In vitro and in vivo studies of dansylated compounds, the putative agonists and antagonists on neuropeptide FF receptors

peptides 27 (2006) 1297–1304

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In vitro and in vivo studies of dansylated compounds, the putative agonists and antagonists on neuropeptide FF receptors Quan Fang, Jia Guo, Ya-li Peng, Min Chang, Feng He, Qiang Chen, Rui Wang * Department of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, PR China

article info

abstract

Article history:

To further evaluate the importance of C-terminal modification of neuropeptide FF (NPFF), in

Received 16 September 2005

the present work, four dansylated NPFF analogues, including two putative agonists (dansyl-

Received in revised form

PQRFamide and dansyl-GSRFamide) and two putative antagonists (dansyl-PQRamide and

25 October 2005

dansyl-GSRamide), were synthesized and investigated to address their potencies and

Accepted 25 October 2005

efficacies in a series of in vitro and in vivo assays. (1) In the isolated mouse colon bioassay,

Published on line 7 December 2005

the four dansylated compounds showed agonistic profiles: both dansyl-GSRFamide (1– 10 mM) and dansyl-GSRamide (1–10 mM) dose-dependently caused colonic contractions,

Keywords:

which were attenuated by pretreatment with BIBP3226; dansyl-PQRFamide and dansyl-

Neuropeptide FF (NPFF)

PQRamide evoked modest colonic contractions at a high dose of 50 mM. (2) In urethane-

Dansylated compound

anaesthetized rats, both dansyl-PQRFamide (50–300 nmol/kg, i.v.) and dansyl-GSRFamide

Colonic contraction

(15–50 nmol/kg, i.v.) dose-dependently increased the mean arterial pressure and heart rate

Mean arterial pressure

in a manner similar to NPFF (50–300 nmol/kg, i.v.); on the contrary, the two putative

Heart rate

antagonists (100–800 nmol/kg, i.v.) decreased blood pressure in a dose-dependent manner. All the results suggest that dansyl-PQRFamide and dansyl-GSRFamide are NPFF full agonists; in contrast, dansyl-GSRamide and dansyl-PQRamide behave as agonists in vitro and antagonists in vivo on NPFF receptors. The findings reveal that the C-terminal Phe might be a crucial residue to determine the efficacy. In addition, the novel analogue dansyl-GSRFamide may be developed as a highly potent agonist to investigate the NPFF system. # 2005 Elsevier Inc. All rights reserved.

1.

Introduction

Neuropeptide FF (NPFF, FLFQPQRFa) was originally isolated from bovine brain through its cross-reaction with antibodies to the molluscan cardioexcitory peptide FMRF-NH2, which possessed similar C-terminal sequence [27]. Recent reports have shown that NPFF belongs to a neuropeptide family including two precursors (pro-NPFFA and pro-NPFFB) and two receptors (NPFF1 and NPFF2) [2,3,8,12,26].

In vitro and in vivo studies have demonstrated that NPFF mediates a variety of biological actions. A summary of findings reported to date indicated that: NPFF and its analogues exhibited either anti-opioid activities or proopioid effects when injected intracerebroventricular or intrathecally in rat and mouse, respectively [19,24]; the increase of NPFF level in the central nervous system might contribute to the development of tolerance and dependence for opioid [24,25]; intravenous administration of NPFF exerted

* Corresponding author. Tel.: +86 931 8912567; fax: +86 931 8912561. E-mail address: [email protected] (R. Wang). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.10.021

