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Effects of neuropeptide FF and related peptides on the antinociceptive activities of VD-hemopressin(α) in naive and cannabinoid-tolerant mice
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Neuropharmacology and analgesia
Effects of neuropeptide FF and related peptides on the antinociceptive activities of VD-hemopressin(α) in naive and cannabinoid-tolerant mice Jia-Xin Pan 1, Zi-Long Wang 1, Ning Li, Nan Zhang, Pei Wang, Hong-Hai Tang, Ting Zhang, Hong-Ping Yu, Run Zhang, Ting Zheng, Quan Fang n, Rui Wang n Key Laboratory of Preclinical Study for New Drugs of Gansu Province, and Institute of Physiology, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou, 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2015 Received in revised form 20 September 2015 Accepted 8 October 2015
Neuropeptide FF (NPFF) system has recently been reported to modulate cannabinoid-induced antinociception. The aim of the present study was to further investigate the roles of NPFF system in the antinociceptive effects induced by intracerebroventricular (i.c.v.) administration of mouse VD-hemopressin(α), a novel endogenous agonist of cannabinoid CB1 receptor, in naive and VD-hemopressin(α)tolerant mice. The effects of NPFF system on the antinociception induced by VD-hemopressin(α) were investigated in the radiant heat tail-flick test in naive mice and VD-hemopressin(α)-tolerant mice. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c. v.) for 5 days and the antinociception was measured on day 6. In naive mice, intracerebroventricular injection of NPFF dose-dependently attenuated central analgesia of VD-hemopressin(α). In contrast, neuropeptide VF (NPVF) and D.NP(N-Me)AFLFQPQRF-NH2 (dNPA), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, enhanced VD-hemopressin(α)-induced antinociception in a dose-dependent manner. In addition, the VD-hemopressin(α)-modulating activities of NPFF and related peptides were antagonized by the Neuropeptide FF receptors selective antagonist 1-adamantanecarbonyl-RF-NH2 (RF9). In VD-hemopressin(α)-tolerant mice, NPFF failed to modify VDhemopressin(α)-induced antinociception. However, both neuropeptide VF and dNPA dose-dependently potentiated the antinociception of VD-hemopressin(α) and these cannabinoid-potentiating effects were reduced by RF9. The present works support the cannabinoid-modulating character of NPFF system in naive and cannabinoid-tolerant mice. In addition, the data suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cannabinoids Neuropeptide FF VD-hemopressin(α) Antinociception Tolerance Mice Chemical compounds studied in this article: NPFF (PubChem CID: 123797) Neuropeptide VF (PubChem CID: 71451611) RF9 (PubChem CID: 53320361) Hemopressin (PubChem CID: 71312009) RVD-hemopressin(α) (PubChem CID: 90488874) WIN55,212-2 (PubChem CID: 6604176) Δ9-tetrahydrocannabinol (PubChem CID: 16078) 2-arachidonoylglycerol (PubChem CID: 5282280) 1DMe (PubChem CID: 71345956) Hu-210 (PubChem CID: 9821569)
1. Introduction Cannabinoid system plays an important role in the treatment of various diseases, including pain management. Two types of cannabinoid receptors, cannabinoid receptor type 1 (CB1) and type 2 (CB2), have been identified and cloned (Matsuda et al., 1990; Munro et al., 1993). The cannabinoid CB1 receptor is primarily expressed in the central nervous system, whereas the cannabinoid CB2 receptor is mainly expressed in immune cells (Howlett et al., 2002). The cannabinoid ligands have been classified into phytocannabinoids n
Corresponding authors. Tel./fax: þ86 931 8912567. E-mail addresses: [email protected] (Q. Fang), [email protected] (R. Wang). 1 Both authors contributed equally to this work.
(e.g., Δ9-tetrahydrocannabinol), synthetic ligands [e.g., (R) (þ ) [2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN55,212-2)] and lipid endocannabinoids (e.g., 2-arachidonoylglycerol) (Bomar and Galande, 2013; Pacher et al., 2006). Recent studies demonstrated that hemopressin and related peptides functioned as the novel endogenous peptide ligands of cannabinoid receptors derived from the hemoglobin (Gomes et al., 2009; Heimann et al., 2007). VD-hemopressin(α), an N-terminally extended peptide of hemopressin, was identified as a highly selective agonist of cannabinoid CB1 receptor (Gomes et al., 2009). The chemical structures of these peptide ligands were distinct from that of the endogenous cannabinoid (eg. 2-arachidonoylglycerol) or the classic cannabinoid receptor agonists [e.g.,
http://dx.doi.org/10.1016/j.ejphar.2015.10.016 0014-2999/& 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Δ8-tetrahydrocannabinol dimethyl heptyl (Hu-210)] (Bomar and Galande, 2013). Moreover, the in vitro assays of ERK1/2 phosphorylation or release of intracellular Gαi16-facilitated Ca2 þ demonstrated that these peptidic agonists might activate different signal transduction pathways (Gomes et al., 2009). These studies demonstrated that the novel cannabinoid peptides were distinct from the lipophilic cannabinoids in signal transduction pathways (Gomes et al., 2009; Sampaio et al., 2015). In our previous study, both supraspinal and spinal administration of VD-hemopressin (α) produced analgesia activities via cannabinoid CB1 receptor in the mouse tail-flick test (Han et al., 2014). In addition, repeated i. c.v. injection of VD-hemopressin(α) resulted in analgesic tolerance in a manner similar to WIN55,212-2 (Han et al., 2014; Pan et al., 2014). Neuropeptide FF (NPFF) was originally isolated from bovine brain and has two G-protein coupled receptors (Neuropeptide FF1 and Neuropeptide FF2) (Bonini et al., 2000; Yang et al., 1985). It is well known that NPFF belongs to an opioid-modulating peptide. To date, the link between NPFF and opioid system has been widely investigated in the cellular and the whole animal levels (Mollereau et al., 2005 a,b; Panula et al., 1996; Rebeyrolles et al., 1996). It is notable that opioid and cannabinoid systems have many common features (Bushlin et al., 2010; Pacher et al., 2006). In addition, our recent study has shown that NPFF and related peptides modulated the antinociception induced by the synthetic cannabinoid agonist WIN55,212–2 in the tail-flick test (Fang et al., 2012; Han et al., 2015). However, it is not yet clear whether a functional interaction exists between NPFF system and endogenous cannabinoids. The present study was conducted to investigate the modulatory effects of NPFF and related peptides on the antinociception induced by VD-hemopressin(α), a novel cannabinoid peptide agonist. In addition, NPFF system has been shown to be involved in opioid tolerance to antinociception (Roumy and Zajac, 1998; Simonin et al., 2006; Stinus et al., 1995). Thus, the effects of NPFF system on the VD-hemopressin(α)-induced antinociception were also evaluated in cannabinoid-tolerant mice.
