Central Administration of Neuropeptide FF and Related Peptides Attenuate Systemic Morphine Analgesia in Mice

Protein & Peptide Letters, 2011, 18, 403-409 403 Central Administration of Neuropeptide FF and Related Peptides Attenuate Systemic Morphine Analgesia in Mice

Quan Fang1,#, Tian-nan Jiang1,#, Ning Li1, Zheng-lan Han1 and Rui Wang1,2,*

1Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Medicine, and Institute of Biochemis- try and Molecular Biology, School of Life Sciences, and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tian Shui South Road, Lanzhou, 730000, PR China; 2State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic Uni- versity, Hong Kong, China

Abstract: Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in the control of pain and analgesia through interactions with the opioid system. However, very few studies examined the effect of supraspinal NPFF system on analgesia induced by opiates administered at the peripheral level. In the present study, intracerebroventricular (i.c.v.) injection of NPFF (1, 3 and 10 nmol) dose-dependently inhibited systemic morphine (0.12 mg, i.p.) analgesia in the mouse tail flick test. Similarly, i.c.v. administration of dNPA and NPVF, two agonists highly selective for NPFF2 and NPFF1 receptors, respectively, decreased analgesia induced by i.p. morphine in mice. Furthermore, these anti-opioid activities of NPFF and related peptides were blocked by pretreatment with the NPFF receptors selective antagonist RF9 (10 nmol, i.c.v.). These results demonstrate that activation of central NPFF1 and NPFF2 receptors has the similar anti-opioid actions on the antinociceptive effect of systemic morphine. Keywords: Analgesia, neuropeptide FF (NPFF), mice, morphine, receptor, intraperitoneal (i.p.) injection.

INTRODUCTION studies. At the cellular level, many pharmacological data suggested that NPFF and related peptides exhibited anti- Neuropeptide FF (NPFF, FLFQPQRFamide) was origi- opioid effects via NPFF and NPFF receptors [14-17]. The nally isolated from bovine brain through its cross-reaction 1 2 biochemical studies recently demonstrated that NPFF recep- with antibodies to the molluscan cardioexcitory peptide 2 tor in the rat spinal cord could exert a molecular anti-opioid FMRF-NH , which possessed the similar C-terminal se- 2 effect [18]. Moreover, at the whole animal level, NPFF ex- quence [1]. Recent reports have shown that NPFF belongs to hibited complex opioid-modulating activities in different a neuropeptide family including two precursors (pro-NPFFA pharmacological studies [13]. Intracerebroventricularly ad- and pro-NPFF ) and two G-protein coupled receptors B ministration of NPFF exhibited opioid-like inhibition of the (NPFF and NPFF ) [2-7]. NPFF and NPFF receptors are 1 2 1 2 mouse colonic bead propulsion time and inhibited small in- about 50 % identical structure [4, 5, 7]. However, several testinal transit [19, 20]. In contrast, the previous reports sug- studies suggested that the pro-NPFF peptides (such as A gested that central administration of NPFF acted as an anti- NPFF and NPA-NPFF) and pro-NPFF peptides (such as B opioid peptide in nociceptive modulation, locomotor activity NPVF) were the preferred ligands for NPFF2 and NPFF1 and feeding behavior [13]. receptors, respectively [7, 8]. In addition, the structure- activities studies and pharmacological assays demonstrated The previous studies indicated that NPFF1 and NPFF2 that NPVF and dNPA (a stable analogue of NPA-NPFF) receptors were distributed very differently in the central were the highest selectivities towards NPFF1 and NPFF2 nervous system [4, 7, 21]. Recently, NPFF related peptides receptors, respectively [7-11] (Table 1). exhibiting different selectivities towards NPFF1 and NPFF2 receptors could evoke different effects on morphine-induced Since the isolation of NPFF in 1985, the biological func- analgesia after i.c.v. co-administration in mice [9, 22]. So, tions suggested for this neuropeptide including pain modula- the results demonstrated that pro- and anti-opioid actions of tion, food intake, gastrointestinal and hormonal modulation, NPFF related peptides were not strictly related to the selec- modulation of opiate tolerance and abstinence and cardio- tivity towards NPFF and NPFF receptors [9, 22]. There- vascular action [12, 13]. Similar to other opioid-modulating 1 2 fore, further in vivo studies are required to define the roles of peptides, the link between the functions of NPFF and opioid NPFF receptor subtypes as it pertains to the modulation of systems has been widely investigated in in vitro and in vivo opioid system.