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pressor and tachycardiac responses [1,23]. In addition, our recent results suggested that NPFF exerted contractions of the isolated mouse colon independently of opioid system [6]. Results obtained from structure–activity relationship studies of NPFF-related peptides suggest that the C-terminal RFamide is essential for NPFF receptors activation and/or occupation but the N-terminus is responsible for binding [7,16,20]. Based on the message/address model, C-terminal RFamide, the active core to activate NPFF receptors, may be considered to be ‘‘message domain’’ of NPFF [16]. Modifications of the C-terminal sequence, especially at the C-terminal Phe, led to the discovery of putative antagonists, such as desaminotyrosyl-FLFQPQRamide and dansyl-PQRamide containing -Ramide in C-terminal domain, which were shortened from the corresponding NPFF agonists [14,15]. In addition, PFR(Tic)amide was derived from the agonist PFRFamide substituting Tic (L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid) for Phe4, which was found as an NPFF putative antagonist because of its depressor response, while it was recently shown to be a ‘‘super-agonist’’ in the [35S]GTPgS binding assay [5,9]. Therefore, further study of the relationship between NPFF C-terminal modification and the efficacy may aid the development of NPFF antagonists. In the present study, we examined the bioactivities of the four dansylated NPFF analogues to evaluate the importance of Phe8. These compounds shared the common lipophilic Nterminal substitutions and were classified into putative agonists (containing -RFamide) and antagonists (containing -Ramide) according to the different C-terminal structural features. In the previous binding experiments, dansylGSRamide, dansyl-PQRFamide and dansyl-PQRamide displayed affinities toward NPFF receptors (Table 1) [15,20,21]. In addition, dansyl-PQRFamide and dansyl-PQRamide have been reported to exert agonistic and antagonistic properties in opioid-dependent tests, respectively [13,15,25]. Systemically injected dansyl-PQRamide behaved as an NPFF antagonist to induce condition place preference [10]. Although currently the function of these four dansylated analogues (dansyl-GSRFamide, dansyl-GSRamide, dansyl-PQRFamide and dansyl-PQRamide) has not been fully characterized, we designed to investigate their bioactivities in the experiments of the isolated mouse colon and in vivo cardiovascular regulation.

Table 1 – Displacement of [125I]YLFQPQRFamide by the dansylated analogues in rat spinal cord membrane preparation Peptides Dansyl-GSRa Dansyl-PQRa Dansyl-GSRFa Dansyl-PQRFa

Ki (nM)a

Reference

1400 13600 –b 6.1

[21] [21] – [20]

Data adapted from the previous reports [20,21]. Ki values were obtained in the competition binding experiments in rat spinal cord membrane preparation. b Ki value has not been reported to date because it is a novel analogue. a

2.

Materials and methods

2.1.

Animals

Male Kunming strain mice and male Wistar rats were obtained from the Experimental Animal Center of Lanzhou University. 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

Dansylated compounds were synthesized on a solid-phase support following the previous report [25]. Peptides were prepared by manual solid-phase synthesis using standard Fmoc chemistry. N-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids (ACT and Fluka, USA) were coupled to a Rink Amide MBHA resin (Tianjin Nankai Hecheng Science & Technology Co. Ltd., China). The following schedule was employed: (1) DMF wash (3); (2) 20% piperidine/DMF (3, 4 min); (3) DMF wash (3); (4) Na-Fmoc-Amino Acid (2.5 eq.)/ HBTU (2.5 eq.)/HOBt (2.5 eq.)/DIPEA (5 eq.) in DMF (1), 1 h; (5) DMF wash (3); (6) Kaiser Test. The dansyl residue was added to the resin bound peptide using dansyl chloride (2.5 eq.)/ DIPEA (3.75 eq.)/catalytic DMAP (0.5 eq.) in DMF (1). The protected peptide-resin was treated with reagent K (TFA/H2O/ phenol/ethanedithiol/thioanisole, 82.5:5:5:2.5:5; v/v; 10 mL/ 0.2 g of resin) for 2 h at room temperature. Gel filtration (Sephadex G-10) was performed to desalt the crude peptides. The purity of the peptide was checked by reversed-phase highperformance liquid chromatography (HPLC). Analytical HPLC analyses were performed on a Waters Delta 600 system coupled to a UV detector with a Delta-pak Analytical column ˚ , 5 mm). Purification was carried C18 (3.9 mm  150 mm, 300 A out by HPLC, if required. NPFF was synthesized manually by the solid-phase peptide synthesis method and was purified by HPLC as described in our previous report [6]. BIBP3226, (R)-N2-(diphenylacetyl)-N[(4-hydroxyphenyl)-methyl]-argininamide was generously provided by Dr. H. Doods (Boehringer-Ingelheim, Biberach, Germany). Dansylated compounds and BIBP3226 were dissolved in 3% DMSO and compared to vehicle control. NPFF was dissolved in sterilized saline and stored at 20 8C.

2.3.