2. Materials and methods 2.1. Animals Male Kunming (KM) mice (18–22 g) were provided by Animal Center of Lanzhou University and were housed in a climatecontrolled room (22 7 1 °C), with food and water available ad libitum. All testing was performed in accordance with European Community guidelines for the use of experimental animals. All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China. 2.2. Drugs Neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-PheNH2), D.NP(N-Me)AFLFQPQRF-NH2 (dNPA, D.Tyr-Pro-(N.Me)AlaPhe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2), neuropeptide VF (NPVF, Val-Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH2), VD-hemopressin(α) (ValAsp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) and 1-adamantanecarbonyl-RF-NH2 (RF9, 1-adamantanecarbonyl-Arg-Phe-NH2) were synthesized as described earlier (Fang et al., 2012; Han et al., 2014). All drugs were dissolved in physiological saline and stored at 20 °C. Lateral ventricle was administered with 4 μl drugs at a constant rate of 10 μl/min, which was followed by 1 μl saline to flush in the drug by using a 25-μl microsyringe.
2.3. Implantation of cannula into lateral ventricle A permanent guide cannula was implanted in an aseptic environment according to the method described by Fang et al. (2012). In short, mice were anesthetized with anesthetic pentobarbital sodium (80 mg/kg, intraperitoneally). A stainless cannula was placed as follows: 3 mm posterior, 1 mm lateral from bregma and 3 mm ventral from the skull surface for the lateral ventricle (i. c.v. injection). All animals were allowed at least 4 days to recover from surgery. To ascertain the exact site of i.c.v. injection, animals were injected with methylene blue dye after behavioral testing. Only the data from those animals with dispersion of the dye throughout the ventricles were used in the study. 2.4. Radiant heat tail-flick test The measurement of the tail-flick latency was conducted using a radiant heat source to focus on the surface of the mouse tail and the time that tail leave the heat source was recorded. The intensity of radiant heat was adjusted so that the baseline tail-flick latency of mouse typically ranged from 3 to 5 s. In addition, a 10 s cut-off time was set to prevent tissue damage. The tail-flick response was elicited by applying radiant heat to the point 1/3 of length away from the tip of the tail. The degree of antinociception expressed as the maximum possible effect (MPE) which was calculated as: MPE (%)¼100 [(post-drug response–baseline response)/(cut-off response–baseline response)]. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c.v.) for 5 days. The effects of NPFF agonists on the antinociception induced by VDhemopressin(α) were measured on day 6 in the radiant heat tailflick test. 2.5. Experimental protocol The present study was designed to examine the effects of the NPFF and related peptides on the central analgesia induced by the cannabinoid peptide VD-hemopressin(α) in naive and cannabinoid-tolerant mice. According to our previous studies (Fang et al., 2012; Han et al., 2014), the doses of VD-hemopressin(α), neuropeptide VF and dNPA were selected in the present work. NPFF and related peptides were injected into lateral ventricle 10 min prior to VD-hemopressin(α) in naive and cannabinoid-tolerant mice. Moreover, to investigate the roles of Neuropeptide FF receptors in the cannabinoid-modulating activities of NPFF and related peptides, Neuropeptide FF receptors antagonist RF9 was co-injected with NPFF, neuropeptide VF and dNPA, respectively. 2.6. Statistical analysis We calculated the area under the curve (AUC) data over the period 0–60 min. Data were expressed as means 7 SEM and analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. Two-way ANOVA with repeated measurements were performed using Dunnett's test with PRISM 5.0 (GraphPad, San Diego). The data were considered statistically significant when the P-value was less than 0.05.
3. Results 3.1. Effects of NPFF agonists on VD-hemopressin(α)-induced antinociception in naive mice We selected a dose of 7.5 nmol VD-hemopressin(α) (i.c.v.), which induced moderate analgesia of 62% MPE at the peak effect,
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 1. Effects of NPFF (45 nmol, i.c.v.), neuropeptide VF (NPVF; 30 nmol, i.c.v.) and dNPA (30 nmol, i.c.v.) on the antinociception induced by 7.5 nmol VD-hemopressin (α) [(m)VD-Hpα] in naive mouse tail-flick assays. Data points represent means 7 S. E.M. (n¼ 8 mice/group). ***P o0.001 significantly different from the value of the Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni posthoc test.
to investigate both potentiation and reversion of the supraspinal analgesia of cannabinoids. As shown in Fig. 1, NPFF (45 nmol, i.c.v.) significantly reduced VD-hemopressin(α)-induced central antinociceptive after i.c.v. administration. In contrast, 30 nmol neuropeptide VF (i.c.v.) and 30 nmol dNPA (i.c.v.), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, markedly enhanced the central analgesia of VD-hemopressin(α) compared to saline þ VD-hemopressin(α) group as shown in Fig. 1. Furthermore, the dose-response relationships of the cannabinoid-modulating activities of NPFF and related peptides were studied. As shown in Fig. 2, i.c.v. administration of NPFF or dNPA alone had no effect on tail-flick latency, while neuropeptide VF alone induced a slight, but not statistically significant, decrease in tail-flick latency compared with the vehicle saline (P4 0.05). Whereas, NPFF (15, 30, 45 nmol) reduced the central analgesia induced by high-dose of VD-hemopressin(α) in a dose-related manner (Fig. 2A). Considering drug dose and time as independent variables, statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F24, 252 ¼12.16, Po0.0001). Both neuropeptide VF (10, 20, 30 nmol) and dNPA (10, 20, 30 nmol) dose-dependently enhanced the central analgesia of VD-hemopressin(α) in Figs. 2B and 2C. Statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F24, 252 ¼ 20.38, P o0.0001 and F24, 234 ¼20.42, P o0.0001, respectively). To characterize whether the modulatory activities of NPFF system are mediated by Neuropeptide FF receptors in the naive mice, the Neuropeptide FF receptors antagonist RF9 was used in the present study. As shown in Fig. 3, RF9 (45 nmol, i.c.v.) itself had no effect on antinociception induced by VD-hemopressin(α) compared to saline. However, co-injection of RF9 significantly blocked the modulating actions of NPFF and related peptides on VD-hemopressin(α)-induced central antinociception (Fig. 3). 3.2. Effects of NPFF agonists on VD-hemopressin(α)-induced analgesia in VD-hemopressin(α)-tolerant mice According to our previous study (Pan et al., 2014), tolerance develops to the antinociception induced by 20 nmol VD-hemopressin(α) at the supraspinal level on day 4, 5 and 6. The present results demonstrated that 20 nmol VD-hemopressin(α) induced approximate 50% MPE antinociception in a mouse model of VD-
Fig. 2. Dose-effects of i.c.v. administration of NPFF-related peptides on the antinociception induced by 7.5 nmol VD-hemopressin(α) [(m)VD-Hpα] in naive mouse tail-flick assays. (A) NPFF (15, 30 and 45 nmol) reduced 15 nmol (m)VD-Hpα-induced central analgesia. (B) neuropeptide VF (NPVF; 10, 20 and 30 nmol) potentiated 3.75 nmol (m)VD-Hpα-induced central analgesia. (C) dNPA (10, 20 and 30 nmol) potentiated 3.75 nmol (m)VD-Hpα-induced central analgesia. Data points represents means 7 S.E.M. (n¼ 7–8 mice/group). **P o 0.01 and ***Po 0.001 significantly different from the value of the Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test.
hemopressin(α)-induced tolerance on day 6 (Fig. 4), which is consistent with our previous results (Pan et al., 2014). In Fig. 4, NPFF (45 nmol, i.c.v.) had no effect on the increases in tail-flick latency elicited by VD-hemopressin(α) in VD-hemopressin(α)tolerant mice (P 40.05). In addition, 45 nmol of NPFF alone did not modify the tail-flick latency compared with the saline control group in the tolerant mice (P 40.05).
