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Effects of intracerebroventricularly administered chimeric peptide of metenkephalin and FMRFa—[D-Ala2]YFa—on antinociception and its modulation in mice
Brain Research Bulletin, Vol. 55, No. 1, pp. 51–57, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00490-7
Effects of intracerebroventricularly administered chimeric peptide of Metenkephalin and FMRFa—[D-Ala2]YFa— on antinociception and its modulation in mice Suparna Gupta,1 Santosh Pasha,1* Y. K. Gupta2 and D. K. Bhardwaj3 1
Peptide Laboratory, Centre for Biochemical Technology (CSIR), Delhi, India; 2Department of Pharmacology, All India Institute of Medical Sciences, Delhi, India; and 3Department of Chemistry, University of Delhi, Delhi, India [Received 18 September 2000; Revised 6 February 2001; Accepted 10 March 2001]
ABSTRACT: An enzymatically stable analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of metenkephalin and FMRFa, was synthesised. The antinociceptive effects of intracerebroventricular injections of this analog—[D-Ala2]YAGFMKKKFMRFamide ([D-Ala2]YFa)—was then investigated using the mouse radiantheat tail-flick test. [D-Ala2]YFa produced modest to good antinociception at 1, 2, and 5 g/mouse (0.64, 1.28, and 3.22 nmol, respectively). This antinociceptive effect was completely reversed by the opioid receptor antagonist naloxone (1.5 g/mouse: 4.12 nmol, intracerebroventricular [i.c.v.]), administered 5 min prior. Pretreatment (5 min) with either neuropeptides FF (1 g/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 g/mouse: 1.69 nmol, i.c.v.) significantly attenuated the antinociceptive effects induced by [D-Ala2]YFa (1 g/mouse, i.c.v.). Intracerebroventricular administration of [D-Ala2]YFa at 1 g/mouse dose with morphine (2 g/ mouse: 5.86 nmol, i.c.v.) produced an additive antinociceptive effect, suggesting that [D-Ala2]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation. © 2001 Elsevier Science Inc.
brain mechanisms [17]. NPFF/FMRFa has been found to decrease the tail-flick latency in rats and attenuate the prolongation of the tail-flick latency induced by morphine [28,31], indicating these peptides may be acting as endogenous anti-opioid peptides in rats and mice. Pretreatment with immunoglobulin G from a NPFF antiserum prevents the naloxone-precipitated abstinence syndrome in morphine-dependent rats [16], and it restores the analgesic response to morphine in morphine-tolerant rats [14]. Moreover, the concentration of NPFF in the cerebrospinal fluid of morphinedependent rats is higher than in naı¨ve rats [16]. Though the detailed mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that NPFF can regulate opioid receptor number [6]. Several studies have shown that chronic infusion of NPFF into the lateral ventricle down regulates opioid receptors [26]. Conversely, administration of an antibody to NPFF increases opioid receptor binding, suggesting tonic regulation of opioid receptor number by NPFF [7]. Evidence from binding studies suggests that NPFF does not bind significantly to opioid receptors [21]. NPFF/FMRFa binding sites, distinct from opioid receptor sites have been demonstrated [16] suggesting that opioid modulating effects of NPFF/FMRFa are most likely mediated through their own set of receptors/ binding sites [1]. Interestingly, NPFF/FMRFa can also display opioid like effects—intrathecal (i.t.) infusion of NPFF/FMRFa produces longlasting analgesia [8]. Similarly, NPFF analogs inhibit intestinal transit after intracerebroventricular (i.c.v.) injection and potentiate opioid effects [20]. Taken together, these experiments reveal the complex interplay existing between NPFF/FMRFa and opioid systems in pain regulatory mechanisms [11,22,25,27]. Methionine-enkephalin-Arg6-Phe7 (MERF) is widely distributed in the central nervous system of different mammals [15,24, 32] and is considered largely to belong to the opioid family [13]. However the most noticeable feature of MERF is the presence of the FMRF sequence within it [9], indicating that MERF and related
KEY WORDS: Chimeric peptide, MERF, Metenkephalin, FMRFa, Antinociception, Morphine, Mice, i.c.v.
