + 35 µg WIN55,212-2 (i.paw) N = 7

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Ipsil Vehicle (i.paw) + 35 µg WIN55,212-2 (i.paw) Ipsil 2.5 µg AM630 (i.paw)+35 µg WIN55,212-2 (i.paw) Contra 2.5 µg AM630 (i.paw)+35 µg WIN55,212-2 (i.paw) Ipsil 7.5 µg AM251 (i.paw)+35 µg WIN55,212-2 (i.paw) Contra 7.5 µg AM251 (i.paw)+35 µg WIN55,212-2 (i.paw) Ipsil 25 µg Naloxone (i.paw)+35 µg WIN55,212-2 (i.paw) Contra 25 µg Naloxone (i.paw)+35 µg WIN55,212-2 (i.paw)

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Ipsil Vehicle (i.paw)+ 35 µg WIN55,212-2 (i.paw) N = 8 Ipsil 7.5 µg AM251 (i.paw)+ Vehicle (i.paw) N=7 Ipsil 7.5 µg AM251 (i.paw)+35 µg WIN55,212-2 (i.paw) N=7 Contra 7.5 µg AM251 (i.paw)+35 µg WIN55,212-2 (i.paw) N=7

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Fig. 7. Effects of i.paw administration of AM630 (2.5 mg), AM251 (7.5 mg) and naloxone (25 mg) on the peripheral antinociception produced by WIN55,212-2 (35 mg, i.paw) in pawwithdrawal test in mice. AM630 (A), AM251 (B) and naloxone (C) were injected into the hindpaw ipsilateral (Ipsil) or contralateral (Contra) to the side of nociceptive testing. (D) Data are expressed as differences in AUC between WIN55,212-2 (35 mg) and WIN55,212-2 co-injected with AM630, AM251 or naloxone, S.E.M. during 0e30 min. Data points represent means  S.E.M. ***P < 0.001 indicating significant differences compared to Vehicle þ 35 mg WIN55,212-2-injected group with one-way ANOVA followed by the Bonferroni’s post-hoc test.

Q. Fang et al. / Neuropharmacology 62 (2012) 855e864

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Fig. 8. Effects of i.c.v. administration of NPFF (30 nmol), dNPA (30 nmol) and NPVF (30 nmol) on the peripheral antinociception produced by WIN55,212-2 (25 mg, i.paw) in paw-withdrawal test in mice. Data points represent means  S.E.M. from experiments conducted on 7e8 mice.

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evidence supports that opioid and cannabinoid systems share common features, including signal transduction, antinociception, sedation and hypothermia, suggesting a similar distribution and mechanism of action (Bushlin et al., 2010; Manzanares et al., 1999; Pacher et al., 2006). Given the lack of information regarding the links between opioid-modulating peptides and cannabinoid systems in nociceptive modulation, the present study was conducted to investigate the modulating role of supraspinal NPFF system in the antinociceptive effect of cannabinoids administered at the central and peripheral level. Initially, the non-selective cannabinoid receptors agonist WIN55,212-2 was used to induce central antinociception in tail-flick test. As expected, i.c.v. injection WIN55,212-2 caused antinociception in a dose-dependent manner. In addition, WIN55,212-2-induced central antinociception were significantly attenuated by the cannabinoid CB1 receptor antagonist AM251 but not the CB2 receptor antagonist AM630. Our data imply that the central antinociceptive response to WIN55,212-2 is mainly mediated by CB1 receptor pathway, which keeps consistent with previous reports (Benamar et al., 2008; Meng et al., 1998). For example, in the cold-water tailflick test, the central antinociception of WIN55,212-2 was shown to be mediated by activation of CB1 receptor but not CB2 receptor (Benamar et al., 2008). These observations agree with the results that the CB1 receptor localized preferentially in the brain and CB2 receptor localized outside the central nervous system (Pacher et al., 2006; Pertwee, 2009). Furthermore, the central antinociception induced by WIN55,212-2 was not changed by pretreatment with classical opioid antagonist naloxone, suggesting that the opioid system may not be involved in the central antinociception of WIN55,212-2. In an attempt to examine the NPFF and cannabinoid interactions, NPFF and WIN55,212-2 were co-administered into the lateral ventricle in the mouse tail-flick test. Our present results demonstrated that i.c.v. administration of NPFF did not modify the nociceptive threshold but dose-dependently reduced the analgesia induced by WIN55,212-2 administered at the supraspinal level. Furthermore, the cannabinoidmodulating effects of NPFF were markedly antagonized by the NPFF receptors selective antagonist RF9 (i.c.v.) (Fang et al., 2008; Simonin et al., 2006), suggesting that the modulatory roles of NPFF in the central antinociception mediated by CB1 receptor were mainly linked to specific activation of NPFF receptors. The previous studies using cells expressing NPFF receptors have revealed that both NPFF1 and NPFF2 receptors recognize NPFF with