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 4. Effects of NPFF (45 nmol, i.c.v.), neuropeptide VF (NPVF; 30 nmol, i.c.v.) and dNPA (30 nmol, i.c.v.) on the antinociception induced by 20 nmol VD-hemopressin (α) [(m)VD-Hpα] in the mouse tolerance to (m)VD-Hpα in the tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day by injecting NPFF and related peptides 10 min before the (m)VD-Hpα injection using the tail-flick assay. Data points represent means 7 S.E.M. (n ¼7–8 mice/group). ***P o 0.001 significantly different from Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test.
RF9 (30 nmol, i.c.v.) significantly reversed the modulating actions of neuropeptide VF and dNPA on VD-hemopressin(α)-induced central antinociception in VD-hemopressin(α)-tolerant mice.
Fig. 3. Effects of NPFF system on VD-hemopressin(α) [(m)VD-Hpα]-induced central antinociception were antagonized by co-injected of RF9 in naive mouse tail-flick assays. 45 nmol NPFF (A), 30 nmol neuropeptide VF (NPVF) and 30 nmol dNPA (B) were i.c.v. injected alone or co-injected with 45 nmol or 30 nmol RF9 10 min prior to administration of (m)VD-Hpα (7.5 nmol, i.c.v.). Each value represents means 7 S.E.M. (n ¼7–8 mice/group). The antinociceptive effects were expressed as AUC values during 0–60 min. **P o0.01 and ***P o0.001 indicating significant differences compared to Saline þ (m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test. ###Po 0.001 significantly different from the modulation of NPFF-related peptides in the absence of RF9.
In contrast, neuropeptide VF (i.c.v., 30 nmol) and dNPA (i.c.v., 30 nmol), markedly increased the central antinociception of VDhemopressin(α) compared to saline þ VD-hemopressin(α) group in the mice which were tolerant to VD-hemopressin(α) (Fig. 4). As shown in Fig. 5, in VD-hemopressin(α)-tolerant mice, both neuropeptide VF and dNPA alone induced slight, but not statistically significant, short lasting antinociception compared with saline (P 40.05). To further investigate the effects of NPFF agonists on the central antinociception of VD-hemopressin(α) in cannabinoid-tolerant mice, the dose-effect relationships of NPFF-related peptides were measured. Both neuropeptide VF (10, 20, 30 nmol) and dNPA (10, 20, 30 nmol) dose-dependently enhanced the central analgesia of VD-hemopressin(α) in VD-hemopressin(α)-tolerant mice. Statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F51, 432 ¼7.889, P o0.0001 and F24, 234 ¼11.19, P o0.0001, respectively). Furthermore, to explore the roles of NPFF receptors in the modulatory activities of neuropeptide VF and dNPA in VD-hemopressin(α)-tolerant mice, RF9 was co-injected with these two NPFF-related peptides. In Fig. 6, the NPFF receptors antagonist RF9 (30 nmol, i.c.v.) itself did not change the nociceptive threshold in VD-hemopressin(α)-tolerant mice. However, co-administration of
4. Discussion Our recent data demonstrated the cannabinoid-modulating activities of NPFF system on the analgesia activities induced by the nonselective cannabinoid receptors agonist WIN55,212–2 following supraspinal and intraplantar routes (Fang et al., 2012). To further characterize the functional interaction between NPFF and cannabinoid systems, the present study was conducted to investigate the modulatory roles of NPFF and related peptides in antinociception induced by VD-hemopressin(α), a novel endogenous cannabinoid peptide. The present study demonstrated that i.c.v. administration of NPFF dose-dependently reduced the analgesia induced by VDhemopressin(α) in naive mice. Moreover, the effect of NPFF on VDhemopressin(α) antinociception was blocked by the Neuropeptide FF receptors selective antagonist RF9 at the supraspinal level. Our findings suggest that NPFF attenuates central analgesia of the endogenous cannabinoid agonist via the Neuropeptide FF receptors in the brain, which keeps consistent with our previous report that the decrease of central analgesia of WIN55,212–2 induced by NPFF are mostly linked to specific activation of Neuropeptide FF receptors (Fang et al., 2012). It is well known that NPFF displays high affinities for both Neuropeptide FF1 and Neuropeptide FF2 receptors (Mollereau et al., 2002). To further explore the interaction between NPFF and cannabinoid, neuropeptide VF and dNPA, two selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, were used in the current study. In contrast to NPFF, both neuropeptide VF and dNPA administered at supraspinal route dose-dependently potentiated the central antinociception of VD-hemopressin(α). Furthermore, the cannabinoidmodulating effects of neuropeptide VF and dNPA were completely blocked by RF9, implying that the modulatory roles of the two selective agonists in the central antinociception induced by VD-hemopressin(α) were mainly mediated through Neuropeptide FF receptors.
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 6. Effects of NPFF-related peptides on VD-hemopressin(α) [(m)VD-Hpα]-induced central analgesia were blocked by co-injected of RF9 in the mice tolerance to (m)VD-Hpα in tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day after the administration of cannabinoid agonist using the tail-flick assay. 30 nmol neuropeptide VF (NPVF) and 30 nmol dNPA were administrated into lateral ventricle alone or co-injected with RF9 10 min prior to administration of (m) VD-Hpα (10 nmol, i.c.v.) on day 6. Each value represents means7 S.E.M. (n¼ 7–8 mice/group). The antinociceptive effects were expressed as AUC values during 0-60 min. ***Po 0.001 indicating significant differences compared to Saline þ (m) VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test. ### Po 0.001 significantly different from the modulatory effects of neuropeptide VF or dNPA in the absence of RF9.
Fig. 5. Dose-effects of i.c.v. administered NPFF-related peptides on the antinociception induced by 20 nmol VD-hemopressin(α) [(m)VD-Hpα] in the mouse tolerance to (m)VD-Hpα in the tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day by injecting NPFF and related peptides 10 min before the (m)VD-Hpα injection using the tail-flick assay. (A) neuropeptide VF (NPVF; 10, 20 and 30 nmol) potentiated 10 nmol (m)VD-Hpα-induced central antinociception. (B) dNPA (10, 20 and 30 nmol) potentiated 10 nmol (m)VD-Hpαinduced central antinociception. Each value represents means 7 S.E.M. (n ¼7–8 mice/group). *P o0.05, **Po 0.01 and ***P o 0.001 indicating neuropeptide VF and dNPA significantly enhanced the central analgesia of (m)VD-Hpα in (m)VD-Hpαtolerant mice, according to one-way ANOVA with the Bonferroni post-hoc test.