INTRODUCTION Neuropeptides FF (NPFF) and AF (NPAF) are homologous amidated peptides that were originally identified on the basis of their similarity to the molluscan neuropeptide FMRFamide [5,31]. Recent cloning of the NPFF gene revealed that the gene encodes for both NPFF and related neuropeptide NPAF [17,19,29,30]. There is considerable pharmacological data which suggest NPFF/FMRFa family of neuropeptides has a significant role in pain modulation as well as in opioid tolerance and dependence in the mammalian central nervous system [11,17,27]. NPFF has been shown to modulate pain sensation and morphine analgesia under both normal and pathological conditions, apparently through spinal as well as
* Address for correspondence: Dr. Santosh Pasha, Peptide Laboratory, Centre for Biochemical Technology (CSIR), Mall Road, Delhi-110007, India. Fax: ⫹ 91-11-766-7471; E-mail: [email protected]
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peptides may not be merely opioid-like peptides. Based on the various reports of NPFF/FMRFa family in pain modulation, it is reasonable to hypothesise that these amphiactive peptides may have a possible role in antinociception and its modulation. Therefore in our earlier study, a chimeric peptide of met-enkephalin and FMRFa was designed as a useful probe [10]. It was demonstrated that intraperitoneal (i.p.) administration of this chimeric peptide, YGGFMKKKFMRFamide (YFa), induced a dose-dependent increase in tail-flick latency in mice, indicating an antinociceptive action. This effect was naloxone reversible, suggesting that opioid receptors are probably involved in the mediation of the antinociceptive effect. Further, YFa potentiated morphine-induced antinociception and attenuated development of tolerance to the antinociceptive action of morphine. These preliminary results indicate a possible role of these types of amphiactive sequences in pain modulation [10]. Following these studies on effects of YFa on antinociception and tolerance development via the peripheral (i.p.) route, an analog of this chimeric peptide [D-Ala2]YAGFMKKKFMRFamide ([DAla2]YFa)—was designed to further investigate the central (i.c.v.) effects of this peptide. This analog was synthesised with N-terminal modification—substitution of Gly2 of parent peptide with D-Ala2—to make it more resistant to degradation by peptidases. The present study evaluated the possible antinociceptive effects of [D-Ala2]YFa via the i.c.v. route, using the tail-flick nociceptive test in mice. Experiments were also carried out to estimate the modulatory effect of this peptide on morphine antinociception. The interaction of [D-Ala2]YFa—with opioid antagonist naloxone as well as the antiopioid peptides- NPFF and FMRFa- was also investigated. MATERIALS AND METHODS Animals Male albino mice weighing 25–30 g were used. They were housed six per cage maintained at room temperature (25–30°C). Food and water was allowed ad libitum. All animals were used only once in the study and were treated in accordance to Indian National Science Academy guidelines for usage of small animals in scientific research. Peptide Synthesis All peptides were synthesised as described earlier [10] by the solid phase method on a mechanical shaker, using the standard chemistry of Fmoc amino acids and HOBt/DCC activation method on Rink amide—MBHA resin. The peptides were purified by semi-preparative reverse phase high-performance liquid chromatography with a 40-min linear gradient from 10 to 50% acetonitrile containing 0.05% tri-fluoroacetic acid in water. The correct peptide sequences synthesised were confirmed through automated peptide sequencing (490 Applied Biosystems) and by MALDI. Chemicals Naloxone hydrochloride and bestatin were purchased from Sigma (St. Louis, MO, USA) and morphine hydrochloride was obtained from Department of Pharmacology, AIIMS. All peptides/ drugs were dissolved in physiological saline and administered i.c.v. [4] in a volume of 5 l. Peptidase inhibitor bestatin (1 M) was included in saline in an attempt to minimise the enzymatic inactivation of the injected neuropeptide(s). A preliminary experiment indicated that i.c.v. injection of bestatin (5 l) did not induce any significant antinociceptive effects.