To date, the abilities of NPFF and the related peptides to *Address correspondence to this author at the Institute of Biochemistry and modulate supraspinal opioid system have been largely stud- Molecular Biology, School of Life Science, Lanzhou University, 222 Tian Shui South Road, Lanzhou, 730000, P.R.China; Tel: +86-931-8912567; ied in rodents after central administration [9, 22]. However, Fax: +86-931-8912567; E-mail: [email protected] very few studies examined the modulatory role of supraspi-

# nal NPFF system in analgesic effect of opioid injected at Both authors contributed equally to this work peripheral level. In addition, no attention has been directed to

Table 1. Affinities (Ki) of NPFF and Related Agonists on Human NPFF1 and NPFF2 Receptors

NPFF1 NPFF2 b a S1/2 Ki (nM) Ki (nM)

NPFF, FLFQPQRF-NH2 2.82 ± 0.06 0.21 ± 0.03 13.4

NPVF, VPNLPQRF-NH2 0.59 ± 0.07 23.0 ± 2.1 0.026

dNPA, D.NP(N-Me)AFLFQPQRF-NH2 2.9 ± 0.5 0.027 ± 0.001 107.4

Data are cited from the previous reports [10]. a b Ki value are expressed as mean ± S.E.M.; S1/2 = Ki (NPFF1)/Ki (NPFF2) for the selectivity index

the exact activities mediated by NPFF1 and NPFF2 receptors Administration of Drugs towards systemic opioid analgesia. Therefore, in the present study, the effects of NPFF and its highly selective agonists The i.c.v. administration was performed according to an adaptation of procedure by Haley and McCormick [24]. The (i.c.v.) on antinociceptive action induced by systemic mor- injection site was 2.5 mm posterior and 1.0 mm lateral to the phine were investigated in the mouse tail flick test. bregma, and 3 mm from the surface of the skull. Drugs were i.c.v. administered uniformly in a volume of 5 l at a con- MATERIALS AND METHODS stant rate of 10 l / min by using a 25-l microsyringe. Animals Proper injection site was verified in pilot experiments by administration and localization of methylene blue dye. Male Kunming strain mice (20-22 g) were obtained from the Experimental Animal Center of Lanzhou University. All In order to test the effects of NPFF and related peptides animals were cared for and experiments were carried out in on morphine-induced analgesia, the peptides were adminis- accordance with the European Community guidelines for the trated 5 min before the intraperitoneal (i.p.) injection of mor- use of experimental animals (86/609/EEC). All the protocols phine. Moreover, to further investigate whether or not the in this study were approved by the Ethics Committee of anti-opioid activities of NPFF and related peptides could be Lanzhou University, China. antagonized by RF9, the agonists were i.c.v. injected alone or co-injected with RF9 (i.c.v.) 5 min before the i.p. admini- Chemicals stration of morphine.

