Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-LysLeu-Ala-Asn-Gln) has been defined as the endogenous ligand for the N/OFQ peptide receptor (NOPR) or ORL1 receptor (Meunier et al., 1995; Reinscheid et al., 1995). Both peripheral and central injections of N/OFQ cause nociceptive as well as antinociceptive effects (Meunier et al., 1995; Reinscheid et al., 1995, 1996; King et al., 1997; Tian et al., 1997; Inoue et al., 1998, 1999; Pan et al., 2000). The metabolism of N/OFQ in mice has recently been reported. Montiel et al. (1997) have reported that incubation of N/OFQ with mouse brain cortical slices led to the generation of a number of fragments, and that four critical sites of enzymatic cleavage characterized by Phe¹-Gly², Ala⁷-Arg⁸, Ala¹¹-Arg¹², and Arg¹²-Lys¹³, form five main fragments of N/OFQ: N/OFQ (1-7), (2-17), (1-11), (12-17), and (13-17). The enzymes responsible were found to be aminopeptidase N and endopeptidase 24.15 and were completely blocked by selective enzyme inhibitors (Montiel et al., 1997; Noble and Roques, 1997). N/OFQ metabolism has also been studied in different tissues (for review, see Terenius et al., 2000). There are many reports on the pharmacological activity of N-terminal fragments of N/OFQ in vivo (Mathis et al., 1998; Sakurada et al., 1999, 2000; Suder et al., 1999). In these reports, the N-terminal fragments, including N/OFQ (1-7) and (1-11), showed antinociceptive activity to N/OFQ-induced nociception, but no nociceptive activation by themselves. Thus, N-terminal fragments may modulate the action of N/OFQ. On the other hand, although it is postulated that the C terminus of N/OFQ plays a less critical role in recognition by the ORL1 receptor than its N-terminal counterparts (Dooley and Houghten, 1996), there is no report on the pharmacological activity of C-terminal fragments of N/OFQ.

Most recently, we reported that N/OFQ at extremely low doses elicited a nociception through substance P (SP) release from nociceptor endings, and that wide ranges of N/OFQ doses produced a typical bell-shaped dose-response relationship both in peripheral and central nociception tests (Inoue et al., 1999). Taking into account that N/OFQ is metabolized by aminopeptidase N and endopeptidase 24.15 to form five main fragments in mouse brain cortical slices and that N-terminal fragments have some pharmacological activity in vivo, the C-terminal fragments could be expected to exert some physiological and pharmacological actions. In this study, we attempted to clarify the pharmacological activities of C-terminal fragments and especially of N/OFQ (13-17), formed after enzymatic cleavage of N/OFQ.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. Male ddY mice or mutant mice weighing 20 to 22 g were used. Mutant mice were homozygotes (NOPR^/) lacking the genomic NOPR gene and its wild-type (NOPR^+/+) counterpart, which have been developed previously (Nishi et al., 1997). The mice were housed in a group of three to four per cage. They were kept in a room maintained at 21 ± 2°C with free access to a standard laboratory diet and tap water. Procedures were approved by Nagasaki University Animal Care Committee and complied with the recommendations of the International Association for the Study of Pain (Zimmermann, 1983).

Drugs. The following drugs were used: N/OFQ (Sawady Technology, Tokyo, Japan), SP (Peptide Institute, Osaka, Japan), capsaicin (Nacalai Tesque, Kyoto, Japan), and pertussis toxin (PTX; Funakoshi, Tokyo, Japan). CP-99994 and CP-100263 were generously provided by Pfizer Central Research (Sandwich, Kent, UK). N/OFQ fragments N/OFQ (1-7), (2-7), (8-17), (12-17), (13-17), (14-17), (15-17), and (16-17) were a generous gift from Dr. Jun Sasaki (Asahi Glass Co., Yokohama, Japan). These peptides were synthesized by solid-phase peptide methodology and purified by high-performance liquid chromatography. N/OFQ, its fragments, SP, PTX, CP-99994, and CP-100263 were dissolved in physiological saline, whereas capsaicin was dissolved in 10% ethanol and 10% Tween 80 in physiological saline.