Measurement of the Antinociceptive Response The antinociceptive response was measured by the radiant-heat tail-flick test as described previously [10]. Briefly, at the beginning of the study, the intensity of heat stimulus in the tail-flick apparatus was so adjusted to elicit a response in control or untreated animals within 3–5 s. To minimize tail skin tissue damage, the cut-off time was set at 10 s. Mice were given three baseline trials, each separated by 10 min, followed by an i.c.v. injection of either physiological saline (5 l) or the peptide/drug and tested 5, 10, 15, 30, and 60 min later. The basal tail-flick latencies were subtracted from the effect induced by the peptide/drug for each mouse. Percent maximum possible effect (% MPE) was calculated for each mouse using the following formula: %MPE ⫽ 100 ⫻ [(test latency ⫺ control latency)/(10 ⫺ control latency)]. Eight mice were used for each treatment group. Intracerebroventricular Injections in Mice The i.c.v. injection procedure was adapted from the method of Zhao and Bhargava [4]. Briefly, the i.c.v. injections were given as follows: under light ether anesthesia, bregma was exposed. An injection volume of 5 l was delivered over a 60-s period, 2 mm lateral and caudal to bregma at a depth of 3 mm by using a 10-ml Hamilton syringe with a 27-gauge needle. To facilitate diffusion into the tissue, the syringe was left in place an additional 15 s [2]. Proper placement was verified in pilot experiments by injection and localization of methylene blue dye. Statistical Analysis All data are expressed as % MPE (mean ⫾ SEM) and analyzed using two-way analysis of variance (ANOVA) with treatment (saline vs. drug/peptide) as a between-subjects variable and time as a within-subject variable. Subsequent post-hoc comparison by Newman-Keul’s test was carried out. Significance level was set at p ⬍ 0.05. RESULTS 2
[D-Ala ]YFa-induced Antinociception The i.c.v. injection of physiological saline had no measurable effect on baseline tail-flick latency. [D-Ala2]YFa produced a significant dose related increase in response latency of mice in the tail-flick test compared to saline-treated mice, with peak effect from 15–30 min after i.c.v. administration (Fig. 1) (two-way ANOVA results for 1 g/mouse (0.64 nmol) dose—Ftreatment (1,84) ⫽ 47.13, p ⬍ 0.0001; Ftime(5,84) ⫽ 4.20, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 3.818, p ⬍ 0.001, 2 g/mouse (1.28 nmol) dose—Ftreatment(1,84) ⫽ 96.52, p ⬍ 0.0001; Ftime(5,84) ⫽ 9.18, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 8.36, p ⬍ 0.001, 5 g/mouse dose (3.22 nmol)—Ftreatment(1,84) ⫽ 117.17, p ⬍ 0.0001; Ftime (5,84) ⫽ 12.29, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 10.94, p ⬍ 0.001). i.c.v. administration of bestatin alone (5 l) showed no significant difference in response latency from saline-treated group (Fig. 1) [Ftreatment(1,84) ⫽ 2.76, n.s.; Ftime(5,84) ⫽ 1.75, n.s.; Ftreatment ⫻ time (5,84) ⫽ 2.29, n.s.]. Naloxone Antagonism of [D-Ala2]YFa-induced Antinociception The i.c.v. administration of the opioid receptor antagonist naloxone (1.5 g/mouse: 4.12 nmol), 5 minutes prior to i.c.v. injection of [D-Ala2]YFa (1 g/mouse: 0.64 nmol) significantly reversed the antinociceptive effect produced by the peptide alone (Fig. 2) [two-way ANOVA showed—Ftreatment(1,84) ⫽ 39.75, p ⬍ 0.0001; Ftime(5,84) ⫽ 5.62, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 2.35, p ⬍ 0.001]. Naloxone at this dose (1.5 g/mouse) did not
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FIG. 1. Effect of the intracerebroventricular (i.c.v.) administration of [D-Ala2]YFa (1, 2, and 5 g/mouse/5 l) or saline (5 l/mouse) in mice in the radiant-heat tail-flick test. [D-Ala2]YFa produced dose-related antinociception which lasted from 60 –90 min (data up to 60 min shown), with peak effect from 15 to 30 min after i.c.v. injection. Effect of bestatin (5 l, i.c.v.) alone on tail-flick values in mice is also represented. Data expressed as mean ⫾ SEM. *Significant difference from the saline-treated group (*p ⬍ 0.05). Abbreviation: MPE, maximum possible effect.
