Introduction

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Nociceptin (NC)/orphanin FQ is the endogenous ligand of a seven-transmembrane domain G protein-coupled receptor, referred to as OP₄. NC and the OP₄ receptor are structurally related to opioid peptides and receptors; however, NC does not interact with classical opioid receptors, and the OP₄ receptor does not bind any selective opioid receptor ligand (for recent reviews, see Calo' et al., 2000c; Meunier, 2000). It has been demonstrated that NC modulates several biological functions, including pain threshold, morphine analgesia, food intake, anxiety, locomotor activity, memory processes, and cardiovascular, renal, respiratory, and gastrointestinal functions (Calo' et al., 2000c; Meunier, 2000). Further investigations of physiological and pathological roles of the NC/OP₄ system require new molecules that potently activate (agonists) or block (antagonists) the OP₄ receptor. Identification of such new molecules (at least of peptide nature) should be facilitated by the knowledge of the amino acid residues of the NC sequence that are critical for receptor occupation and activation. Over the last 5 years, several structure-activity relationship studies have been performed on NC (Dooley and Houghten, 1996; Reinscheid et al., 1996; Butour et al., 1997; Guerrini et al., 1997; Calo' et al., 1998a; Guerrini et al., 2000a). These studies suggested that Phe¹ and Phe⁴ represent the critical residues of the message domain of NC (Phe¹-Gly²-Gly³-Phe⁴), which should be involved in receptor binding and activation, whereas the positively charged residues that are present in the address domain of the molecule (Arg^8,12, Lys^9,13) appear to be crucial for receptor occupation. Indeed, they are expected to interact with the acidic amino acids that are present in the second extracellular loop of the OP₄ receptor (for a review, see Guerrini et al., 2000b). The importance of positively charged amino acids was further supported by the fact that the OP₄ receptor ligands, identified by screening of peptide combinatorial libraries (Dooley et al., 1997; Becker et al., 1999), are enriched in cationic (Arg/Lys) residues.

Based on these considerations, Okada et al. (2000) recently synthesized analogs in which a Arg-Lys dipeptide was introduced in positions 6-7, 10-11, or 14-15 of NC. Among these analogs, [Arg¹⁴,Lys¹⁵]NC was found to be 3- and 17-fold more potent than NC in binding and functional assays performed on cells expressing the human recombinant OP₄ receptor (Okada et al., 2000).

In the present study, we further characterized the action of [Arg¹⁴,Lys¹⁵]NC in vitro on native OP₄ receptors expressed in isolated tissues from various species. To pharmacologically characterize the effects of [Arg¹⁴,Lys¹⁵]NC, we used the selective OP₄ receptor antagonists [Nphe¹]NC(1-13)NH₂ (Calo' et al., 2000b; Guerrini et al., 2000a), J-113397 (Kawamoto et al., 1999), and the nonselective opioid receptor antagonist naloxone. The effects of [Arg¹⁴,Lys¹⁵]NC were also evaluated in vivo, in the locomotor activity and in the tail-withdrawal assays after i.c.v. injection of NC, which has been shown to inhibit motility (Rizzi et al., 2001) and evoke pronociceptive effects (Calo' et al., 1998b) in the mouse.

	Materials and Methods

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In Vitro Studies. Tissues were taken from male Swiss mice (30-35 g), albino guinea pigs (300-350 g), and Sprague-Dawley rats (300-350 g). The mouse vas deferens and colon, the guinea pig ileum, and the rat vas deferens were prepared as previously described (Bigoni et al., 1999; Rizzi et al., 1999). The mouse vas deferens, guinea pig ileum, and rat vas deferens were continuously stimulated through two platinum ring electrodes with supramaximal voltage rectangular pulses of 1-ms duration and 0.05 Hz frequency. The electrically evoked contractions (twitches) were measured isotonically with a strain gauge transducer [Basile 7006; Ugo Basile Biological Research Apparatus, Comerio (VA), Italy] and recorded with the personal computer-based acquisition system Autotrace 2.2 (RCS, Florence, Italy). After an equilibration period of about 60 min, the contractions induced by electrical field stimulation were stable; at this time, cumulative concentration-response curves to NC or [Arg¹⁴,Lys¹⁵]NC were performed (0.5 log-unit step) in the absence or in the presence of [Nphe¹]NC(1-13)NH₂ (10 µM), J-113397 (0.1 µM), or naloxone (1 µM), added to the medium 15 min before performing concentration-response curve of the agonists. In one series of experiments, in the rat vas deferens, concentration-response curves to NC and [Arg¹⁴,Lys¹⁵]NC were performed in the absence and in the presence of a mixture of peptidase inhibitors (amastatin, bestatin, phosphoramidon, and captopril at the final concentration of 30 µM), incubated for 30 min.