Evaluation of Nociceptive Flexor Responses All experiments were performed in compliance with the relevant laws and institutional guidelines. Nociceptive flexor responses induced by intraplantar injection (2 µl) of nociceptive substances were evaluated in mice as previously described (Inoue et al., 1998; Ueda and Inoue, 2000; Ueda et al., 2001). Briefly, mice were lightly anesthetized with ether and held in a cloth sling with their four limbs hanging free through holes. The sling was suspended on a metal bar. All limbs were tied with soft thread strings. Then three limbs were fixed to the floor, while the other one (right hind limb) was connected to an isotonic transducer and recorder. Two polyethylene cannulas (0.61 mm in outer diameter) filled with drug solution were connected to separate microsyringes. One cannula was filled with SP or saline and the other was filled with tests drugs. All experiments were started after complete recovery (20-30 min) from the light ether anesthesia and when intraplantar (i.pl.) injection of saline did not show any significant flexor responses. The nociceptive activity of different test drugs was represented as the percentage of maximal reflex. The biggest response among the spontaneous and nonspecific flexor responses occurred immediately following cannulation was considered as maximal reflex. In most experiments with N/OFQ (or fragments), we evaluated the average of flexor responses induced by two consecutive challenges per each dose, and at most four different doses were tested in each mouse. In some experiments, the antinociceptive effect of N/OFQ or N/OFQ (13-17) was expressed as the ratio of the response observed over the average of two repeated control SP-induced responses in the beginning of the experiments (i.e., as percentage of control response). In this case, SP was given i.pl. at 10 and 5 min prior to and 5, 10, 20, and 30 min after N/OFQ or N/OFQ (13-17) injection. The median effective dose (ED₅₀) was calculated from the linear regression curve of the percentage of maximal reflex response against log i.pl. dose of N/OFQ or its fragments.

Evaluation of Nociceptive Scratching, Biting, and Licking (SBL) Responses. The nociceptive responses characterized by reciprocal hind limb scratching and caudally directed biting and licking (SBL behavior) observed after intrathecal (i.t.) injection of N/OFQ and its fragments were measured as reported elsewhere (Hylden and Wilcox, 1981, 1983). The intrathecal injections were performed according to the method of Hylden and Wilcox (1980). All drugs were given slowly in a volume of 5 µl. One hour prior to i.t. injection, animals were adapted to an individual plastic cage, which also served as the observation chamber. Neurokinin 1 (NK1) receptor antagonist or saline was injected (i.t.) 5 min prior to the injection of N/OFQ (13-17). All animals were used for only one experiment by the observer who did not know what kind of pretreatment had been given.

Capsaicin Treatment. Capsaicin was injected subcutaneously into the back of newborn (P4) ddY mice at a dose of 50 mg/kg. This treatment is known to cause a degeneration of small-diameter afferent sensory neurons (Hiura and Ishizuka, 1989). As a control, vehicle (10% ethanol and 10% Tween 80 in physiological saline) used for dissolving capsaicin was injected into mice. For the nociception tests, capsaicin- or vehicle-treated mice weighing 20 to 22 g were used. No gross behavioral changes were observed in such treated mice.