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Fig. 9. Dose-related effects of i.c.v. administration of NPFF-related peptides on the peripheral antinociception produced by WIN55,212-2 (i.paw) in paw-withdrawal test in mice. (A) Reduction of 35 mg WIN55,212-2-induced peripheral antinociception by NPFF (3, 10 and 30 nmol). (B) and (C) Potentiation of 15 mg WIN55,212-2-induced peripheral antinociception by dNPA (3, 10 and 30 nmol) and NPVF (3, 10 and 30 nmol). Data points represent means  S.E.M. from experiments conducted on 7e8 mice.

affinities in the nanomolar range (Bonini et al., 2000; Gouarderes et al., 2007). To further explore the roles of two NPFF receptor subtypes in the cannabinoid-modulating effects, NPVF and dNPA, two selective agonists for NPFF1 and NPFF2 receptors, were used in this study, respectively. dNPA, a stable analogue of pro-NPFFA

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Q. Fang et al. / Neuropharmacology 62 (2012) 855e864

Saline (i.c.v.) + 35 µg WIN55,212-2 (i.paw) N = 7 30 nmol RF9 (i.c.v.) + 35 µg WIN55,212-2 (i.paw) N = 7 30 nmol NPFF (i.c.v.) + 35 µg WIN55,212-2 (i.paw) N = 7 30 nmol (NPFF+RF9) (i.c.v.) + 35 µg WIN55,212-2 (i.paw) N = 7

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B Saline (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 8 30 nmol RF9 (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 7 30 nmol dNPA (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 7 30 nmol (dNPA+RF9) (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 7 30 nmol NPVF (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 7 30 nmol (NPVF+RF9) (i.c.v.) + 15 µg WIN55,212-2 (i.paw) N = 7 1800 ***

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300 0 Fig. 10. The effects of NPFF-related peptides (30 nmol, i.c.v.) on WIN55,212-2-induced peripheral antinociception were antagonized by co-administration of RF9 (30 nmol, i.c.v.) in paw-withdrawal test in mice. (A) Data are expressed as differences in AUC between WIN55,212-2 (35 mg, i.paw) and WIN55,212-2 co-injected with NPFF, RF9 or NPFF plus RF9, S.E.M. during 0e30 min. (B) Data are expressed as differences in AUC between WIN55,212-2 (15 mg, i.paw) and WIN55,212-2 co-injected with NPFF receptors selective agonists (dNPA and NPVF), RF9 or agonists plus RF9, S.E.M. during 0e30 min. Data points represent means  S.E.M. from experiments conducted on 7e8 mice. ***P < 0.001, versus Saline þ WIN55,212-2-injected group; # # #P < 0.001 indicating significant differences from the modulatory effects of NPFF-related peptides in the absence of RF9 with one-way ANOVA followed by the Bonferroni’s post-hoc test.

peptide NPA-NPFF, has at least 100 times higher affinity for the NPFF2 than for the NPFF1 receptor (Gouarderes et al., 2007; Roussin et al., 2005), whereas the pro-NPFFB peptide NPVF has 30 times more affinity for the NPFF1 than for the NPFF2 receptor (Gouarderes et al., 2007; Mollereau et al., 2002). To our surprise, either dNPA or NPVF administered at supraspinal route enhanced WIN55,212-2induced central antinociception in a dose-dependent manner. In addition, the NPFF receptors antagonist RF9 (i.c.v.) significantly reduced the cannabinoid-modulating activities induced by these two highly selective agonists for NPFF1 and NPFF2 receptors, indicating that the pharmacological effects of dNPA and NPVF were mediated through its receptors. Thus, our findings might be taken to suggest that dNPA, a highly selective NPFF2 receptor agonist,