Mouse isolated colon bioassays

Experiments were performed, as described earlier [6]. Briefly, adult mice (weighing 25–30 g) were sacrificed by cervical dislocation. Segments of mouse colon (1.5 cm in length) were obtained from distal (1 cm proximal to the anus) regions of large intestine, flushed of their contents and trimmed of mesentery. Preparations were suspended in the axis of the longitudinal muscle under 1 g tension with fine thread in 10 ml siliconised organ baths containing Krebs’ solution (NaCl, 118 mM; KCl, 4.74 mM; CaCl2, 2.54 mM; KH2PO4, 1.19 mM; MgSO4, 1.20 mM; NaHCO3, 25 mM; glucose, 11 mM;) maintained at 37 8C and bubbled with 95% O2 and 5% CO2, then allowed to

peptides 27 (2006) 1297–1304

equilibrate for 60 min prior to drug addition, with changes of Krebs’ solution every 15 min. Isometric responses were recorded using a strain gauge transducer (Machine Equipment Corporation of GaoBeiDian, China) linked to a recorder system (model BL-420E+, Taimeng Technology Corporation of Chengdu, China). The concentration–response curves of drugs were determined non-cumulatively. Carbachol (1 mM) was added as an internal contractile control at the end of each assay. Drugs induced maximal increase in basal tone (within 3 min of agonist addition) were pooled and quoted as increase in g tension throughout (means  S.E.M.). The antagonist (BIBP3226) was incubated for 15 min prior to addition of drugs.

2.4.

Cardiovascular measurement

Male Wistar rats weighing 240–280 g were anesthetized with urethane (l.2 g/kg, i.p.). Supplemental doses of urethane were given as needed to maintain a uniform level of anesthesia. The trachea was incised to get rid of mucus. The animals spontaneously breathed room air. Polyethylene catheters were inserted into the external jugular vein for i.v. administration of drugs and into the carotid artery for measurement of mean arterial pressure (MAP) with an YP100 pressure transducer (Machine Equipment Corporation of GaoBeiDian, China). MAP and heart rate (HR) were derived by electronic averaging and recorded on a recorder system (model BL-420E+, Taimeng Technology Corporation of Chengdu, China). Peptides were injected intravenously within 30 s. It was found in preliminary experiments that a 15-min interval between injections of NPFF and its analogues was sufficient to allow the blood pressure and HR to recover to the pre-injection level. Therefore, increasing doses of the dansylated compounds and NPFF were injected in the experiments with a 30-min interval. Changes in MAP and HR were expressed as the means  S.E.M.

2.5.

Statistical analysis

Data are given as means  S.E.M. One-way ANOVA followed by the Dunnett’s post hoc test was used to establish statistical significance at a probability level of P < 0.05.

3.

Results

3.1. Colonic contractile responses to the dansylated NPFF analogues Mouse colonic preparations exerted spontaneous phasic contractions in vitro. Dansyl-GSRFamide (1–10 mM) and dansyl-GSRamide (1–10 mM) evoked contractile effects in mouse distal colon in a concentration-dependent manner. While addition of 1–10 mM dansyl-PQRFamide and dansyl-PQRamide failed to elicit significant contractions in colonic preparations (data not shown), they induced mild contractile responses at a high dose of 50 mM (n = 4). Their phasic contractions were similar to those of NPFF. As shown in Fig. 1E, dansyl-GSRFamide caused sustained contractions with a pEC50 value of 5.13 (range 1–10 mM) and an Emax of 60.69% of the control tissue response elicited with 1 mM carbachol. The contractile potency of dansyl-GSRFamide was

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similar to that obtained with NPFF in the recent report (range 1–15 mM; pEC50 = 5.44; Emax = 43.36%) [6]. The Emax of dansylGSRamide was 18.73% of the control tissue response, which was relatively weaker than that of NPFF (Fig. 1E).

3.2. Inhibitory effects of BIBP3226 on the responses induced by dansyl-GSRFamide and dansyl-GSRamide To further examine the contractions of dansylated NPFF analogues, inhibitory effects of BIBP3226 on the contractile responses induced by dansyl-GSRFamide and dansyl-GSRamide were investigated. As shown in Fig. 2, colonic contractions induced by the maximal concentrations of dansylGSRFamide (10 mM) and dansyl-GSRamide (10 mM) could be reduced by pretreatment of 30 mM BIBP3226 (n = 4), which was sufficient to abolish the contractions of NPFF (3 mM) and NPVF (3 mM) [6].