Generation of Recombinant Human NOPR Baculovirus with Recombinant G Proteins and in Vitro [³⁵S]GTPS Binding. The NOPR baculovirus was constructed as follows. The NotI fragment containing the human NOPR coding region was excised from pThNOPR and was inserted at NotI sites of pFASTBac1 (pFhNOPR). The pFhNOPR was transformed into DH10Bac for transposition of human NOPR plasmid to a bacmid. The recombinant bacmid DNA was transfected by using CELLFECTIN reagent (Invitrogen, Carlsbad, CA) into Spodoptera frugiperda (sf21) cells, which had been seeded at a density of 9 × 10⁵ cells/well on a six-well plate with EX-CELL 400-GM medium (JRH Biosciences, Lenexa, KS) and incubated in EX-CELL 400 medium with penicillin and streptomycin for 1 h at 28°C. The generated recombinant virus was amplified by infection and the amplified virus (1 × 10⁸ plaque-forming units/ml) was stored at 4°C. Sf21 cells (1.0 × 10⁷ cells) were infected with recombinant viruses at a multiplicity of infection of 5 for ORL1 and G₁/₂- and G_i1-subunits. Baculoviruses for these G protein subunits have been prepared, as previously reported (Yoshida and Ueda, 1999). Cells were harvested ~2 to 3 days after infection at 27°C. For membrane preparation, the sf21 cells were washed once and homogenized using a glass-Teflon homogenizer in 1 ml of TES buffer (0.32 M sucrose, 0.1 mM EDTA, 25 mM Tris-HCl, pH 7.5) and centrifuged at 17,600g for 40 min at 4°C. The pellet was resuspended with buffer A (50 mM HEPES-KOH, 1 mM EGTA, 1 mM dithiothreitol, 100 mM NaCl, and 5 mM MgCl₂, pH 7.4) and centrifuged at 400g for 5 min. The supernatant was stored on ice until use. In vitro [³⁵S]GTPS binding assays were essentially carried out as reported previously (Traynor and Nahorski, 1995; Yoshida and Ueda, 1999).

Statistical Analysis. Statistical evaluations were performed using Student's t test after one-way analysis of variance performing Bonferroni's test. In the time course experiments, statistical evaluations were also performed using Student's t test after one-way analysis of variance at each time (5, 10, 20, or 30 min) after drug application. Data are expressed as mean ± S.E.M. Significance was established at *p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Peripheral Nociceptive Flexor Responses Produced by N/OFQ and Its Fragments. We synthesized eight fragments of N/OFQ and tested the biological activity of these fragments in the in vivo experiments (Table 1). When N/OFQ was injected into the plantar surface (i.pl.) of mouse hind limb, it induced very short-lasting but constant nociceptive flexor responses upon repeated challenges, as previously reported (Inoue et al., 1998). N/OFQ-induced nociceptive responses were dose-dependent in a wide range of doses from 10 amol to 10 fmol (i.pl.), as shown in Fig. 1. The median effective dose, ED₅₀ (±S.E.M.), was 0.38 ± 0.08 fmol (n = 6), as shown in Table 1. Similar dose-dependent nociceptive responses were also observed with C-terminal fragments N/OFQ (13-17) and N/OFQ (12-17) (Fig. 1). However, N/OFQ (13-17) was about 20 times more potent than N/OFQ. As shown in Table 1, N/OFQ (8-17) also showed weak nociceptive responses with an ED₅₀ of 4706.2 ± 366.9 fmol (n = 6). On the other hand, N-terminal fragments, N/OFQ (1-7), and N/OFQ (2-7) showed no significant nociceptive responses even at a dose of 100,000 times higher (100 pmol) than the nociceptive dose of N/OFQ (1 fmol), as shown in Fig. 1. Furthermore, we synthesized more deleted C-terminal fragments, N/OFQ (14-17), (15-17), and (16-17) to identify the critical amino acid sequence responsible for the potent nociceptive activity of N/OFQ (13-17). However, N/OFQ (14-17) showed only weak nociceptive activity, whereas N/OFQ (15-17) and N/OFQ (16-17) showed no significant nociceptive activity in the nociceptor endings (Table 1). These results suggest that the C-terminal five amino acids, KLANQ, are necessary to cause the potent nociception of N/OFQ (13-17).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Nociceptive flexor responses induced by N/OFQ and its C-terminal fragments but not by N-terminal fragments. N/OFQ and its C-terminal fragments induced nociception over a wide range of doses, whereas the N-terminal fragments were unable to produce any nociception even at higher doses. Results represent the percentage of maximal reflex. Details are described under Materials and Methods. All data are the mean ± S.E.M. from six separate experiments.