NPFF, NPVF, dNPA and RF9 were synthesized on a Nociceptive Test solid support following the recent report [23]. Peptides were prepared by manual solid-phase synthesis using standard N- Experiments were performed, as described earlier [25, fluorenylmethoxycarbonyl (Fmoc) chemistry. Fmoc- 26]. The nociceptive response was assessed by the radiant protected amino acids (GL Biochem (Shanghai) Ltd.) were heat tail-flick test. Every mouse was used only once. Briefly, coupled to a Rink Amide MBHA resin (Tianjin Nankai He- the animals were gently restrained by hand, and a light beam cheng Science & Technology Co.,Ltd ,China). The following was focused onto the tail. At the beginning of the study, the schedule was employed: (1) DMF wash (3); (2) 20% lamp intensity was adjusted to elicit a response in control piperidine/DMF (3, 4min); (3) DMF wash (3); (4) N- animals within 3 - 5 sec. A cut-off time was set at 10 sec to Fmoc-Amino Acid (2.5eq.)/HBTU (2.5eq.)/HOBt minimize tissue damage. Tail-flick time was determined be- (2.5eq.)/DIPEA (5eq.) in DMF (1), 1 h; (5) DMF wash fore injection and then at 10, 20, 30, 45, 60, 75 and 90 min (3); (6) Kaiser Test. RF9 was obtained after acylation of the after i.c.v. injection. N-terminus with 1-adamantanecarboxylic acid (3eq.) /HBTU (3eq.)/HOBt (3eq.)/DIPEA (6eq.) in DMF (1), 1 h. The Statistical Analysis protected peptide-resin was treated with reagent K Data are expressed as the percent maximum possible (TFA/H O/phenol/ethanedithiol/thioanisole, 82.5:5:5:2.5:5) 2 effect (% MPE) calculated as: % MPE = 100 [(post-drug for 2 h at room temperature. Gel filtration (Sephadex G-10) response – baseline response)/(cut-off response – baseline was performed to desalt the crude peptides. The desalted response)]. The raw data from each animal were converted to peptide was purified by preparative reversed-phase HPLC area under the curve (AUC). We calculated the AUC data using a Waters Delta 600 system coupled to a UV detector. over the period 0 to 60 min. Data were statistically compared Fractions containing the purified peptides were pooled and by means of one-way ANOVA followed by the Dunnett’s or lyophilized. The purity of the peptide was established by Bonferroni’s post-hoc test. P-values less than 0.05 were con- analytical HPLC. The molecular weight of the peptide was sidered to be significant. confirmed by an electrospray ionization mass spectrometer (Mariner ESI-TOF MS, Applied Biosystems, CA). RESULTS In addition, morphine hydrochloride was the product of Shenyang First Pharmaceutical Factory, China. All drugs Effects of i.c.v. Injection of NPFF and Related Peptides on Systemic Morphine Analgesia were dissolved in sterilized saline, and the solutions were divided into aliquots and stored at –20 oC. The aliquots were As shown in Fig. (1), co-administration of i.c.v. saline thawed and used on the day of the experiment. with systemic saline (i.p.) did not significantly alter the noci- Neuropeptide FF Attenuates Systemic Morphine Analgesia Protein & Peptide Letters, 2011, Vol. 18, No. 4 405

80 Saline(i.c.v.)+Saline(i.p.) (n=9) Saline(i.c.v.)+0.12 mg Mor(i.p.) (n=16) (A) 70 10 nmol NPFF(i.c.v.)+0.12 mg Mor(i.p.) (n=13) 10 nmol NPFF(i.c.v.)+Saline(i.p.) (n=7) 60

50 40

MPE (%) 30

-10 0 102030405060708090 Time after i.p. injection (min)

80 Saline(i.c.v.)+Saline(i.p.) (n=9) (B) Saline(i.c.v.)+0.12 mg Mor(i.p.) (n=16) 70 10 nmol dNPA(i.c.v.)+0.12 mg Mor(i.p.) (n=11) 10 nmol dNPA(i.c.v.)+Saline(i.p.) (n=10) 60 50

MPE (%) 30

-10 0 102030405060708090 Time after i.p. injection (min) 80 Saline(i.c.v.)+Saline(i.p.) (n=9) (C) Saline(i.c.v.)+0.12 mg Mor(i.p.) (n=16) 70 20 nmol NPVF(i.c.v.)+0.12 mg Mor(i.p.) (n=10) 20 nmol NPVF(i.c.v.)+Saline(i.p.) (n=10) 60