In Vivo Studies. Male Swiss albino mice weighing 20 to 25 g were used. The animals were handled according to guidelines published in the European Communities Council directives (86/609/EEC). They were housed under standard conditions (22°C, 12-h light/dark cycle) with food and water ad libitum for at least 2 days before experiments began. Each animal was used once. Intracerebroventricular injections (2 µl/mouse) were given in the left ventricle, according to the procedure described by Laursen and Belknap (1986).

Locomotor Activity Assay. Experiments were carried out between 2:00 and 6:00 PM, following the procedure described by Rizzi et al. (2001). Briefly, the animals were routinely tested 3 min after i.c.v. injection. Locomotor activity was assessed using Basile activity cages, which consist of a four-channel resistance detector circuit that converts the bridges "broken" by the animal paws into pulses that are summed up by an electronic counter every 5 min. Total number of impulses was recorded every 5 min for 30 min. Animals were not accustomed to the cages before drug treatments and the experiment was performed in a quiet and dimly illuminated room. For each experiment, three mice were randomly assigned to each treatment (three mice per drug dose) and the experiments were repeated at least five times; therefore, each experimental point is the mean of the results obtained in 15 mice.

Tail-Withdrawal Assay. All experiments were started at 10:00 AM and performed according to the procedure described by Calo' et al. (1998b). Briefly, the animals were placed in a holder and the distal half of the tail was immersed in water at 48°C; the withdrawal latency time was measured by an experienced observer blind to drug treatment. A cutoff time of 20 s was chosen to avoid tissue damage. Four mice were randomly assigned to each experimental group. Tail-withdrawal time was determined immediately before and 5, 15, 30, and 60 min after i.c.v. injection of 2 µl of saline (control), NC, or [Arg¹⁴,Lys¹⁵]NC.

Drugs. The peptides used in this study were prepared and purified as previously described (Guerrini et al., 1997). J-113397 was prepared as a racemic mixture, according to Bigoni et al. (2000); naloxone was obtained from Tocris Cookson (Bristol, UK). The peptidase inhibitors amastatin, bestatin, phosphoramidon, and captopril (from Sigma, Milan, Italy) were solubilized in water and stocked at 10 mM concentrations. For in vitro experiments, the compounds were solubilized in physiological solution, and stock solutions (1 mM) were kept at 20°C until use; for in vivo studies, the substances were solubilized in physiological medium just before performing the experiment.

Data Analysis and Terminology. All data are expressed as means ± standard error of the mean of n experiments. Data have been statistically analyzed with Student's t test or one-way ANOVA followed by the Dunnett test, as specified in table and figure legends; p values less than 0.05 were considered to be significant. The pharmacological terminology adopted in this paper is consistent with the International Union of Pharmacology recommendations (Jenkinson et al., 1995). The agonist potencies were measured as pEC₅₀, which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. The E_max is the maximal effect that an agonist can elicit in a given tissue. In the electrically stimulated tissues, the E_max of agonists is expressed as percentage of inhibition of the control twitch, whereas in the mouse colon it is expressed as percentage of the contraction elicited by 10 µM carbachol. Antagonist potencies are expressed in terms of pA₂, which is the negative logarithm to base 10 of the antagonist molar concentration that makes it necessary to double the agonist concentration to elicit the original submaximal response. pA₂ values of antagonists were calculated by Schild analysis in the mouse vas deferens and, in the other preparations, with the Gaddum Schild equation pA₂ = log((CR-1)/[Antagonist]), assuming a slope equal to unity. CR is the ratio of the EC₅₀ of an agonist in the presence and in the absence of an antagonist.