3.3. Cardiovascular effects of the dansylated NPFF analogues The values of basal mean arterial pressure and heart rate were 91.1  1.7 mmHg and 363.4  7.8 bpm (n = 26), respectively, in urethane-anesthetized rats. The vehicle control (3% DMSO, i.v.) significantly failed to affect blood pressure and heart rate. Fig. 3 showed typical recordings of pulsatile arterial pressure (PAP) and mean arterial pressure before and after injections of NPFF and its putative agonists. NPFF (300 nmol/ kg, i.v.), dansyl-GSRFamide (50 nmol/kg, i.v.) and dansylPQRFamide (300 nmol/kg, i.v.) caused transient pressor responses, which were sustained for 2–3 min and their peaks were reached within 1 min. NPFF increased MAP and HR maximally by about 45 mmHg and 60 bpm, respectively; both maximal effects were obtained at a dose of 300 nmol/kg. The dose–response curves of dansyl-PQRFamide for both cardiovascular parameters were virtually coincident with those of NPFF in the same dose range. Dansyl-GSRFamide displayed higher potency and induced the similar increase in MAP and HR at much lower doses than NPFF and dansyl-PQRFamide, and its maximal effects were obtained at a dose of 50 nmol/kg (Fig. 4). The doses of the two NPFF putative antagonists tested ranged from 100 to 800 nmol/kg. Unlike the putative agonists, they lowered significantly the mean arterial pressure, which returned to baseline level within 3 min and the peaks were reached about 1 min later (Fig. 5). Our results showed that the depressor effects of the putative antagonists on MAP were in a dose-dependent manner (Table 2). As to the depressor responses, dansyl-PQRamide was more potent than dansylGSRamide. Table 2 illustrated the effects of dansyl-GSRamide and dansyl-PQRamide on HR. Compared with the vehicle control (3% DMSO, i.v.), the putative antagonists significantly failed to induce changes in HR (P > 0.05).

4.

Discussion

The structure–activity relationship studies of NPFF reveal that the C-terminal RFamide is essential for NPFF receptors activation and/or occupation but the N-terminus is respon-

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Fig. 1 – Effects of dansylated NPFF analogues on the mouse distal colon in vitro. Representative tracings of isolated colonic contractile responses to dansyl-GSRamide (DNS-GSRa; 10 mM, (A)), dansyl-GSRFamide (DNS-GSRFa; 10 mM, (B)), dansylPQRamide (DNS-PQRa; 50 mM, (C)) and dansyl-PQRFamide (DNS-PQRFa; 50 mM, (D)). Tracings shown were from a single animal, respectively. Contraction evoked by 1 mM carbachol represents the maximum contractile activity of this organ. W: washing of tissue with fresh physiological salt solution. Arrows: indicating the time of compound addition to the bath. (E) Dansyl-GSRamide (1, 3, 5 and 10 mM) and dansyl-GSRFamide (1, 3, 5 and 10 mM) concentration–response curves of the distal mouse colon. Data points represent means W S.E.M. from experiments conducted on five to nine mice.

sible for binding [7,16,20]. The suggestion was supported by the data obtained from analogues, which were modified at the C-terminus of NPFF and the related tetrapeptide FMRFamide [16,20]. Shortening of the NPFF sequence from the N-terminius

produced only a moderate decrease in affinity until NPFF(4-8) reached [7]. The analogues PMRFamide and PQRFamide displayed high affinities for NPFF receptors and stimulated Eu-GTP and [35S]GTPgS binding to membranes of CHO cells

Fig. 2 – Inhibitory effects of 30 mM BIBP3226 on the isolated colonic contractile responses of dansyl-GSRamide (DNS-GSRa; 10 mM, (A)) and dansyl-GSRFamide (DNS-GSRFa; 10 mM, (B)). The putative antagonist was incubated for 15 min before dansylated analogues were tested. Tracings in the figure (A and B) were from a single animal, respectively. The meaning of W and arrows is the same as in Fig. 1.

peptides 27 (2006) 1297–1304

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Fig. 3 – Typical pressor responses to the maximum dose of NPFF (300 nmol/kg, i.v., (A)), dansyl-GSRFamide (DNS-GSRFa; 50 nmol/kg, i.v., (B)) and dansyl-PQRFamide (DNS-PQRFa; 300 nmol/kg, i.v., (C)). Top and bottom tracings represent pulsatile arterial pressure (PAP) and mean arterial pressure (MAP), respectively. Arrows indicate time of injection.