MPE (%) 30

10 0 -10 0 102030405060708090 Time after i.p. injection (min)

Figure 1. Time course of the effects of i.c.v. injection of NPFF (A, 10 nmol), dNPA (B, 10 nmol) and NPVF (C, 20 nmol) on analgesia induced by morphine (Mor, 0.12 mg, i.p.) in the mouse tail flick test. Saline, NPFF and related peptides were i.c.v. injected 5 min prior to peripheral administration of saline or morphine (i.p.). Data points represent means ± S.E.M. from experiments conducted on 7 - 16 mice. Area under the curve (AUC) during 0 - 60 min calculated from these data are statistically analyzed and are presented in Fig. (2). 406 Protein & Peptide Letters, 2011, Vol. 18, No. 4 Fang et al.

ceptive threshold (AUC: -81.14 ± 57.77). Morphine (0.12 failed to induce significant effects on the antinociceptive mg, i.p.) given after i.c.v. saline produced significant antino- responses to i.p. injection of morphine (0.12 mg) (AUC: ciception, reaching maximal antinociceptive responses at 30 1795.3 ± 368.7; P > 0.05, vs. saline + morphine group). min after the injection in conscious mice (AUC: 2406.6 ± Moreover, effects of RF9 on the anti-opioid activities of 369.2, P > 0.05, vs. saline + saline group). NPFF and related peptides were further investigated in the NPFF (1, 3 and 10 nmol, i.c.v.) administered 5 min be- tail-flick test. Interestingly, pretreatment with 10 nmol of fore i.p. injection of morphine (0.12 mg) reduced the mor- RF9 (i.c.v.) significantly prevented the anti-opioid actions of phine-induced analgesia in a dose-dependent manner Fig. NPFF and related peptides in this nociceptive test (Table 2). (2). At a dose of 10 nmol, NPFF did not modify the tail-flick latency but significantly attenuated the analgesic effect elic- DISCUSSION ited by systemic morphine (P < 0.05) Figs. (1 and 2). How- NPFF represents a neurotransimitter system acting as a ever, 1 and 3 nmol NPFF did not significantly modify anal- modulator of opioid functions [13]. A great deal of evidence gesia of morphine injected at the peripheral level (P > 0.05) Fig. (2). suggests that NPFF and related analogues play important roles in nociceptive regulation through interactions with the Similar to NPFF, both dNPA (1, 3 and 10 nmol) and opioid system [27-29]. The present study was conducted to NPVF (3, 10 and 20 nmol) administered into the lateral ven- further investigate the modulatory role of supraspinal NPFF tricle, reduced the antinociception of systemic morphine system in antinociceptive effect of opiates administered at (0.12 mg, i.p.) compared to that induced by injection at the the peripheral level. Thus, we measured the effect of central same dose of morphine alone. Fig. (1B and C) illustrated that administration of NPFF and its highly selective ligands, central administration of highest dose of dNPA (10 nmol, dNPA and NPVF, on the systemic morphine analgesia in the i.c.v.) and NPVF (20 nmol, i.c.v.) did not change the noci- mouse tail flick test. ceptive threshold but induced marked decrease of systemic Our present results demonstrated that i.c.v. administra- morphine analgesia. The lower doses of dNPA and NPVF (1 tion of NPFF did not modify the nociceptive threshold but and 3 nmol) could not exert significant effects on analgesia. According to the dose-response relationships for their anti- dose-dependently reduced the analgesia induced by morphine (i.p.) administered at the peripheral level. These obser- morphine activities, the order of the potency of these three vations agree with the results obtained in the recent study by peptides to reduce systemic morphine analgesia was: dNPA using the mouse tail immersion test [30]. Furthermore, the NPFF > NPVF. anti-morphine effects induced by NPFF were markedly antagonized by the NPFF receptors selective antagonist RF9 Effects of RF9 on the Anti-Morphine Actions of NPFF and Related Peptides (i.c.v.), suggesting that the anti-opioid activities of NPFF were mainly linked to specific activation of NPFF receptors As shown in Fig. (3), i.c.v. injection of RF9 (10 nmol) [31, 32]. Thus, these biological functions of NPFF may be did not significantly alter the tail-flick latency compared to consistent with the distribution of abundant NPFF1 and the vehicle control (AUC: -6.17 ± 87.90; P > 0.05, vs. saline NPFF2 receptors in central nervous system, especially in the + saline group). Additionally, administration of 10 nmol RF9 brain [7, 21].