In the present work, our findings demonstrated that central administration of NPFF reduced VD-hemopressin(α)-induced antinociception, whereas the selective agonists neuropeptide VF and dNPA enhanced analgesia of VD-hemopressin(α). These data further confirm a functional interaction between NPFF and cannabinoid systems. It is worthy to note that NPFF system induced the same cannabinoid-modulating effects on WIN55,212–2-induced central and peripheral antinociception in previous study (Fang et al., 2012). Taken together the above findings, these data indicate that activation of central Neuropeptide FF receptors not only interferes with the antinociception of exogenous cannabinoids, but also modulates the analgesia induced by the endogenous peptide agonist of cannabinoid CB1 receptor. In addition, the similar effects
of NPFF system on WIN55,212–2- and VD-hemopressin(α)-induced central analgesia indirectly suggested that a same mechanism might be involved in the central antinociception of these two cannabinoid agonists. In fact, the reported results also demonstrated that both WIN55,212–2 and VD-hemopressin(α) produced their central analgesia via cannabinoid CB1 receptor in the brain (Fang et al., 2012; Han et al., 2014; Pertwee, 2001). In the present study, both neuropeptide VF and dNPA, the Neuropeptide FF1 and Neuropeptide FF2 receptors selective agonists, potentiated the antinociception of VD-hemopressin(α). In contrast, NPFF dose-dependently decreased the central antinociception of VD-hemopressin(α). These findings imply that NPFF and related peptides exert a complex modulatory role in cannabinoid antinociception. In fact, the previous results have shown that NPFF system exerted different modulating effects on the central antinociception mediated by different receptors, such as mu-opioid receptor and α2-adrenoceptors (Kontinen and Kalso, 1995). The recent results also demonstrated that NPFF could reduce the central antinociception mediated by mu-opioid receptor, while enhance the central antinociception of kappa-opioid receptor (Wang et al., 2014). In addition, it is notable that the pro- and anti-opioid actions induced by NPFF related peptides were not strictly related to their selectivities towards Neuropeptide FF1 and Neuropeptide FF2 receptors (Quelven et al., 2005; Roussin et al., 2005). The previous studies have shown that [D.Tyr1,(N.Me)Phe3]NPFF (1DMe) and [Nic-Pro]1DMe (Nic-1DMe) with similar selectivities towards Neuropeptide FF receptors could induce different opioid-modulating effects (Quelven et al., 2002). Nic-1DMe (32 nmol) potentiates morphine antinociception, whereas 1DMe (3–36 nmol) decreased opioid analgesia (Quelven et al., 2002). Therefore, further studies are needed to explore the mechanism involved in the interaction between NPFF and cannabinoid systems. There is considerable evidence describing the similar pharmacological activities of cannabinoid and opioid systems (Pacher et al., 2006; Shapira et al., 2003). Both opioid and cannabinoid receptors have been reported to play important roles in analgesia and antinociceptive tolerance (Gonzalez et al., 2005; Pertwee, 2001; Zubieta et al., 2001). The previous studies indicated that NPFF system exerted the different modulating properties between naive and opioid-tolerant mice. For example, high doses of 1DMe, an analog of NPFF significantly reduced the analgesic activity of morphine in naive mice in the tail-flick test (Gelot et al., 1998). However, the anti-opioid effect of 1DMe was no longer observed in
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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morphine-tolerant mice (Gelot et al., 1998). Thus, the further studies need to investigate whether NPFF system causes a different modulating effect on cannabinoid analgesia in cannabinoid-tolerant mice. Our results demonstrated that NPFF reduced the analgesia induced by VD-hemopressin(α) in naive mice, whereas the cannabinoid-modulating activity of NPFF was not observed in VD-hemopressin(α)-tolerant mice. It is difficult to explore the different modulating effects of NPFF. However, there are two possible explanations for the findings as to why the cannabinoid-modulating activities of NPFF are altered in cannabinoid-tolerant mice. It is possible that chronic treatment of cannabinoids causes the physiological changes in mice, which results in the difference in cannabinoid-modulating activities of NPFF in naive and cannabinoid-tolerant mice. In fact, a considerable number of studies have shown that repeated administration of cannabinoid agonist could lead to the desensitization and downregulation of cannabinoid CB1 receptor (Corchero et al., 1999; Oviedo et al., 1993; Rodriguez de Fonseca et al., 1994; Sim et al., 1996). Similarly, during the development of VD-hemopressin(α) tolerance, several transcription factors and cellular signal molecules related to cannabinoid system might also be changed. Another possibility is that the repeated treatment of VD-hemopressin(α) reduced the degree of cannabinoid antinociception, which might also alter the cannabinoidmodulating activities of NPFF. In the previous studies, the opioidmodulating effects of NPFF agonists depended on the degree of morphine-induced antinociception in mice (Quelven et al., 2005; Roussin et al., 2005). In the VD-hemopressin(α)-tolerant mice, the decrease in cannabinoid-induced antinociception might also modify the endogenous NPFF system and its cannabinoid-modulating activities. In contrast to NPFF, both neuropeptide VF and dNPA dose-dependently enhanced VD-hemopressin(α)-induced antinociception in cannabinoid-tolerant mice. Additionally, their cananbinoid-potentiating effects were completely blocked by RF9 in cannabinoidtolerant mice, indicating that the cannabinoid-modulating actions are also mediated by Neuropeptide FF receptors. Taken together, the present works suggest that a chronic VD-hemopressin(α) treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. At present, it is difficult to explain the different effects of NPFF and these two selective agonists on VD-hemopressin(α)-induced antinociception in cannabinoid-tolerant mice. However, the results imply that the canabinoid-modulating activities of NPFF and the two selective agonists might be mediated by different pathways in the tolerant mice. Some observations further support this assumption. NPFF alone did not modify the tail-flick latency in naive or tolerant mice, whereas neuropeptide VF and dNPA alone caused slight increases in tail-flick latency by themselves in cannabinoid-tolerant mice. These findings indicated that several pathways of the two selective agonists of Neuropeptide FF receptors might be distinct from those activated by NPFF. Indeed, the previous studies demonstrated that several G-protein-coupled receptors could exert functional selectivity properties and lead to differential activation of downstream signaling (Mou et al., 2011; Perez and Karnik, 2005; Urban et al., 2007). In addition, some endogenous peptides were shown to activate the same receptor and induce differential pharmacological profiles. For example, the antinociceptive effects of endomorphin-1 and endomorphin-2 were mediated by the activation of mu-opioid receptor, whereas different mechanisms were subsequently involved in their analgesic activities (Sakurada et al., 1999; Tseng et al., 2000). However, further studies with the highly selective antagonists towards Neuropeptide FF1 and Neuropeptide FF2 receptors could shed some light on the interaction of NPFF and cannabinoid systems. In summary, the body of data derived from the present findings
investigated, for the first time, the regulatory effects of NPFF system on the antinociception induced by the endogenous cannabinoid CB1 receptor agonist, VD-hemopressin(α) in naive and cannabinoid-tolerant mice, which further supports the cannabinoidmodulating character of NPFF system. Moreover, the present works suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. In addition, the cannabinoid-potentiating effects of neuropeptide VF and dNPA provided a novel strategy of combination treatment to reduce the therapeutic dose of cannabinoids and to separate the antinociceptive activity of cannabinoids from unwanted effects.
Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (Nos. 81273355, 81473095), Program for New Century Excellent Talents in University (NCET13-0257), the Natural Science Foundation of Gansu Province, China (1208RJYA001), and the Fundamental Research Funds for the Central Universities (lzujbky-2014-141).