	Results

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In Vitro Studies. NC and [Arg¹⁴,Lys¹⁵]NC inhibited in a concentration-dependent manner the electrically induced contraction of the mouse vas deferens with similar efficacy (E_max about 90% inhibition control twitch) but different potencies. Indeed, [Arg¹⁴,Lys¹⁵]NC was 17-fold more potent than NC (Fig. 1). The effects of the NC analog were also tested in other preparations, such as the guinea pig ileum and rat vas deferens, in which NC inhibits the twitch response to electrical field stimulation (Bigoni et al., 1999), and in the mouse colon, in which NC induces concentration-dependent contractions (Osinski et al., 1999). In all these tissues, [Arg¹⁴,Lys¹⁵]NC elicited similar maximal effects to NC but displayed higher potency by 5-fold in the guinea pig ileum and mouse colon and by 10-fold in the rat vas deferens (Table 1). In these isolated tissues, the kinetics of action of NC and [Arg¹⁴,Lys¹⁵]NC were similar; in fact, the effects of both peptides took place immediately after adding the peptide to the bath were rapidly reversible after washing, and could be repeated in the same tissue (data not shown), thus demonstrating lack of tachyphylaxis.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of NC and [Arg14,Lys15]NC in the electrically stimulated mouse vas deferens. Points indicate the means and vertical lines indicate the S.E.M. of at least four experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Left panels, effects of [Nphe1]NC(1-13)NH2 and J-113397 versus [Arg14,Lys15]NC in the electrically stimulated mouse vas deferens. Right panels, Schild plots. CR is the ratio of the EC50 of an agonist in the presence and in the absence of an agonist. Points indicate the means, and vertical lines indicate the S.E.M. of at least five experiments.

In Vivo Studies. [Arg¹⁴,Lys¹⁵]NC was administered i.c.v. in mice at doses ranging from 0.01 to 0.3 nmol. Intracerebroventricular injections of [Arg¹⁴,Lys¹⁵]NC at 0.01 nmol did not induce any obvious behavioral effects. In contrast, mice injected with 0.1 and 0.3 nmol showed a decrease in locomotor activity and muscular tone (especially in the hindpaws), ataxia, and loss of the righting reflex; however, a normal tail-pinch reflex was maintained. These behavioral effects of [Arg¹⁴,Lys¹⁵]NC were very similar to those evoked by high doses (i.e., 10 nmol) of NC, as reported by several investigators (Reinscheid et al., 1995; Devine et al., 1996; Noble and Roques, 1997; Calo' et al., 1998b). However, whereas the effects of NC (10 nmol) were fully reversible after 90 min, those elicited by [Arg¹⁴,Lys¹⁵]NC (0.3 nmol) were evident for several hours. Indeed, more than 6 h were needed for normal behavior to return in [Arg¹⁴,Lys¹⁵]NC-treated animals.

Locomotor Activity Assay. As shown in Fig. 3, mice treated with saline (2 µl/mouse, i.c.v.) displayed a progressive reduction in spontaneous locomotor activity from 195 ± 17 impulses/5 min to 51 ± 8 impulses/5 min during the 30 min of the experiment. [Arg¹⁴,Lys¹⁵]NC (0.01-0.3 nmol) caused a dose-dependent reduction of locomotor activity compared with saline. This effect was statistically significant for 0.1 and 0.3 nmol (p < 0.05 according to ANOVA followed by the Dunnett test). In the same series of experiments, the effect of 1 nmol of NC was evaluated; as reported previously (Rizzi et al., 2001), at this dose the peptide caused a significant inhibition of locomotor activity only in the first 15 min after i.c.v. injection (data not shown). In the insert of Fig. 3, the dose-response curve of [Arg¹⁴,Lys¹⁵]NC is compared with that of NC (data taken from Rizzi et al., 2001); [Arg¹⁴,Lys¹⁵]NC was about 30-fold more potent than the natural ligand.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of different doses (0.01-0.3 nmol, i.c.v.) of [Arg14,Lys15]NC on spontaneous locomotor activity in mice. Points indicate the means and vertical lines indicate the S.E.M. of at least six experiments. The effects of [Arg14,Lys15]NC at 0.1 and 0.3 nmol are significantly different from saline, according to ANOVA followed by the Dunnett test. Insert, dose-response curve to NC and [Arg14,Lys15]NC on spontaneous locomotor activity in mice. The data are expressed as percentage of the data obtained in saline-injected animals. Points indicate the means, and vertical lines indicate the S.E.M. of at least six experiments.