3500 Saline(i.c.v.) + 0.12 mg Morphine(i.p.) NPFF(i.c.v.) + 0.12 mg Morphine(i.p.)

3000 dNPA(i.c.v.) + 0.12 mg Morphine(i.p.) # # # NPVF(i.c.v.) + 0.12 mg Morphine(i.p.)

2500

2000

1500 * * Antinociception(AUC) 1000 *

500

0 013 10 1310131020 Doses of NPFF and related peptides (nmol, i.c.v.)

Figure 2. Dose-response relationships for the actions of NPFF and related peptides pretreatment on the antinociception induced by morphine (0.12 mg/kg, i.p.) in mouse tail-flick test. Saline, NPFF and related peptides were i.c.v. injected 5 min prior to peripheral administration of morphine (i.p.). The values for anti-nociceptive response, expressed as AUC, ± S.E.M. during 0 - 60 min (n = 8 - 16). # # # P < 0.001, compared with the response of saline-treated mice; * P < 0.05 indicating significant differences from the action of morphine alone with one-way ANOVA followed by the Bonferroni’s post-hoc test. Neuropeptide FF Attenuates Systemic Morphine Analgesia Protein & Peptide Letters, 2011, Vol. 18, No. 4 407

Table 2. Effects of NPFF Receptors Antagonist RF9 on the Anti-Opioid Actions of NPFF and Related Peptides in the Mouse Tail Flick Test

Treatments MPE % (Time After i.p. Injection) AUC n i.c.v. i.p. 10 Min 20 Min 30 Min 45 Min (0-60 Min)

Saline Mor 15.34 ± 6.17 51.60 ± 11.43 70.64 ± 8.93 47.74 ± 10.65 16 2406.6 ± 369.2 NPFF Mor 4.07 ± 1.33 10.53 ± 2.88 34.29 ± 9.21 18.56 ± 4.38 13 943.84 ± 225.47 (RF9+NPFF) Mor 19.09 ± 7.58 78.10 ± 10.00 76.95 ± 9.43 54.77 ± 11.42 12 2926.6 ± 427.7 b

dNPA Mor 14.01 ± 8.88 29.82 ± 10.60 15.50 ± 2.28 9.60 ± 3.23 11 809.69 ± 99.03 (RF9+dNPA) Mor 27.45 ± 10.57 49.15 ± 10.98 66.61 ± 10.99 38.97 ± 10.15 14 2352.6 ± 471.9 a

NPVF Mor 4.67 ± 1.38 17.93 ± 9.30 30.98 ± 12.24 14.63 ± 4.60 10 897.87 ± 207.14 (RF9+NPVF) Mor 34.29 ± 14.38 49.97 ± 14.33 55.39 ± 12.93 42.71 ± 11.55 10 2530.8 ± 539.9 a

NPFF and related peptides (NPFF, 10 nmol; dNPA, 10 nmol; NPVF, 20 nmol) were i.c.v. injected alone or co-injected with RF9 (10 nmol, i.c.v.) 5 min prior to peripheral administration of morphine (Mor, 0.12 mg, i.p.). Data are means ± S.E.M. The values for anti-nociceptive response, expressed as area under the time-effect curve (AUC), ± S.E.M. during 0 - 60 min. a P < 0.05 and b P < 0.001 indicating significant differences from anti-opioid actions of NPFF and related peptides in the absence of RF9 with one-way ANOVA followed by the Bonferroni’s post-hoc test