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Neuropharmacology and analgesia
Effects of neuropeptide FF and related peptides on the antinociceptive activities of VD-hemopressin(α) in naive and cannabinoid-tolerant mice Jia-Xin Pan 1, Zi-Long Wang 1, Ning Li, Nan Zhang, Pei Wang, Hong-Hai Tang, Ting Zhang, Hong-Ping Yu, Run Zhang, Ting Zheng, Quan Fang n, Rui Wang n Key Laboratory of Preclinical Study for New Drugs of Gansu Province, and Institute of Physiology, School of Basic Medical Sciences, Lanzhou University, 199 Donggang West Road, Lanzhou, 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2015 Received in revised form 20 September 2015 Accepted 8 October 2015
Neuropeptide FF (NPFF) system has recently been reported to modulate cannabinoid-induced antinociception. The aim of the present study was to further investigate the roles of NPFF system in the antinociceptive effects induced by intracerebroventricular (i.c.v.) administration of mouse VD-hemopressin(α), a novel endogenous agonist of cannabinoid CB1 receptor, in naive and VD-hemopressin(α)tolerant mice. The effects of NPFF system on the antinociception induced by VD-hemopressin(α) were investigated in the radiant heat tail-flick test in naive mice and VD-hemopressin(α)-tolerant mice. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c. v.) for 5 days and the antinociception was measured on day 6. In naive mice, intracerebroventricular injection of NPFF dose-dependently attenuated central analgesia of VD-hemopressin(α). In contrast, neuropeptide VF (NPVF) and D.NP(N-Me)AFLFQPQRF-NH2 (dNPA), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, enhanced VD-hemopressin(α)-induced antinociception in a dose-dependent manner. In addition, the VD-hemopressin(α)-modulating activities of NPFF and related peptides were antagonized by the Neuropeptide FF receptors selective antagonist 1-adamantanecarbonyl-RF-NH2 (RF9). In VD-hemopressin(α)-tolerant mice, NPFF failed to modify VDhemopressin(α)-induced antinociception. However, both neuropeptide VF and dNPA dose-dependently potentiated the antinociception of VD-hemopressin(α) and these cannabinoid-potentiating effects were reduced by RF9. The present works support the cannabinoid-modulating character of NPFF system in naive and cannabinoid-tolerant mice. In addition, the data suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cannabinoids Neuropeptide FF VD-hemopressin(α) Antinociception Tolerance Mice Chemical compounds studied in this article: NPFF (PubChem CID: 123797) Neuropeptide VF (PubChem CID: 71451611) RF9 (PubChem CID: 53320361) Hemopressin (PubChem CID: 71312009) RVD-hemopressin(α) (PubChem CID: 90488874) WIN55,212-2 (PubChem CID: 6604176) Δ9-tetrahydrocannabinol (PubChem CID: 16078) 2-arachidonoylglycerol (PubChem CID: 5282280) 1DMe (PubChem CID: 71345956) Hu-210 (PubChem CID: 9821569)
1. Introduction Cannabinoid system plays an important role in the treatment of various diseases, including pain management. Two types of cannabinoid receptors, cannabinoid receptor type 1 (CB1) and type 2 (CB2), have been identified and cloned (Matsuda et al., 1990; Munro et al., 1993). The cannabinoid CB1 receptor is primarily expressed in the central nervous system, whereas the cannabinoid CB2 receptor is mainly expressed in immune cells (Howlett et al., 2002). The cannabinoid ligands have been classified into phytocannabinoids n
Corresponding authors. Tel./fax: þ86 931 8912567. E-mail addresses: [email protected] (Q. Fang), [email protected] (R. Wang). 1 Both authors contributed equally to this work.
(e.g., Δ9-tetrahydrocannabinol), synthetic ligands [e.g., (R) (þ ) [2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN55,212-2)] and lipid endocannabinoids (e.g., 2-arachidonoylglycerol) (Bomar and Galande, 2013; Pacher et al., 2006). Recent studies demonstrated that hemopressin and related peptides functioned as the novel endogenous peptide ligands of cannabinoid receptors derived from the hemoglobin (Gomes et al., 2009; Heimann et al., 2007). VD-hemopressin(α), an N-terminally extended peptide of hemopressin, was identified as a highly selective agonist of cannabinoid CB1 receptor (Gomes et al., 2009). The chemical structures of these peptide ligands were distinct from that of the endogenous cannabinoid (eg. 2-arachidonoylglycerol) or the classic cannabinoid receptor agonists [e.g.,
http://dx.doi.org/10.1016/j.ejphar.2015.10.016 0014-2999/& 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Δ8-tetrahydrocannabinol dimethyl heptyl (Hu-210)] (Bomar and Galande, 2013). Moreover, the in vitro assays of ERK1/2 phosphorylation or release of intracellular Gαi16-facilitated Ca2 þ demonstrated that these peptidic agonists might activate different signal transduction pathways (Gomes et al., 2009). These studies demonstrated that the novel cannabinoid peptides were distinct from the lipophilic cannabinoids in signal transduction pathways (Gomes et al., 2009; Sampaio et al., 2015). In our previous study, both supraspinal and spinal administration of VD-hemopressin (α) produced analgesia activities via cannabinoid CB1 receptor in the mouse tail-flick test (Han et al., 2014). In addition, repeated i. c.v. injection of VD-hemopressin(α) resulted in analgesic tolerance in a manner similar to WIN55,212-2 (Han et al., 2014; Pan et al., 2014). Neuropeptide FF (NPFF) was originally isolated from bovine brain and has two G-protein coupled receptors (Neuropeptide FF1 and Neuropeptide FF2) (Bonini et al., 2000; Yang et al., 1985). It is well known that NPFF belongs to an opioid-modulating peptide. To date, the link between NPFF and opioid system has been widely investigated in the cellular and the whole animal levels (Mollereau et al., 2005 a,b; Panula et al., 1996; Rebeyrolles et al., 1996). It is notable that opioid and cannabinoid systems have many common features (Bushlin et al., 2010; Pacher et al., 2006). In addition, our recent study has shown that NPFF and related peptides modulated the antinociception induced by the synthetic cannabinoid agonist WIN55,212–2 in the tail-flick test (Fang et al., 2012; Han et al., 2015). However, it is not yet clear whether a functional interaction exists between NPFF system and endogenous cannabinoids. The present study was conducted to investigate the modulatory effects of NPFF and related peptides on the antinociception induced by VD-hemopressin(α), a novel cannabinoid peptide agonist. In addition, NPFF system has been shown to be involved in opioid tolerance to antinociception (Roumy and Zajac, 1998; Simonin et al., 2006; Stinus et al., 1995). Thus, the effects of NPFF system on the VD-hemopressin(α)-induced antinociception were also evaluated in cannabinoid-tolerant mice.
2. Materials and methods 2.1. Animals Male Kunming (KM) mice (18–22 g) were provided by Animal Center of Lanzhou University and were housed in a climatecontrolled room (22 7 1 °C), with food and water available ad libitum. All testing was performed in accordance with European Community guidelines for the use of experimental animals. All the protocols in this study were approved by the Ethics Committee of Lanzhou University, China. 2.2. Drugs Neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-PheNH2), D.NP(N-Me)AFLFQPQRF-NH2 (dNPA, D.Tyr-Pro-(N.Me)AlaPhe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2), neuropeptide VF (NPVF, Val-Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH2), VD-hemopressin(α) (ValAsp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) and 1-adamantanecarbonyl-RF-NH2 (RF9, 1-adamantanecarbonyl-Arg-Phe-NH2) were synthesized as described earlier (Fang et al., 2012; Han et al., 2014). All drugs were dissolved in physiological saline and stored at 20 °C. Lateral ventricle was administered with 4 μl drugs at a constant rate of 10 μl/min, which was followed by 1 μl saline to flush in the drug by using a 25-μl microsyringe.