causes the same modulating effect on WIN55,212-2-induced central antinociception as the NPFF1 receptor agonist NPVF at the comparable doses and in contrast, the non-selective NPFF receptor agonist (NPFF) exerted opposite action. These features imply that activation of central NPFF1 and NPFF2 receptors exerts complex modulating actions on central antinociception of WIN55,212-2 mediated by CB1 receptor, which is not strictly related to the selectivities and affinities of the agonists used. It is difficult to explore the complex modulating effects of NPFF and related peptides on the central antinociception of cannabinoids at present. However, there are at least two possible mechanisms to explain the phenomena in the present study. The first reason involves a heteromerization between NPFF and CB1 receptors. Some evidences support this assumption. CB1 receptor and NPFF receptors are classified as G-protein coupled receptor family. Moreover, cannabinoid CB1 receptor was found in several brain regions known to participate in antinociception (Pacher et al., 2006; Pertwee, 2009). It is worthy to note that the recent results showed that NPFF2 receptors could affect mu-opioid receptor by physical interaction (Roumy et al., 2007). However, the oligomerization of NPFF and CB1 receptors might alter NPFF ligand properties and affect receptor trafficking, which result in the complex cannabinoid-modulating actions of NPFF agonists. Indeed, the dimerization of opioid receptors has been shown to display novel pharmacology and signalling regulation properties (George et al., 2000; Levac et al., 2002; Waldhoer et al., 2005). The second is that there may be some separate subtypes of NPFF1 and/or NPFF2 receptors in the brain. It raises the possibility that different subtype receptors were stimulated by NPFF-related peptides and involved in the complex modulation of antinociceptive effects mediated by CB1 receptor. Therefore, further pharmacological studies are required to prove the detailed mechanism involved in the interaction between NPFF and cannabinoid systems. In addition, taking into account the potencies of the two highly selective NPFF agonists, in this study, NPVF displayed a much higher potency than dNPA. The observation could be partially explained by the activation of other receptors besides NPFF receptors, which also leads to potentiation of WIN55,212-2-induced central antinociception. This deduction is further supported by the present results that the modulating effect of NPVF on WIN55,212-2-induced central antinociception was partially antagonized by a high dose of RF9. However, at the same dose, RF9 is sufficient to completely block the modulating action of NPVF on the peripheral antinociception of cannabinoids. Indeed, the previous data demonstrated that BIBP3226, a mixed antagonist of NPY Y1 and NPFF receptors, did not modify the pro-opioid effect of NPVF after supraspinal co-administration (Fang et al., 2006). The second important point worthy of comment is the effect of central administration of NPFF and the highly selective ligands, dNPA and NPVF, on the peripheral analgesia of cannabinoids. Thus, in pawwithdrawal assay, WIN55,212-2 injected into one paw induced peripheral antinociception in a dose-dependent manner when a stimulus was applied to the same paw. As expected, an equivalent dose of WIN55,212-2 did not produce antinociception when administrated into the contralateral hindpaw, indicating the local, peripheral nature of the antinociception induced by i.paw WIN55,212-2. In the previous studies, both CB1 and CB2 receptors were reported in peripheral tissues (Pacher et al., 2006; Pertwee, 2009). In the present work, AM630 prevented the WIN55,212-2induced antinociceptive effect, indicating that the activation of cannabinoid CB2 receptor in the hindpaw mediates the peripheral antinociception of cannabinoids. In contrast, AM251 did not affect the peripheral analgesia of WIN55,212-2, suggesting that WIN55,212-2indcued antinociception is insensitive to CB1 receptor activation at peripheral level. Taken together, the peripheral antinociception of WIN55,212-2 appears to be due to activation of CB2 receptor, which is