Blockade by Pertussis Toxin and a Neurokinin 1 Receptor Antagonist of Peripheral Nociception Induced by N/OFQ (13-17). As in the case with N/OFQ (Fig. 2B), when i.pl. injection of 10 ng/2 µl of PTX was given after the second N/OFQ (13-17) challenge, the following N/OFQ (13-17)-induced responses rapidly attenuated, and complete loss of N/OFQ (13-17) responses was observed in 10 min after the PTX treatment (Fig. 2A). Furthermore, we found that N/OFQ (13-17) given i.pl. exerts nociceptive responses through an SP release from nociceptor endings. As shown in Fig. 2C, N/OFQ (13-17) (10 amol i.pl.)-induced nociceptive flexor responses were blocked by 1 pmol of CP-99994, a neurokinin 1 receptor antagonist (McLean et al., 1993), but not by 1 pmol of CP-100263, an inactive isomer, which is similar to the case with N/OFQ (Fig. 2D). These findings suggest that both N/OFQ and N/OFQ (13-17) have similar mechanisms as amplification through local SP release from nociceptors.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Antagonism by pertussis toxin or NK1 receptor antagonist of both N/OFQ (13-17)- and N/OFQ-induced nociception. A and C, effects on N/OFQ (13-17)-induced nociception. Results represent the percentage of control responses produced by 10 amol (i.pl.) of N/OFQ (13-17) at various periods after the challenge of 10 ng of PTX (A) or 1 pmol of CP-99994 or its inactive isomer CP-100263 (C). Saline was treated instead of toxin or antagonist as a control (Veh). Abscissa represents the time after the toxin or antagonist challenge. B and D, effects on N/OFQ-induced nociception. Results represent the data obtained 30 min after the toxin or antagonist (or saline) challenge. Toxin or antagonist was given i.pl. through another cannula 5 min after the twice control N/OFQ (13-17) or N/OFQ challenges. *p < 0.05. Each point is the mean ± S.E.M. from five to eight separate experiments.

N/OFQ- and N/OFQ (13-17)-Induced Nociceptive and Antinociceptive Actions through Nociceptors. As shown in Fig. 3A, N/OFQ-induced nociceptive responses were dose-dependent in a wide range of doses from 0.01 to 100 fmol (i.pl.), whereas they started declining from 1 pmol to 1 nmol (i.pl.). N/OFQ (13-17) also produced a typical bell-shaped dose-response relationship in a wide range of doses in the peripheral nociceptors (Fig. 3A). Again, when N/OFQ (13-17) was given i.pl. at a dose of 1 nmol 5 min after the twice challenge of SP (10 pmol i.pl.), the following SP-induced nociceptive responses were rapidly attenuated and were completely abolished at 10 min after the N/OFQ (13-17) challenge (Fig. 3B). This antinociception against SP-induced nociception in the nociceptors was more potent than that in the case with N/OFQ (Fig. 3B). Thus, the bell-shaped dose-response relationship observed with N/OFQ (13-17) in our experiments may be due to its dose-related opposite modulatory effects on SP-induced nociception in nociceptor endings like N/OFQ as reported previously (Inoue et al., 1999).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Nociceptive and antinociceptive effects of N/OFQ (13-17) and N/OFQ in the peripheral nociceptive flexor test. A, dose-response curves of N/OFQ (13-17)- and N/OFQ-induced flexor responses in ddY mice. Results represent the percentage of each N/OFQ (13-17)- (or N/OFQ)-induced response to the maximal reflex as described under Materials and Methods. B, time course for the inhibitory effects of N/OFQ (13-17) or N/OFQ at 1-nmol (i.pl.) doses on SP (10 pmol)-induced flexor responses in ddY mice. SP challenges for control responses were performed twice at 5-min interval, followed immediately by N/OFQ (13-17) or N/OFQ injection through another cannula. The results represent the percentage ratio of SP response at the indicated time after N/OFQ (13-17) or N/OFQ injection to the average of control SP responses obtained at the beginning of each experiment. *p < 0.05, compared with vehicle-treated mice. Note: data for N/OFQ were reproduced from our earlier observations (Inoue et al., 1999) for the purpose of comparison with N/OFQ (13-17).