2.3. Implantation of cannula into lateral ventricle A permanent guide cannula was implanted in an aseptic environment according to the method described by Fang et al. (2012). In short, mice were anesthetized with anesthetic pentobarbital sodium (80 mg/kg, intraperitoneally). A stainless cannula was placed as follows: 3 mm posterior, 1 mm lateral from bregma and 3 mm ventral from the skull surface for the lateral ventricle (i. c.v. injection). All animals were allowed at least 4 days to recover from surgery. To ascertain the exact site of i.c.v. injection, animals were injected with methylene blue dye after behavioral testing. Only the data from those animals with dispersion of the dye throughout the ventricles were used in the study. 2.4. Radiant heat tail-flick test The measurement of the tail-flick latency was conducted using a radiant heat source to focus on the surface of the mouse tail and the time that tail leave the heat source was recorded. The intensity of radiant heat was adjusted so that the baseline tail-flick latency of mouse typically ranged from 3 to 5 s. In addition, a 10 s cut-off time was set to prevent tissue damage. The tail-flick response was elicited by applying radiant heat to the point 1/3 of length away from the tip of the tail. The degree of antinociception expressed as the maximum possible effect (MPE) which was calculated as: MPE (%)¼100 [(post-drug response–baseline response)/(cut-off response–baseline response)]. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c.v.) for 5 days. The effects of NPFF agonists on the antinociception induced by VDhemopressin(α) were measured on day 6 in the radiant heat tailflick test. 2.5. Experimental protocol The present study was designed to examine the effects of the NPFF and related peptides on the central analgesia induced by the cannabinoid peptide VD-hemopressin(α) in naive and cannabinoid-tolerant mice. According to our previous studies (Fang et al., 2012; Han et al., 2014), the doses of VD-hemopressin(α), neuropeptide VF and dNPA were selected in the present work. NPFF and related peptides were injected into lateral ventricle 10 min prior to VD-hemopressin(α) in naive and cannabinoid-tolerant mice. Moreover, to investigate the roles of Neuropeptide FF receptors in the cannabinoid-modulating activities of NPFF and related peptides, Neuropeptide FF receptors antagonist RF9 was co-injected with NPFF, neuropeptide VF and dNPA, respectively. 2.6. Statistical analysis We calculated the area under the curve (AUC) data over the period 0–60 min. Data were expressed as means 7 SEM and analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test. Two-way ANOVA with repeated measurements were performed using Dunnett's test with PRISM 5.0 (GraphPad, San Diego). The data were considered statistically significant when the P-value was less than 0.05.
3. Results 3.1. Effects of NPFF agonists on VD-hemopressin(α)-induced antinociception in naive mice We selected a dose of 7.5 nmol VD-hemopressin(α) (i.c.v.), which induced moderate analgesia of 62% MPE at the peak effect,
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 1. Effects of NPFF (45 nmol, i.c.v.), neuropeptide VF (NPVF; 30 nmol, i.c.v.) and dNPA (30 nmol, i.c.v.) on the antinociception induced by 7.5 nmol VD-hemopressin (α) [(m)VD-Hpα] in naive mouse tail-flick assays. Data points represent means 7 S. E.M. (n¼ 8 mice/group). ***P o0.001 significantly different from the value of the Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni posthoc test.
to investigate both potentiation and reversion of the supraspinal analgesia of cannabinoids. As shown in Fig. 1, NPFF (45 nmol, i.c.v.) significantly reduced VD-hemopressin(α)-induced central antinociceptive after i.c.v. administration. In contrast, 30 nmol neuropeptide VF (i.c.v.) and 30 nmol dNPA (i.c.v.), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, markedly enhanced the central analgesia of VD-hemopressin(α) compared to saline þ VD-hemopressin(α) group as shown in Fig. 1. Furthermore, the dose-response relationships of the cannabinoid-modulating activities of NPFF and related peptides were studied. As shown in Fig. 2, i.c.v. administration of NPFF or dNPA alone had no effect on tail-flick latency, while neuropeptide VF alone induced a slight, but not statistically significant, decrease in tail-flick latency compared with the vehicle saline (P4 0.05). Whereas, NPFF (15, 30, 45 nmol) reduced the central analgesia induced by high-dose of VD-hemopressin(α) in a dose-related manner (Fig. 2A). Considering drug dose and time as independent variables, statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F24, 252 ¼12.16, Po0.0001). Both neuropeptide VF (10, 20, 30 nmol) and dNPA (10, 20, 30 nmol) dose-dependently enhanced the central analgesia of VD-hemopressin(α) in Figs. 2B and 2C. Statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F24, 252 ¼ 20.38, P o0.0001 and F24, 234 ¼20.42, P o0.0001, respectively). To characterize whether the modulatory activities of NPFF system are mediated by Neuropeptide FF receptors in the naive mice, the Neuropeptide FF receptors antagonist RF9 was used in the present study. As shown in Fig. 3, RF9 (45 nmol, i.c.v.) itself had no effect on antinociception induced by VD-hemopressin(α) compared to saline. However, co-injection of RF9 significantly blocked the modulating actions of NPFF and related peptides on VD-hemopressin(α)-induced central antinociception (Fig. 3). 3.2. Effects of NPFF agonists on VD-hemopressin(α)-induced analgesia in VD-hemopressin(α)-tolerant mice According to our previous study (Pan et al., 2014), tolerance develops to the antinociception induced by 20 nmol VD-hemopressin(α) at the supraspinal level on day 4, 5 and 6. The present results demonstrated that 20 nmol VD-hemopressin(α) induced approximate 50% MPE antinociception in a mouse model of VD-
Fig. 2. Dose-effects of i.c.v. administration of NPFF-related peptides on the antinociception induced by 7.5 nmol VD-hemopressin(α) [(m)VD-Hpα] in naive mouse tail-flick assays. (A) NPFF (15, 30 and 45 nmol) reduced 15 nmol (m)VD-Hpα-induced central analgesia. (B) neuropeptide VF (NPVF; 10, 20 and 30 nmol) potentiated 3.75 nmol (m)VD-Hpα-induced central analgesia. (C) dNPA (10, 20 and 30 nmol) potentiated 3.75 nmol (m)VD-Hpα-induced central analgesia. Data points represents means 7 S.E.M. (n¼ 7–8 mice/group). **P o 0.01 and ***Po 0.001 significantly different from the value of the Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test.
hemopressin(α)-induced tolerance on day 6 (Fig. 4), which is consistent with our previous results (Pan et al., 2014). In Fig. 4, NPFF (45 nmol, i.c.v.) had no effect on the increases in tail-flick latency elicited by VD-hemopressin(α) in VD-hemopressin(α)tolerant mice (P 40.05). In addition, 45 nmol of NPFF alone did not modify the tail-flick latency compared with the saline control group in the tolerant mice (P 40.05).