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consistent with the results obtained in the previous studies (Ibrahim et al., 2005, 2006; Malan et al., 2001). Furthermore, this peripheral antinociception of cannabinoid was sensitive to the classical opioid receptors antagonist naloxone, which is in agreement with the data obtained in the recent study by using the CB2 receptor selective agonist AM1241. Ibrahim et al. reported that activation of keratinocyte CB2 receptor results in the release of endogenous opioid peptides, which then acts on primary afferent neurons to produce peripheral antinociception (Ibrahim et al., 2005). In addition, the inability of both AM630 and naloxone to affect WIN55,212-2-induced peripheral antinociception when injected into the hindpaw contralateral to the side of testing supports a local site of the peripheral antinociception of WIN55,212-2. Similarly, central administration of NPFF reduced peripheral analgesia of cannabinoids, whereas the selective agonists dNPA and NPVF dose-dependently enhanced WIN55,212-2-induced peripheral antinociception meditated by CB2 receptor. Additionally, the NPFF receptors antagonist RF9 completely prevented the regulatory effects of NPFF and related peptides on WIN55,212-2-induced peripheral antinociception, indicating that the modulating actions of these NPFF agonists are mediated by NPFF1 and NPFF2 receptors. However, further studies are required to define the roles of these two types NPFF receptors in the cannabinoid-modulating effects. Taken together the above findings, NPFF system in the brain not only interacts with the central antinociception of WIN55,212-2, but also alters the peripheral analgesia of cannabinoids. It is notable that the central and peripheral antinociception induced by WIN55,212-2 were mediated by CB1 and CB2 receptor, respectively. As to the different localization of CB1 receptor and CB2 receptor, two different mechanisms might be involved in modulating effects of NPFF system on the central and peripheral antinociception of cannabinoids. NPFF and its related peptides modulate the local, peripheral antinociception of cannabinoids through the activation of NPFF receptors in the brain, and might influence the nociceptive processing of cannabinoids-induced peripheral antinociception via neuronal circuit. However, the detailed nature of the interaction of NPFF and cannabinoid systems needs to be further characterized. In contrast to NPFF, dNPA and NPVF displayed additive effects on the central and peripheral antinociception induce by cannabinoids. It is worthy to note that the peripheral antinociception of WIN55,212-2 resulted from the release of endogenous opioids. Moreover, the previous data demonstrated that dNPA only exerts an anti-opioid effect after supraspinal administration (Fang et al., 2011; Roussin et al., 2005). Thus, it seems difficult to explain why the highly selective NPFF2 receptor agonist dNPA enhanced cannabinoid-induced peripheral analgesia related to the release of opioid peptides. However, this implies that the antinociceptive effects of opioid and cannabinoid might be modulated by NPFF agonists via different pathways. In the present work, central administration of NPFF dosedependently attenuated both supraspinal and peripheral antinociception, which might be helpful to understand the development of cross-tolerance between opioid and cannabinoid agonists in nociceptive assays (Bushlin et al., 2010). In other words, the analgesic action of cannabinoids could be reduced when there are elevated levels of NPFF, which frequently occurs after morphine treatment. Indeed, chronic or acute morphine treatment was reported to be associated with an increase of NPFF-like immunoreactivity in brain (Stinus et al., 1995). In recent years, cannabinoid receptor-specific agonists are considered potent and exciting analgesics (Pacher et al., 2006; Pertwee, 2009). With respect to the cannabinoid-potentiating effects of dNPA and NPVF, co-administration of low doses of cannabinoids with NPFF agonists may provide ways to separate the analgesic activity of cannabinoids from their side effects, though additive