Lack of NOPR Involvement in N/OFQ (13-17)-Induced Responses. The responses to both N/OFQ and N/OFQ (13-17) were similar in wild-type (NOPR^+/+) (Fig. 4A) and in ddY mice used in the other experiments (Fig. 1). Although N/OFQ-induced responses were completely lost in mice lacking the NOPR gene (NOPR^/) at all doses tested (0.1-10 fmol), the N/OFQ (13-17)-induced responses were not affected in these mice (Fig. 4A). This result suggests that N/OFQ (13-17)-induced nociceptive actions were mediated by a novel mechanism independent of activation of NOPR. This finding was further confirmed by in vitro [³⁵S]GTPS binding experiments with baculovirus/sf21 cells expressing NOPR together with G protein _i1-, ₁-, and ₂-subunits. As shown in Fig. 4B, N/OFQ at 10 nM to 10 µM stimulated the [³⁵S]GTPS binding in a concentration-dependent manner. However, N/OFQ (13-17) at a concentration 10 µM showed no significant binding activity in such membrane preparations. Together, these findings suggest that N/OFQ (13-17) does not have an affinity for NOPR.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Lack of NOPR involvement in N/OFQ (13-17)-induced responses. A, dose-response curves of N/OFQ (13-17)- and N/OFQ-induced flexor responses in NOPR knockout mice in the peripheral nociception test. Results represent the percentage of maximal reflex. *p < 0.05. B, N/OFQ but not N/OFQ (13-17) stimulated [35S]GTPS binding to membranes containing NOPR with G protein subunits in the baculovirus/sf21 cell expression system. Each point is the mean ± S.E.M. of the data from five separate experiments in triplicate.

N/OFQ (13-17) (i.t.)-Induced Nociceptive SBL Responses through an SP Release from Spinal Synapses. As we reported previously (Inoue et al., 1999), N/OFQ given intrathecally produces nociceptive activities characterized by SBL behaviors through SP release from spinal synapses. Thus, it is expected that N/OFQ (13-17) may also induce nociceptive SBL responses through an SP release. Accordingly, N/OFQ (13-17) was given i.t. to see such SBL responses. As shown in Fig. 5A, N/OFQ (13-17) showed dose-dependent SBL responses in a wide range of doses between 0.03 and 30 amol, which were about 100 times lower than the SBL nociceptive doses of N/OFQ. Furthermore, another C-terminal fragment N/OFQ (12-17) also showed dose-dependent SBL responses (Fig. 5A). Similar to the case with peripheral nociceptive responses, 3 amol of N/OFQ (13-17) (i.t.)-induced SBL responses were blocked by CP-99994 (1 nmol i.t.), but not by the inactive isomer CP-100263 (1 nmol i.t.), as shown in Fig. 5B. This finding was further supported by experiments with capsaicin-treated mice, where small-diameter SP-containing primary afferent fibers are degenerated (Hiura and Ishizuka, 1989; Inoue et al., 1999). In such mice, 3 amol of N/OFQ (13-17)-induced SBL responses were almost completely abolished (Fig. 5C). Again, N/OFQ showed a bell-shaped dose-response relationship in this central nociception test, whereas N/OFQ (13-17) did not produce such a dose-response curve (Fig. 5A). Together, these findings suggest that N/OFQ (13-17) has no effect in the postsynapses of the spinal cord.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Nociceptive effects of N/OFQ (13-17) and N/OFQ evaluated as SBL responses. A, dose-response curves of N/OFQ (13-17), N/OFQ (12-17), and N/OFQ-induced SBL responses in ddY mice. Each peptide was administered i.t. in a volume of 5 µl into mice. B and C, N/OFQ (13-17)-induced SBL responses through an SP release. B, blockade of N/OFQ (13-17) (3 amol i.t.)-induced SBL responses by NK1 receptor antagonist in ddY mice. Saline (Veh, 5 µl), CP-99994 (1 nmol), or CP-100263 (1 nmol) was administered i.t. 5 min before N/OFQ (13-17) injection (i.t.). Data are the mean ± S.E.M. from six to eight separate experiments. *p < 0.05, compared with Veh. C, loss of N/OFQ (13-17) (3 amol i.t.)-induced SBL responses in capsaicin-treated (+) ddY mice. Data are the mean ± S.E.M. from six to eight separate experiments. *p < 0.05, compared with capsaicin-untreated mice ().