Fig. 4 – Dose–response curves of NPFF, dansyl-GSRFamide (DNS-GSRFa) and dansyl-PQRF-amide (DNS-PQRFa) on mean arterial pressure and heart rate in anaesthetized rats. (A) Dose–response curves of NPFF (50, 100, 200 and 300 nmol/kg, i.v., n = 6), dansyl-GSRFamide (15, 30, 40 and 50 nmol/kg, i.v., n = 6) and dansyl-PQRFamide (50, 100, 200 and 300 nmol/kg, i.v., n = 6) on mean arterial pressure (MAP). (B) Dose–response curves of NPFF (50, 100, 200 and 300 nmol/kg, i.v., n = 6), dansylGSRFamide (15, 30, 40 and 50 nmol/kg, i.v., n = 6) and dansyl-PQRFamide (50, 100, 200 and 300 nmol/kg, i.v., n = 6) on heart rate (HR). Data points represent means W S.E.M.

Fig. 5 – Typical depressor responses to the maximum dose of dansyl-GSRamide (DNS-GSRa; 800 nmol/kg, i.v., (A)) and dansyl-PQRamide (DNS-PQRa; 800 nmol/kg, i.v., (B)). Top and bottom tracings represent pulsatile arterial pressure (PAP) and mean arterial pressure (MAP), respectively. Arrows indicate time of injection.

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Table 2 – Changes in mean arterial pressure (MAP) and heart rate (HR) induced by i.v. injections of dansylGSRamide (DNS-GSRa) and dansyl-PQRamide (DNSPQRa) in anesthetized rats Peptides

Dose (nmol/kg)

DMAP (mmHg)

DHR (bpm)

DNS-GSRa

0 100 200 400 800

3.07  1.27 4.01  1.29* 8.66  2.14** 11.18  0.85** 16.17  1.78**

4.07  2.36 3.39  4.01 3.10  2.91 5.16  2.38 5.80  4.56

DNS-PQRa

0 100 200 400 800

3.37  0.56 15.83  0.77** 18.56  1.24** 20.27  3.13** 31.35  3.84**

6.65  1.91 9.21  5.56 6.23  2.91 7.99  2.89 3.56  3.25

Data are expressed as means  S.E.M. from experiments conducted on four rats, respectively. To establish statistical significance, the data were statistically analyzed by one-way ANOVA followed by the Dunnett’s post hoc test. * P < 0.05 (compared with the vehicle control group). ** P < 0.01 (compared with the vehicle control group).

stably expressing the NPFF2 receptor [4]. Surprisingly, the shorter tripeptide FRFamide was sufficient to activate the NPFF2 receptor in the Eu-GTP and [35S]GTPgS binding assays [4]. While the subtle substitutions for the C-terminal RFamide of NPFF induced a significant loss in affinity [16]. Based on the message/address model, modifications of ‘‘message domain’’ (the C-terminal RFamide) of NPFF might induce changes in the efficacy. To date, several compounds have been reported as the putative antagonists towards NPFF receptors, which were mainly modified in the C-terminal domain, for example desaminotyrosyl-FLFQPQRamide, dansyl-PQRamide and PFR(Tic)amide [9,14,15]. However, the corresponding analogues of these putative antagonists containing the C-terminal RFamide showed agonist activities, namely desaminotyrosyl-FLFQPQRFamide, dansyl-PQRFamide and PFRFamide [9,13–15]. Interestingly, PFR(Tic)amide was found as an NPFF putative antagonist because of its depressor responses in anaesthetized rats, while it was recently shown to be a ‘‘super-agonist’’ in the [35S]GTPgS binding assay [5,9]. These findings imply that the relationship between modification of the C-terminus and the efficacy is very complex. To further investigate the importance of C-terminal modification of NPFF, the structure–activity relationship study was performed in the present work to evaluate the importance of Phe8. These four analogues had the same N-terminal dansyl group, which was reported to enhance potency and hydrophobicity [20,21], and they were classified into putative agonists (dansyl-PQRFamide and dansyl-GSRFamide) and putative antagonists (dansyl-PQRamide and dansyl-GSRamide) according to the C-terminal structural features. In the previous reports, the [125I]Y8Famide binding to NPFF receptors in spinal cord membranes was displaced by dansyl-PQRFamide, dansyl-PQRamide and dansyl-GSRamide (Table 1) [15,20,21]. In addition, dansyl-PQRFamide and dansyl-PQRamide have been reported to exert agonistic and antagonistic properties in opioid-dependence tests, respectively [13,15,25]. Systemically injected dansyl-PQRamide acted as an NPFF