80 Saline(i.c.v.)+Saline(i.p.) (n=9) Saline(i.c.v.)+0.12 mg Mor(i.p.) (n=16) 70 10 nmol RF9(i.c.v.)+0.12 mg Mor(i.p.) (n=12) 10 nmol RF9(i.c.v.)+Saline(i.p.) (n=8) 60

MPE (%) 30

20 10

-10

0 102030405060708090 Time after injection (min)

Figure 3. Time course of the effects of i.c.v. injection of RF9 (10 nmol) on analgesia induced by morphine (Mor, 0.12 mg, i.p.) in the mouse tail flick test. Saline and RF9 were i.c.v. injected 5 min prior to peripheral administration of saline or morphine (i.p.). Data points represent means ± S.E.M. from experiments conducted on 8 - 16 mice. AUC during 0 - 60 min calculated from these data are statistically analyzed and are presented in the text.

The previous studies using cells expressing NPFF recep- and NPFF2 receptors. Therefore, our data indicate that acti- tors have revealed that both NPFF1 and NPFF2 receptors vation of central NPFF1 and NPFF2 receptors exerts the simi- recognize NPFF with affinities in the nanomolar range [4, lar anti-morphine actions on antinociception of morphine 10]. To further explore the roles of two NPFF receptor sub- injected at the peripheral level. types in the opioid-modulating effects, NPVF and dNPA, Taking account the absolute affinity and the rank order of two selective agonists for NPFF1 and NPFF2 receptors, re- selectivity of these agonists, it appears dNPA and NPFF ex- spectively, were used in this study. dNPA, a stable analogue hibit the higher affinity towards NPFF receptor (Table 1). It of pro-NPFF peptide NPA-NPFF, has at least 100 times 2 A is worthy to note that the NPFF receptor agonist dNPA and higher affinity for the NPFF than for the NPFF receptor [9, 2 2 1 NPFF display much higher potency than NPVF in the pre- 10], whereas the pro-NPFF peptide NPVF has 30 times B sent work. The result seems to be in agreement with the ob- more affinity for the NPFF1 than for the NPFF2 receptor [8, servation that NPFF2 receptor is much more abundant than 10]. Interestingly, both dNPA and NPVF administered into NPFF receptor in central nervous system [21]. the lateral ventricle reduced systemic morphine analgesia in 1 a manner similar to NPFF. In addition, the NPFF receptors In the previous work, the link between the functions of antagonist RF9 (i.c.v.) reversed the anti-opioid activities NPFF and opioid systems has been widely studied at the induced by these two highly selective agonists for NPFF1 cellular level. In acutely dissociated neurons, NPFF and 408 Protein & Peptide Letters, 2011, Vol. 18, No. 4 Fang et al.

NPVF attenuated the magnitude of the inhibitory effect of an ABBREVIATION opioid receptor agonist on the [Ca2+] transient triggered by i 1DMe = D.YL(N-Me)FQPQRFamide depolarization [14, 15]. By using a model of SH-SY5Y cells, 1DMe (a stable agonist of NPFF) and NPVF were reported dNPA = D.NP(N-Me)AFLFQPQRFamide to exert the neuronal anti-opioid activities via activation of NPA-NPFF = NPAFLFQPQRFamide NPFF2 and NPFF1 receptors, respectively [16, 17]. NPFF reduced the inhibitory effect of mu-opioid receptor agonist NPVF = VPNLPQRFamide on the C-fiber evoked firing of rat spinal cord neurons [33]. BIBP3226 = (R)-N2-(diphenylacetyl)-N-[(4- In addition, the molecular mechanism of the cellular anti- hydroxyphenyl)-methyl]-argininamide opioid effect of NPFF receptors was studied in SH-SY5Y human neuroblastoma cells. The results showed that NPFF REFERENCES receptors could block opioid receptor by physical interaction [16, 17]. Recently, fluorescence resonance energy transfer [1] Yang, H. 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,Received: September 08, 2010 Revised: November 20, 2010 Accepted: November 25, 2010