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 4. Effects of NPFF (45 nmol, i.c.v.), neuropeptide VF (NPVF; 30 nmol, i.c.v.) and dNPA (30 nmol, i.c.v.) on the antinociception induced by 20 nmol VD-hemopressin (α) [(m)VD-Hpα] in the mouse tolerance to (m)VD-Hpα in the tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day by injecting NPFF and related peptides 10 min before the (m)VD-Hpα injection using the tail-flick assay. Data points represent means 7 S.E.M. (n ¼7–8 mice/group). ***P o 0.001 significantly different from Saline þ(m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test.
RF9 (30 nmol, i.c.v.) significantly reversed the modulating actions of neuropeptide VF and dNPA on VD-hemopressin(α)-induced central antinociception in VD-hemopressin(α)-tolerant mice.
Fig. 3. Effects of NPFF system on VD-hemopressin(α) [(m)VD-Hpα]-induced central antinociception were antagonized by co-injected of RF9 in naive mouse tail-flick assays. 45 nmol NPFF (A), 30 nmol neuropeptide VF (NPVF) and 30 nmol dNPA (B) were i.c.v. injected alone or co-injected with 45 nmol or 30 nmol RF9 10 min prior to administration of (m)VD-Hpα (7.5 nmol, i.c.v.). Each value represents means 7 S.E.M. (n ¼7–8 mice/group). The antinociceptive effects were expressed as AUC values during 0–60 min. **P o0.01 and ***P o0.001 indicating significant differences compared to Saline þ (m)VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test. ###Po 0.001 significantly different from the modulation of NPFF-related peptides in the absence of RF9.
In contrast, neuropeptide VF (i.c.v., 30 nmol) and dNPA (i.c.v., 30 nmol), markedly increased the central antinociception of VDhemopressin(α) compared to saline þ VD-hemopressin(α) group in the mice which were tolerant to VD-hemopressin(α) (Fig. 4). As shown in Fig. 5, in VD-hemopressin(α)-tolerant mice, both neuropeptide VF and dNPA alone induced slight, but not statistically significant, short lasting antinociception compared with saline (P 40.05). To further investigate the effects of NPFF agonists on the central antinociception of VD-hemopressin(α) in cannabinoid-tolerant mice, the dose-effect relationships of NPFF-related peptides were measured. Both neuropeptide VF (10, 20, 30 nmol) and dNPA (10, 20, 30 nmol) dose-dependently enhanced the central analgesia of VD-hemopressin(α) in VD-hemopressin(α)-tolerant mice. Statistical analysis using two-way ANOVA (dose time) revealed a statistical significance (F51, 432 ¼7.889, P o0.0001 and F24, 234 ¼11.19, P o0.0001, respectively). Furthermore, to explore the roles of NPFF receptors in the modulatory activities of neuropeptide VF and dNPA in VD-hemopressin(α)-tolerant mice, RF9 was co-injected with these two NPFF-related peptides. In Fig. 6, the NPFF receptors antagonist RF9 (30 nmol, i.c.v.) itself did not change the nociceptive threshold in VD-hemopressin(α)-tolerant mice. However, co-administration of
4. Discussion Our recent data demonstrated the cannabinoid-modulating activities of NPFF system on the analgesia activities induced by the nonselective cannabinoid receptors agonist WIN55,212–2 following supraspinal and intraplantar routes (Fang et al., 2012). To further characterize the functional interaction between NPFF and cannabinoid systems, the present study was conducted to investigate the modulatory roles of NPFF and related peptides in antinociception induced by VD-hemopressin(α), a novel endogenous cannabinoid peptide. The present study demonstrated that i.c.v. administration of NPFF dose-dependently reduced the analgesia induced by VDhemopressin(α) in naive mice. Moreover, the effect of NPFF on VDhemopressin(α) antinociception was blocked by the Neuropeptide FF receptors selective antagonist RF9 at the supraspinal level. Our findings suggest that NPFF attenuates central analgesia of the endogenous cannabinoid agonist via the Neuropeptide FF receptors in the brain, which keeps consistent with our previous report that the decrease of central analgesia of WIN55,212–2 induced by NPFF are mostly linked to specific activation of Neuropeptide FF receptors (Fang et al., 2012). It is well known that NPFF displays high affinities for both Neuropeptide FF1 and Neuropeptide FF2 receptors (Mollereau et al., 2002). To further explore the interaction between NPFF and cannabinoid, neuropeptide VF and dNPA, two selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, were used in the current study. In contrast to NPFF, both neuropeptide VF and dNPA administered at supraspinal route dose-dependently potentiated the central antinociception of VD-hemopressin(α). Furthermore, the cannabinoidmodulating effects of neuropeptide VF and dNPA were completely blocked by RF9, implying that the modulatory roles of the two selective agonists in the central antinociception induced by VD-hemopressin(α) were mainly mediated through Neuropeptide FF receptors.
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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Fig. 6. Effects of NPFF-related peptides on VD-hemopressin(α) [(m)VD-Hpα]-induced central analgesia were blocked by co-injected of RF9 in the mice tolerance to (m)VD-Hpα in tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day after the administration of cannabinoid agonist using the tail-flick assay. 30 nmol neuropeptide VF (NPVF) and 30 nmol dNPA were administrated into lateral ventricle alone or co-injected with RF9 10 min prior to administration of (m) VD-Hpα (10 nmol, i.c.v.) on day 6. Each value represents means7 S.E.M. (n¼ 7–8 mice/group). The antinociceptive effects were expressed as AUC values during 0-60 min. ***Po 0.001 indicating significant differences compared to Saline þ (m) VD-Hpα-injected group in one-way ANOVA with the Bonferroni post-hoc test. ### Po 0.001 significantly different from the modulatory effects of neuropeptide VF or dNPA in the absence of RF9.
Fig. 5. Dose-effects of i.c.v. administered NPFF-related peptides on the antinociception induced by 20 nmol VD-hemopressin(α) [(m)VD-Hpα] in the mouse tolerance to (m)VD-Hpα in the tail-flick assays. Animals received intracerebroventricular injections of 20 nmol (m)VD-Hpa once daily for 5 days. Analgesia was measured on the sixth day by injecting NPFF and related peptides 10 min before the (m)VD-Hpα injection using the tail-flick assay. (A) neuropeptide VF (NPVF; 10, 20 and 30 nmol) potentiated 10 nmol (m)VD-Hpα-induced central antinociception. (B) dNPA (10, 20 and 30 nmol) potentiated 10 nmol (m)VD-Hpαinduced central antinociception. Each value represents means 7 S.E.M. (n ¼7–8 mice/group). *P o0.05, **Po 0.01 and ***P o 0.001 indicating neuropeptide VF and dNPA significantly enhanced the central analgesia of (m)VD-Hpα in (m)VD-Hpαtolerant mice, according to one-way ANOVA with the Bonferroni post-hoc test.