Tail-Withdrawal Assay. Results summarized in Fig. 4 show that tail-withdrawal reaction time of saline-injected mice were stable at values around 4 s over the time course of the experiment. [Arg¹⁴,Lys¹⁵]NC was ineffective at 0.01 nmol, but it elicited a statistically significant pronociceptive effect that peaked at 15 min and lasted for 30 min at 0.1 nmol. [Arg¹⁴,Lys¹⁵]NC produced a long-lasting reduction in tail-withdrawal latencies, which was still evident 1 h after the injection of 0.3 nmol (Fig. 4). In some experiments of this series, we tested the effects of 1 nmol of NC and confirmed previous findings (Calo' et al., 1998b); NC caused a significant reduction of tail-withdrawal latencies in the first 15 min after injection (data not shown). Comparison of the effects of [Arg¹⁴,Lys¹⁵]NC with those elicited by NC under the same experimental conditions (Calo' et al., 1998b) indicate that, also in this assay, [Arg¹⁴,Lys¹⁵]NC is about 30-fold more potent than the natural ligand and produces longer lasting effects.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of [Arg14,Lys15]NC in the dose range of 0.01 to 0.3 nmol on tail-withdrawal latency (TW) in mice. Points indicate the means, and vertical lines indicate the S.E.M. of at least five experiments. The effects of [Arg14,Lys15]NC at 0.1 and 0.3 nmol are significantly different from saline, according to ANOVA followed by the Dunnett test.

	Discussion

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The results presented above demonstrate that [Arg¹⁴,Lys¹⁵]NC behaves as a highly potent and selective agonist of the OP₄ receptor, and its effects in vivo are lasting longer than those of the natural peptide NC.

[Arg¹⁴,Lys¹⁵]NC mimicked the effects of NC by inhibiting in vitro the electrically induced twitches of the mouse vas deferens, rat vas deferens, and guinea pig ileum and by inducing contraction of the mouse colon. In all preparations, the kinetics of action of the two peptides were similar, and their effects were observed immediately after adding the peptide to the bath, were rapidly reversible after washing, and could be repeated several times in the same tissue. In the present study, [Arg¹⁴,Lys¹⁵]NC displayed higher potencies (5 to 17-fold) than NC, in line with the findings by Okada et al. (2000), who measured an increased binding affinity of 3-fold and a potency increment of 17-fold in the guanosine 5' -3-O -(thio)triphosphate binding assay performed on human embryonic kidney 293 cells expressing the recombinant human OP₄ receptor.

To characterize the functional site of action of [Arg¹⁴,Lys¹⁵]NC in isolated tissues, the effects of the compound were measured in the presence of the classical opioid receptor antagonist, naloxone, and in the presence of two selective OP₄ receptor antagonists, [Nphe¹]NC(1-13)NH₂ and J-113397. [Nphe¹]NC(1-13)NH₂ is a peptide OP₄ receptor ligand, the selectivity of action and antagonist behavior of which have been demonstrated in a wide variety of in vitro and in vivo assays (see Calo' et al., 2000a). J-113397 is a nonpeptide compound recently identified by investigators at Banyu as a selective antagonist of the recombinant human OP₄ receptor (Kawamoto et al., 1999). The antagonist properties and selectivity of action of J-113397 have later been confirmed in vitro on the native OP₄ receptor expressed in isolated tissues (Bigoni et al., 2000; Corboz et al., 2000) and in several in vivo studies (Ozaki et al., 2000; Ueda et al., 2000; McLeod et al., 2001). Naloxone (1 µM) was completely inactive against [Arg¹⁴,Lys¹⁵]NC, although both [Nphe¹]NC(1-13)NH₂ and J-113397 competitively antagonized the effects of the NC analog, showing pA₂ values in the range of 5.83 to 6.56 and 7.63 to 8.06, respectively. These pA₂ values are similar to those obtained by testing these antagonists against NC (Rizzi et al., 1999; Bigoni et al., 2000; Calo' et al., 2000b) and therefore demonstrate that the receptor mediating the effects of both NC and [Arg¹⁴,Lys¹⁵]NC is of the OP₄ type.