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effects may be compound specific and correct pairing of the most effective combinations is essential. In short, the strategy using combination treatment of cannabinoid and NPFF agonists is an attractive avenue of research that needs to be more fully investigated. In conclusion, the body of data derived from our present experiments provides, for the first time, the in vivo evidence for a functional interaction between NPFF and cannabinoid systems. Interestingly, NPFF system demonstrates weak intrinsic activity but exerts complex effects on the central and peripheral analgesia mediated by CB1 and CB2 receptors, respectively. The effects are due to the activation of NPFF receptors in the brain. These results should facilitate the investigation of the role of NPFF system in acute pain and may open novel pharmacological interventions. NPFF is not only an opioidmodulating peptide but also a novel modulator of the activity of cannabinoids. Acknowledgements This study was supported by the grants from the National Natural Science Foundation of China (Nos. 20932003 and 20902041), the Key National S&T Program of the Ministry of Science and Technology (2009ZX09503-017), the Specialized Research Fund for the Doctoral Program in Higher Education Institutions (No. 200807301028), and the Fundamental Research Funds for the Central Universities. References Benamar, K., Geller, E.B., Adler, M.W., 2008. First in vivo evidence for a functional interaction between chemokine and cannabinoid systems in the brain. J. Pharmacol. Exp. Ther. 325, 641e645. Bonini, J.A., Jones, K.A., Adham, N., Forray, C., Artymyshyn, R., Durkin, M.M., Smith, K.E., Tamm, J.A., Boteju, L.W., Lakhlani, P.P., Raddatz, R., Yao, W.J., Ogozalek, K.L., Boyle, N., Kouranova, E.V., Quan, Y., Vaysse, P.J., Wetzel, J.M., Branchek, T.A., Gerald, C., Borowsky, B., 2000. Identification and characterization of two G proteincoupled receptors for neuropeptide FF. J. Biol. Chem. 275, 39324e39331. Bushlin, I., Rozenfeld, R., Devi, L.A., 2010. Cannabinoid-opioid interactions during neuropathic pain and analgesia. Curr. Opin. Pharmacol. 10, 80e86. Chen, X., Bing, F., Dai, P., Hong, Y., 2006. Involvement of protein kinase C in 5-HTevoked thermal hyperalgesia and spinal fos protein expression in the rat. Pharmacol. Biochem. Behav. 84, 8e16. Chhatwal, J.P., Gutman, A.R., Maguschak, K.A., Bowser, M.E., Yang, Y., Davis, M., Ressler, K.J., 2009. Functional interactions between endocannabinoid and CCK neurotransmitter systems may be critical for extinction learning. Neuropsychopharmacology 34, 509e521. Cichewicz, D.L., 2004. Synergistic interactions between cannabinoid and opioid analgesics. Life Sci. 74, 1317e1324. Corchero, J., Avila, M.A., Fuentes, J.A., Manzanares, J., 1997. delta-9Tetrahydrocannabinol increases prodynorphin and proenkephalin gene expression in the spinal cord of the rat. Life Sci. 61, PL 39ePL 43. Elshourbagy, N.A., Ames, R.S., Fitzgerald, L.R., Foley, J.J., Chambers, J.K., Szekeres, P.G., Evans, N.A., Schmidt, D.B., Buckley, P.T., Dytko, G.M., Murdock, P.R., Milligan, G., Groarke, D.A., Tan, K.B., Shabon, U., Nuthulaganti, P., Wang, D.Y., Wilson, S., Bergsma, D.J., Sarau, H.M., 2000. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J. Biol. Chem. 275, 25965e25971. Fang, Q., Guo, J., He, F., Peng, Y.L., Chang, M., Wang, R., 2006. In vivo inhibition of neuropeptide FF agonism by BIBP3226, an NPY Y1 receptor antagonist. Peptides 27, 2207e2213. Fang, Q., Jiang, T.N., Li, N., Han, Z.L., Wang, R., 2011. Central administration of neuropeptide FF and related peptides attenuate systemic morphine analgesia in mice. Protein Pept. Lett. 18, 403e409. Fang, Q., Wang, Y.Q., He, F., Guo, J., Guo, J., Chen, Q., Wang, R., 2008. Inhibition of neuropeptide FF (NPFF)-induced hypothermia and anti-morphine analgesia by RF9, a new selective NPFF receptors antagonist. Regul. Pept. 147, 45e51. George, S.R., Fan, T., Xie, Z., Tse, R., Tam, V., Varghese, G., O’Dowd, B.F., 2000. Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. J. Biol. Chem. 275, 26128e26135. Gicquel, S., Fioramonti, J., Bueno, L., Zajac, J.M., 1993. Effects of F8Famide analogs on intestinal transit in mice. Peptides 14, 749e753. Gouarderes, C., Mazarguil, H., Mollereau, C., Chartrel, N., Leprince, J., Vaudry, H., Zajac, J.M., 2007. Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding. Neuropharmacology 52, 376e386. Harrison, L.M., Kastin, A.J., Zadina, J.E., 1998. Opiate tolerance and dependence: receptors, G-proteins, and antiopiates. Peptides 19, 1603e1630.

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