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

N/OFQ has been reported to be metabolized by aminopeptidase N and endopeptidase 24.15 in mouse brain cortical slices, forming five main fragments, N/OFQ (1-7), (2-17), (1-11), (12-17), and (13-17) (Montiel et al., 1997). Although there are many reports on the pharmacological activities of N-terminal fragments of N/OFQ in vivo (Mathis et al., 1998; Sakurada et al., 1999, 2000; Suder et al., 1999), there are no reports on the activity of the C-terminal fragments of N/OFQ. Here, we attempted to clarify the pharmacological activities of the C-terminal fragments of N/OFQ, formed after enzymatic cleavage. The present report demonstrated that C-terminal fragments of N/OFQ possess nociceptive activity in peripheral nociceptors as well as in spinal cord in mice. Furthermore, we found that C-terminal fragment N/OFQ (13-17) induced an antinociception in the peripheral nociceptors like N/OFQ.

Recently, we reported that i.pl. application of N/OFQ at extremely low doses elicited nociceptive responses through an SP release from nociceptor endings via the activation of G_i and phospholipase C (PLC) and that N/OFQ may work in the amplification of pain signaling (Inoue et al., 1998). In the present study, i.pl. administration of C-terminal fragments of N/OFQ, N/OFQ (8-17), (12-17), (13-17), and (14-17) elicited nociceptive actions (Fig. 1; Table 1). Of the C-terminal fragments, N/OFQ (13-17), was found to be most potent and the potency was about 20 times higher than that of N/OFQ (Table 1). Like N/OFQ, N/OFQ (13-17)-induced nociception was completely abolished by PTX, indicating the involvement of G_i in this signaling mechanism. Moreover, NK1 receptor antagonist CP-99994 effectively blocked the nociception induced by i.pl. application of N/OFQ (13-17), suggesting that N/OFQ (13-17) may elicit the nociceptive responses through an SP release from nociceptor endings via activation of G_i. Because the potency of N/OFQ (13-17) was extremely high compared with that of SP (ED₅₀ = 1350 ± 340 fmol) in nerve endings (Inoue et al., 1998), we are speculating the working hypothesis that like N/OFQ, one molecule of N/OFQ (13-17) may release many molecules of SP and that this mechanism works as an amplification in pain signaling. And N/OFQ (13-17) might be more sensitive to this amplification mechanism than N/OFQ. In contrast, the N-terminal fragments of N/OFQ, N/OFQ (1-7) and (2-7), showed no significant nociception (Fig. 1; Table 1). These data are consistent with the reports where N/OFQ N-terminal fragments did not have any significant effect by themselves or on SP-induced nociception (King et al., 1997; Sakurada et al., 1999). Similar to the case with N/OFQ as reported previously (Inoue et al., 1999), in the present study wide ranges of intraplantar N/OFQ (13-17) doses also produced a typical bell-shaped dose-response relationship in the nociceptor endings (Fig. 3A) and this peptide fragment at higher doses also inhibited the SP-induced nociception (Fig. 3B). Recently, we reported that N/OFQ produced a dose-related opposite modulatory effect of SP nociception in nociceptors and spinal cord (Inoue et al., 1999). In that report, we proposed the hypothesis that the antinociceptive signaling at higher doses of N/OFQ may be due to release of -subunits from G_i/o through NOPR, inhibiting the production of free G_q/11 through the NK1 receptor as postreceptor cross talk. This possibility was based on the fact that the stoichiometry of receptor/G_q/11 coupling is quite low compared with that of receptor/G_i/o (Pang and Sternweis, 1990). Alternatively, G_i possesses a weaker activity to stimulate PLC and may competitively inhibit G_q, which possesses a stronger affinity for PLC, as reported elsewhere (Ueda et al., 1995). However, we do not know whether the N/OFQ (13-17) fragment also shared a similar mechanism with N/OFQ in producing the bell-shaped dose-response relationship at the nociceptors.