antagonist to induce condition place preference [10]. Based on the chemical structure of dansyl-PQRamide, dansylGSRamide was obtained as an NPFF antagonist with high affinity from combinatorial lead optimization [21]. However, the bio-effects of this compound in vitro and in vivo were poorly reported. Dansyl-GSRFamide was synthesized as a novel NPFF agonist and combined the dansylated N-terminus of dansyl-GSRamide with the C-terminal RFamide of NPFF. Collectively, the function of these four dansylated analogues has not been fully characterized, so they were examined in two NPFF assays: in vitro activity in the isolated mouse colon and in vivo cardiovascular regulation. In the isolated mouse colon, NPFF and NPVF have been showed to induce dose-dependent contractions via NPFF receptors in our previous report [6]. To our surprise, all the four dansylated analogues exerted NPFF-like contractile responses in this study. However, dansyl-GSRamide was more potent than dansyl-PQRamide in the isolated mouse colon; dansylGSRFamide exerted more significant contractile effects than those of dansyl-PQRFamide. The results seem to agree with the binding data obtained from the previous experiments. The Ki values in the previous radioligand-binding assay showed that dansyl-GSRamide had about a 10-fold higher affinity for NPFF receptors than dansyl-PQRamide (Ki = 1.4 mM versus Ki = 13.6 mM) [21]. The result suggests that the N-terminal structure of dansyl-GSRamide is responsible for this high affinity. It is worthy to note that dansyl-GSRFamide has a similar N-terminal structural feature as dansyl-GSRamide, and dansyl-PQRFamide shares the same N-terminus with dansyl-PQRamide. Therefore, dansyl-GSRFamide could be speculated to display much higher affinity than dansylPQRFamide (Ki = 6.1 nM) [20], though the affinity of dansylGSRFamide for NPFF receptors has not yet been determined in ligand-receptor binding assay. The results in the isolated mouse colon experiment support this deduction. In order to further investigate their colonic contractions, the effects of BIBP3226 on the contractions induced by both dansyl-GSRamide and dansyl-GSRFamide were tested. BIBP3226, an NPFF antagonist [6,17,18], attenuated the contractile effects of both dansyl-GSRamide and dansyl-GSRFamide. The observation demonstrates that the putative NPFF agonists and antagonist induce colonic contraction via NPFF receptors. However, it seems difficult to explain why dansylGSRamide displays a higher potency than danyl-PQRFamide in the isolated mouse colon because dansyl-GSRamide possessed much lower affinity than dansyl-PQRFamide (Ki = 1.4 mM versus 6.1 nM) [20,21]. It is notable that PFR(Tic)amide produced a larger agonistic responses on NPFF2 receptor than the stable NPFF analogues (1DMe)Y8Famide ([D-Tyr1, NMePhe3]NPFF) [5]. This implies that NPFF receptors might be activated via different pathways. It is likely that the two putative antagonists interact with NPFF receptors in a similar manner to PFR(Tic)amide. These findings suggest that the putative NPFF agonists and antagonists induce mouse colonic contractions via NPFF receptors, but their interactions with NPFF receptors are mediated via different pathways. When administrated i.v. to rats, both the putative NPFF agonists increased MAP and HR in a dose-dependent manner, while the two putative NPFF antagonists induced a dosedependent decrease in MAP. These observations were