In the present work, our findings demonstrated that central administration of NPFF reduced VD-hemopressin(α)-induced antinociception, whereas the selective agonists neuropeptide VF and dNPA enhanced analgesia of VD-hemopressin(α). These data further confirm a functional interaction between NPFF and cannabinoid systems. It is worthy to note that NPFF system induced the same cannabinoid-modulating effects on WIN55,212–2-induced central and peripheral antinociception in previous study (Fang et al., 2012). Taken together the above findings, these data indicate that activation of central Neuropeptide FF receptors not only interferes with the antinociception of exogenous cannabinoids, but also modulates the analgesia induced by the endogenous peptide agonist of cannabinoid CB1 receptor. In addition, the similar effects
of NPFF system on WIN55,212–2- and VD-hemopressin(α)-induced central analgesia indirectly suggested that a same mechanism might be involved in the central antinociception of these two cannabinoid agonists. In fact, the reported results also demonstrated that both WIN55,212–2 and VD-hemopressin(α) produced their central analgesia via cannabinoid CB1 receptor in the brain (Fang et al., 2012; Han et al., 2014; Pertwee, 2001). In the present study, both neuropeptide VF and dNPA, the Neuropeptide FF1 and Neuropeptide FF2 receptors selective agonists, potentiated the antinociception of VD-hemopressin(α). In contrast, NPFF dose-dependently decreased the central antinociception of VD-hemopressin(α). These findings imply that NPFF and related peptides exert a complex modulatory role in cannabinoid antinociception. In fact, the previous results have shown that NPFF system exerted different modulating effects on the central antinociception mediated by different receptors, such as mu-opioid receptor and α2-adrenoceptors (Kontinen and Kalso, 1995). The recent results also demonstrated that NPFF could reduce the central antinociception mediated by mu-opioid receptor, while enhance the central antinociception of kappa-opioid receptor (Wang et al., 2014). In addition, it is notable that the pro- and anti-opioid actions induced by NPFF related peptides were not strictly related to their selectivities towards Neuropeptide FF1 and Neuropeptide FF2 receptors (Quelven et al., 2005; Roussin et al., 2005). The previous studies have shown that [D.Tyr1,(N.Me)Phe3]NPFF (1DMe) and [Nic-Pro]1DMe (Nic-1DMe) with similar selectivities towards Neuropeptide FF receptors could induce different opioid-modulating effects (Quelven et al., 2002). Nic-1DMe (32 nmol) potentiates morphine antinociception, whereas 1DMe (3–36 nmol) decreased opioid analgesia (Quelven et al., 2002). Therefore, further studies are needed to explore the mechanism involved in the interaction between NPFF and cannabinoid systems. There is considerable evidence describing the similar pharmacological activities of cannabinoid and opioid systems (Pacher et al., 2006; Shapira et al., 2003). Both opioid and cannabinoid receptors have been reported to play important roles in analgesia and antinociceptive tolerance (Gonzalez et al., 2005; Pertwee, 2001; Zubieta et al., 2001). The previous studies indicated that NPFF system exerted the different modulating properties between naive and opioid-tolerant mice. For example, high doses of 1DMe, an analog of NPFF significantly reduced the analgesic activity of morphine in naive mice in the tail-flick test (Gelot et al., 1998). However, the anti-opioid effect of 1DMe was no longer observed in
Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i
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morphine-tolerant mice (Gelot et al., 1998). Thus, the further studies need to investigate whether NPFF system causes a different modulating effect on cannabinoid analgesia in cannabinoid-tolerant mice. Our results demonstrated that NPFF reduced the analgesia induced by VD-hemopressin(α) in naive mice, whereas the cannabinoid-modulating activity of NPFF was not observed in VD-hemopressin(α)-tolerant mice. It is difficult to explore the different modulating effects of NPFF. However, there are two possible explanations for the findings as to why the cannabinoid-modulating activities of NPFF are altered in cannabinoid-tolerant mice. It is possible that chronic treatment of cannabinoids causes the physiological changes in mice, which results in the difference in cannabinoid-modulating activities of NPFF in naive and cannabinoid-tolerant mice. In fact, a considerable number of studies have shown that repeated administration of cannabinoid agonist could lead to the desensitization and downregulation of cannabinoid CB1 receptor (Corchero et al., 1999; Oviedo et al., 1993; Rodriguez de Fonseca et al., 1994; Sim et al., 1996). Similarly, during the development of VD-hemopressin(α) tolerance, several transcription factors and cellular signal molecules related to cannabinoid system might also be changed. Another possibility is that the repeated treatment of VD-hemopressin(α) reduced the degree of cannabinoid antinociception, which might also alter the cannabinoidmodulating activities of NPFF. In the previous studies, the opioidmodulating effects of NPFF agonists depended on the degree of morphine-induced antinociception in mice (Quelven et al., 2005; Roussin et al., 2005). In the VD-hemopressin(α)-tolerant mice, the decrease in cannabinoid-induced antinociception might also modify the endogenous NPFF system and its cannabinoid-modulating activities. In contrast to NPFF, both neuropeptide VF and dNPA dose-dependently enhanced VD-hemopressin(α)-induced antinociception in cannabinoid-tolerant mice. Additionally, their cananbinoid-potentiating effects were completely blocked by RF9 in cannabinoidtolerant mice, indicating that the cannabinoid-modulating actions are also mediated by Neuropeptide FF receptors. Taken together, the present works suggest that a chronic VD-hemopressin(α) treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. At present, it is difficult to explain the different effects of NPFF and these two selective agonists on VD-hemopressin(α)-induced antinociception in cannabinoid-tolerant mice. However, the results imply that the canabinoid-modulating activities of NPFF and the two selective agonists might be mediated by different pathways in the tolerant mice. Some observations further support this assumption. NPFF alone did not modify the tail-flick latency in naive or tolerant mice, whereas neuropeptide VF and dNPA alone caused slight increases in tail-flick latency by themselves in cannabinoid-tolerant mice. These findings indicated that several pathways of the two selective agonists of Neuropeptide FF receptors might be distinct from those activated by NPFF. Indeed, the previous studies demonstrated that several G-protein-coupled receptors could exert functional selectivity properties and lead to differential activation of downstream signaling (Mou et al., 2011; Perez and Karnik, 2005; Urban et al., 2007). In addition, some endogenous peptides were shown to activate the same receptor and induce differential pharmacological profiles. For example, the antinociceptive effects of endomorphin-1 and endomorphin-2 were mediated by the activation of mu-opioid receptor, whereas different mechanisms were subsequently involved in their analgesic activities (Sakurada et al., 1999; Tseng et al., 2000). However, further studies with the highly selective antagonists towards Neuropeptide FF1 and Neuropeptide FF2 receptors could shed some light on the interaction of NPFF and cannabinoid systems. In summary, the body of data derived from the present findings
investigated, for the first time, the regulatory effects of NPFF system on the antinociception induced by the endogenous cannabinoid CB1 receptor agonist, VD-hemopressin(α) in naive and cannabinoid-tolerant mice, which further supports the cannabinoidmodulating character of NPFF system. Moreover, the present works suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA. In addition, the cannabinoid-potentiating effects of neuropeptide VF and dNPA provided a novel strategy of combination treatment to reduce the therapeutic dose of cannabinoids and to separate the antinociceptive activity of cannabinoids from unwanted effects.
Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (Nos. 81273355, 81473095), Program for New Century Excellent Talents in University (NCET13-0257), the Natural Science Foundation of Gansu Province, China (1208RJYA001), and the Fundamental Research Funds for the Central Universities (lzujbky-2014-141).
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Please cite this article as: Pan, J.-X., et al., Effects of neuropeptide FF and related peptides on the antinociceptive activities of VDhemopressin(α) in naive and cannabinoid-tolerant mice. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.10.016i