We also demonstrate that the N/OFQ (13-17)-induced nociception was not mediated through activation of NOPR. In our experiment, N/OFQ (13-17)-induced nociception was unaffected in NOPR knockout mice, although the N/OFQ-induced ones were completely abolished in such mice (Fig. 4A). This finding was also confirmed in the in vitro [³⁵S]GTPS binding experiments. N/OFQ showed significant binding activity in baculovirus/sf21 cells expressing NOPR together with G protein _i1-, ₁-, and ₂-subunits in a concentration-dependent manner, whereas N/OFQ (13-17) showed no significant stimulation (Fig. 4B). Similar to N/OFQ (13-17), N/OFQ (12-17) also showed no significant binding activity in such cells (data not shown). These data are consist with previous findings that NOPR requires the N-terminal tetrapeptide Phe-Gly-Gly-Phe for its activation (Guerrini et al., 1997). It has also been reported that the amidated form of N/OFQ and N/OFQ (1-13) can activate the NOPR with similar potencies as the natural ligand in both in vivo and in vitro experiments (Calo et al., 2000). In addition, the amidated form of the other N-terminal fragments N/OFQ (1-12), N/OFQ (1-11), and N/OFQ (1-9) have significant binding affinity for NOPR expressed in Chinese hamster ovary cells (Guerrini et al., 2000). However, there is no report on the functional affinities of the C-terminal fragment N/OFQ (13-17) on the ORL1 receptor. In our experiments, the presence of almost all the nociceptive activities of N/OFQ (13-17) in NOPR knockout mice indicates that all the metabolic fragments of N/OFQ are unlikely to contribute to the effects of the N/OFQ, and that N/OFQ (13-17) may give its actions through a novel mechanism independent of the activation of NOPR.

Moreover, we examined the pharmacological activities of the C-terminal fragments in spinal cord level. In the present study measuring nociceptive responses characterized by SBL behaviors, N/OFQ (13-17) administered intrathecally produced potent nociceptive actions (Fig. 5A). N/OFQ (12-17) also induced nociceptive SBL responses, and the potency of this peptide was between that of N/OFQ (13-17) and N/OFQ (Fig. 5A), just like the cases with peripheral nociceptive responses. Similar to peripheral mechanism, N/OFQ (13-17)-induced SBL responses were found to be mediated through an SP release from spinal primary afferent terminals because they were abolished by NK1 receptor antagonist (Fig. 5B), and in capsaicin-treated mice (Fig. 5C). However, in contrast to the case with nociceptor endings, a wide range of N/OFQ (13-17) doses did not follow a typical bell-shaped dose-response relationship in the spinal cord, whereas a wide range of N/OFQ doses did (Fig. 5A). We previously reported that N/OFQ inhibited the SP-induced nociception in the capsaicin-treated mice, which are devoid of presynaptic mechanisms, indicating that the postsynaptic mechanisms contribute to the antinociceptive actions of N/OFQ administered centrally (Inoue et al., 1999). The fact that spinal N/OFQ (13-17) was unable to produce a bell-shaped dose-response relationship and that the N/OFQ (13-17)-induced SBL responses were abolished in capsaicin-treated mice indicates that the physiological and pharmacological activities of N/OFQ (13-17) and N/OFQ in the spinal cord level are mediated through different mechanisms. It also raises the possibility that the N/OFQ (13-17) does not act on the postsynapse in spinal cord, and that N/OFQ (13-17)-mediated central action is only related to SP release from the spinal primary afferent terminals (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Schematic model of in vivo pain regulatory role of N/OFQ (13-17) in spinal dorsal horn. Details are described in the text.

In summary, the present results suggest that the C-terminal fragments of N/OFQ possess potent pronociceptive activities, and N/OFQ (13-17) is the most potent among them. We also demonstrate that N/OFQ (13-17) elicits its actions through a novel mechanism independent of the activation of NOPR. Further biochemical studies, including binding experiments for the putative targets of these fragments, should be the next step.