peptides 27 (2006) 1297–1304

consistent with the previous reports that dansyl-PQRFamide and dansyl-PQRamide exerted in vivo agonistic and antagonistic profiles on NPFF receptors in opioid-dependence models [13,15,25]. Compared with NPFF, dansyl-PQRFamide possessed a similar potency while dansyl-GSRFamide displayed a much higher potency in cardiovascular regulation. These observations agree with the above-mentioned deduction that dansyl-GSRFamide displays a high affinity for NPFF receptors. The two putative agonists displayed much higher agonistic potencies compared with the results obtained from the assay in the isolated mouse colon. It was worthy to note that dansyl-PQRamide has a half-life over 300 times longer than NPFF under aminopeptidase digestion [15]. So, this high potency of the putative agonists in vivo could be partially explained by the nature of dansylated compounds for their resistance against proteolytic enzymes. In contrast, the putative antagonists induced effects on MAP opposite to that of NPFF, which suggests that they could be NPFF antagonists. As to their potency in blood pressure, dansyl-GSRamide was less potent than dansyl-PQRamide, which seems to disagree with the data obtained in binding experiments and in the isolated mouse colon assay. It is difficult to explain the discrepancy at present. However, the results imply that their changes in MAP do not depend directly or not only upon the affinities of the compounds used. A similar hypothesis was proposed by Quelven et al. in mouse tail-flick test [22]. To gain a full scope of interaction between these compounds and NPFF receptors, further experimentation to investigate their selectivities towards NPFF1 and NPFF2 receptors would be necessary. Additionally, we cannot dismiss the possibility that there was a still unidentified closely related receptor in the cardiovascular system to mediate the cardiovascular effects we observed. Furthermore, the putative antagonists had no significant effects on heart rate. A recent report demonstrated that the putative NPFF antagonist PFR(Tic)amide produced both MAP and HR attenuation [9], which seems to disagree with changes in HR induced by the dansylated putative antagonists. Their different regulation of HR might be relevant to the abilities of dansylated compounds for crossing the blood–brain barrier [15,21]. The previous reports showed that micro-injection of NPFF into the commissural nucleus tractus solitarius (NTS) produced bradycardia, while administrated i.v. of NPFF increased HR [1,11]. It is very likely that dansyl-PQRamide and dansyl-GSRamide could activate NPFF receptors within both CNS and peripheral systems, which lead to the dual changes in HR at the same time. In summary, the putative NPFF agonists (dansyl-PQRFamide and dansyl-GSRFamide) are NPFF full agonists in vitro and in vivo; in contrast, the putative antagonists (dansylGSRamide and dansyl-PQRamide) behave as NPFF agonists in vitro and antagonists in vivo. The findings highlight the role of C-terminal Phe as a crucial residue for determining the efficacy. Further modification in Phe8 should be helpful to discover more NPFF antagonists. The NPFF C-terminal conversion from -RFamide to -Ramide could not be attributed to a simple relationship between one C-terminal modification and an agonist/antagonist. In addition, the novel analogue dansylGSRFamide exerts highly potent agonistic profiles in the present study. Moreover, it possesses other important proper-

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ties, such as the high fluorescence, hydrophobicity and resistance to enzymatic breakdown [13,15,21]. Thus, it is expected that in future dansyl-GSRFamide may play a broader role in pharmacological researches of the NPFF system.

Acknowledgements We wish to thank Dr. Henri Doods (Boehringer-Ingelheim, Biberach, Germany) for his generous supply of BIBP3226. This study was supported by the grants from the National Natural Science Foundation of China (Nos. 20372028, 20472026), the Chang Jiang Program of the Ministry of Education of China, and the Ministry of Science and Technology of China. Thanks to Dr. P.G. Long (Visiting Professor-Cuiying Scholar Program, Lanzhou University) for revising the language.

references

[1] Allard M, Labrouche S, Nosjean A, Laguzzi R. Mechanisms underlying the cardiovascular responses to peripheral administration of NPFF in the rat. J Pharmacol Exp Ther 1995;274:577–83. [2] Bonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, et al. Identification and characterization of two G protein-coupled receptors for neuropeptide FF. J Biol Chem 2000;275:39324–31. [3] Elshourbagy NA, Ames RS, Fitzgerald LR, Foley JJ, Chambers JK, Szekeres PG, et al. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J Biol Chem 2000;275:25965–71. [4] Engstrom M, Narvanen A, Savola JM, Wurster S. Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay. Peptides 2004;25: 2099–104. [5] Engstrom M, Wurster S, Savola JM, Panula P. Functional properties of PRF(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors. Peptides 2003;24: 1947–54. [6] Fang Q, Guo J, Chang M, Chen LX, Chen Q, Wang R. Neuropeptide FF receptors exert contractile activity via inhibition of nitric oxide release in the mouse distal colon. Peptides 2005;26:791–7. [7] Gicquel S, Mazarguil H, Desprat C, Allard M, Devillers JP, Simonnet G, et al. Structure-activity study of neuropeptide FF: contribution of N-terminal regions to affinity and activity. J Med Chem 1994;37:3477–81. [8] Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, et al. New neuropeptides containing carboxyterminal RFamide and their receptor in mammals. Nat Cell Biol 2000;2:703–8. [9] Huang EY, Li JY, Tan PP, Wong CH, Chen JC. The cardiovascular effects of PFRFamide and PFR(Tic)amide, a possible agonist and antagonist of neuropeptide FF (NPFF). Peptides 2000;21:205–10. [10] Huang EY, Li JY, Wong CH, Tan PP, Chen JC. DansylPQRamide, a possible neuropeptide FF receptor antagonist, induces conditioned place preference. Peptides 2002;23:489–96. [11] Laguzzi R, Nosjean A, Mazaguil H, Allard M. Cardiovascular effects induced by the stimulation of neuropeptide FF receptors in the dorsal vagal complex: an autoradiographic and pharmacological study in the rat. Brain Res 1996;711:193–202.