These data indicate that the addition of two positively charged residues, Arg¹⁴ and Lys¹⁵, to the sequence of NC leads to increased potency, possibly through an increase in receptor affinity. However, Okada et al. (2000) reported that [Arg¹⁴,Lys¹⁵]NC binds to OP₄ receptor sites expressed in human embryonic kidney 293 cells with a K_i value (0.32 nM) that is only 3-fold higher than that of NC (0.93 nM). Therefore, we performed a series of experiments to investigate if other mechanisms, such as differences in susceptibility to enzymatic degradation, may account for the different potencies of [Arg¹⁴,Lys¹⁵]NC and NC. Indeed, a cocktail of peptidase inhibitors produced a statistically significant increase in the potency of NC but not of [Arg¹⁴,Lys¹⁵]NC. The fact that the pEC₅₀ of [Arg¹⁴,Lys¹⁵]NC is not modified by the inhibitors suggests that the compound is more metabolically stable than the natural peptide. This is not surprising because it has been demonstrated that NC can be inactivated by the action of an endopeptidase, which generates the fragments 1-13 and 14-17 (Sandin et al., 1999; Vlaskovska et al., 1999). The addition of positively charged residues (Arg, Lys) in position 14 and 15 may reduce the activity of this enzyme. We therefore suggest that the increased potency of [Arg¹⁴,Lys¹⁵]NC may not only result from increased binding affinity but also from lower susceptibility to enzymatic degradation.

[Arg¹⁴,Lys¹⁵]NC mimicked the effects of NC also in vivo, by inhibiting the locomotor behavior and by producing pronociceptive effects after i.c.v. administration. Moreover, in both the assays, [Arg¹⁴,Lys¹⁵]NC was found to be about 30-fold more potent than NC and to produce long-lasting effects. Indeed, the pronociceptive effects of 0.1 nmol of [Arg¹⁴,Lys¹⁵]NC were similar to those elicited by NC at 10 nmol (Calo' et al., 1998b), and the same can be said for the motor inhibiting action of [Arg¹⁴,Lys¹⁵]NC (0.3 nmol) and NC (10 nmol) (Rizzi et al., 2001). In addition, the pronociceptive effect of [Arg¹⁴,Lys¹⁵]NC (0.3 nmol) lasted more than 60 min, whereas that of the natural peptide (at 10 nmol) persisted for only 30 min (Calo' et al., 1998b). Finally, the general behavioral effects of [Arg¹⁴,Lys¹⁵]NC [decrease in muscular tone (especially in the hindpaws), ataxia, and loss of the righting reflex] were more pronounced and longer lasting than those elicited by NC at 30-fold higher doses. Therefore, the ratio of potency between [Arg¹⁴,Lys¹⁵]NC and NC seems to be higher in vivo than in vitro. This further corroborates the hypothesis that the chemical modifications introduced in the [Arg¹⁴,Lys¹⁵]NC sequence may confer higher metabolic stability to the compound, resulting in the higher potency of the analog in vivo, in which peptidase activity is more relevant.

Taken together, the present data, as well as those of Okada et al. (2000), indicate that [Arg¹⁴,Lys¹⁵]NC is the first OP₄ receptor agonist more potent than the natural ligand NC. Its effects are long-lasting in vivo. This analog can be considered a novel pharmacological tool for the investigation of the neurobiology of the NC/OP₄ receptor system, in particular for clarifying the therapeutic potential of OP₄ receptor agonists as anxiolytics (Jenck et al., 1997), antianorectics (Ciccocioppo et al., 2001), and antitussives (McLeod et al., 2001), or as novel agents to be used for the treatment of drug abuse (Ciccocioppo et al., 2000), cardiovascular (Salis et al., 2000) or renal (Kapusta, 2000) diseases, and urinary incontinence (Lecci et al., 2000; Lazzeri et al., 2001).