1304

peptides 27 (2006) 1297–1304

[12] Liu Q, Guan XM, Martin WJ, McDonald TP, Clements MK, Jiang Q, et al. Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception. J Biol Chem 2001;276:36961–9. [13] Malin DH, Lake JR, Arcangeli KR, Deshotel KD, Hausam DD, Witherspoon WE, et al. Subcutaneous injection of an analog of neuropeptide FF precipitates morphine abstinence syndrome. Life Sci 1993;53: L261–6. [14] Malin DH, Lake JR, Leyva JE, Hammond MV, Rogillio RB, Arcangeli KR, et al. Analog of neuropeptide FF attenuates morphine abstinence syndrome. Peptides 1991;12: 1011–4. [15] Malin DH, Lake JR, Smith DA, Jones JA, Morel J, Claunch AE, et al. Subcutaneous injection of an analog of neuropeptide FF prevents naloxoneprecipitated morphine abstinence syndrome. Drug Alcohol Depend 1995;40:37–42. [16] Mazarguil H, Gouarderes C, Tafani JA, Marcus D, Kotani M, Mollereau C, et al. Structure–activity relationships of neuropeptide FF: role of C-terminal regions. Peptides 2001;22:1471–8. [17] Mollereau C, Gouarderes C, Dumont Y, Kotani M, Detheux M, Doods H, et al. Agonist and antagonist activities on human NPFF2 receptors of the NPY ligands GR231118 and BIBP3226. Br J Pharmacol 2001;133:1–4. [18] Mollereau C, Mazarguil H, Marcus D, Quelven I, Kotani M, Lannoy V, et al. Pharmacological characterization of human NPFF1 and NPFF2 receptors expressed in CHO cells by using NPY Y1 receptor antagonists. Eur J Pharmacol 2002;451:245–56.

[19] Panula P, Kalso E, Nieminen M, Kontinen VK, Brandt A, Pertovaara A. Neuropeptide FF and modulation of pain. Brain Res 1999;848:191–6. [20] Payza K, Akar CA, Yang HY. Neuropeptide FF receptors: structure–activity relationship and effect of morphine. J Pharmacol Exp Ther 1993;267:88–94. [21] Prokai L, Prokai-Tatrai K, Zharikova A, Li X, Rocca JR. Combinatorial lead optimization of a neuropeptide FF antagonist. J Med Chem 2001;44:1623–6. [22] Quelven I, Roussin A, Zajac JM. Comparison of pharmacological activities of neuropeptide FF1 and Neuropeptide FF2 receptor agonists. Eur J Pharmacol 2005;508:107–14. [23] Roth BL, Disimone J, Majane EA, Yang HY. Elevation of arterial pressure in rats by two new vertebrate peptides FLFQPQRF–NH2 and AGEGLSSPFWSLAAPQRF–NH2 which are immunoreactive to FMRF–NH2 antiserum. Neuropeptides 1987;10:37–42. [24] Roumy M, Zajac JM. Neuropeptide FF, pain and analgesia. Eur J Pharmacol 1998;345:1–11. [25] Tan PP, Chen JC, Li JY, Liang KW, Wong CH, Huang EY. Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs. Peptides 1999;20:1211–7. [26] Vilim FS, Aarnisalo AA, Nieminen ML, Lintunen M, Karlstedt K, Kontinen VK, et al. Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol Pharmacol 1999;55:804–11. [27] Yang HY, Fratta W, Majane EA, Costa E. Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82:7757–61.