induce any effect on tail-flick latency. Two-way ANOVA did not reveal a significant difference in response latency between the groups receiving naloxone and the saline-treated group [Ftreatment (1,84) ⫽ 0.97, n.s.; Ftime(5,84) ⫽ 0.31, n.s.; Ftreatment ⫻ time(5,84) ⫽ 0.75, n.s.]. [D-Ala2]YFa Potentiation of Morphine-induced Antinociception The i.c.v. administration of morphine (2 g/mouse: 5.86 nmol) alone produced a clear antinociceptive effect in the tail-flick test (Fig. 3) (Ftreatment(1,84) ⫽ 50.29, p ⬍ 0.0001; Ftime(5,84) ⫽ 3.08, p ⬍ 0.01; Ftreatment ⫻ time(5,84) ⫽ 3.77, p ⬍ 0.001). [D-Ala2]YFa (1 g/mouse: 0.64 nmol) coinjected i.c.v. with morphine (2 g/ mouse) demonstrated an additive antinociceptive effect [two-way ANOVA: Ftreatment(1,84) ⫽ 16.95, p ⬍ 0.0001; Ftime(5,84) ⫽ 8.13, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 4.88, p ⬍ 0.001]. Effects of Antiopioid Peptides NPFF and FMRFa on [DAla2]YFa-induced Antinociception The effects of i.c.v. NPFF and FMRFa on [D-Ala2]YFa-induced antinociception are shown in Figs. 4a and 4b, respectively. NPFF (1 g/mouse: 0.92 nmol) or FMRFa (1 g/mouse: 1.69 nmol) alone produced no significant change in the tail-flick values
in mice [Two-way ANOVA results for NPFF—Ftreatment(1,84) ⫽ 0.22, n.s.; Ftime(5,84) ⫽ 1.02, n.s.; Ftreatment ⫻ time(5,84) ⫽ 0.24, n.s., FMRFa—Ftreatment(1,84) ⫽ 1.18, n.s.; Ftime(5,84) ⫽ 1.82, n.s.; Ftreatment ⫻ time(5,84) ⫽ 1.68, n.s.]. However, pretreatment (5 min) with NPFF (1 g/mouse) or FMRFa (1 g/mouse) significantly attenuated [D-Ala2]YFa (1 g/mouse: 0.64 nmol) antinociception from 63.78 ⫾ 11.72 (% MPE, peak effect) to 39.33 ⫾ 13.07 and 31.19 ⫾ 10.87, respectively [two-way ANOVA showed—NPFF (1 g/mouse) Ftreatment(1,84) ⫽ 15.43, p ⬍ 0.0001; Ftime(5,84) ⫽ 4.11, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 3.71, p ⬍ 0.001, FMRFa (1 g/mouse) Ftreatment(1,84) ⫽ 7.08, p ⬍ 0.001; Ftime(5,84) ⫽ 3.99, p ⬍ 0.0001; Ftreatment ⫻ time (5,84) ⫽ 2.5, p ⬍ 0.01]. DISCUSSION In our earlier study [10], a chimeric peptide of met-enkephalin and FMRFa—YGGFMKKKFMRFa (YFa) was designed, based on the endogenous amphiactive peptide MERF and various reports on the role of NPFF/FMRFa peptides in pain modulation [11,22, 25,27]. The rationale of the study was to explore the possibility of a role of these sequences in antinociception and its modulation. Briefly, i.p. YFa in mice produced naloxone reversible, dose-
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FIG. 2. Influence of naloxone on the antinociceptive effect of [D-Ala2]YFa in the radiant-heat tail-flick test in mice. Naloxone (1.5 g/mouse, intracerebroventricular [i.c.v.]) administered 5 min prior reversed the antinociceptive effect of [D-Ala2]YFa. Naloxone (1.5 g/mouse, i.c.v.) alone had no significant antinociceptive effect. Antinociception (% maximum possible effect [MPE]) values for [D-Ala2]YFa (1 g/mouse, i.c.v.) are also represented (from Fig. 1). Data expressed as mean ⫾ SEM. *Significant difference from [D-Ala2]YFa-treated group (*p ⬍ 0.05).
dependent antinociception; potentiated morphine-induced antinociception and attenuated tolerance development—indicating a possible role of these amphiactive sequences in pain modulation [10]. Further, several reports indicate that NPFF, FMRFa and related peptides display either analgesia or blockade of opiate analgesia depending on such experimental conditions as route of administration. For example, intraventricular (i.v.) NPFF attenuates the antinociceptive effect of morphine administered i.c.v. [28,31], whereas NPFF administered i.t. produces a dose-dependent, longlasting antinociceptive effect that is partially naloxone reversible [8]. Because YFa contains FMRFa sequence [10], it would be interesting to examine if YFa administered i.v. also displays effects distinct from those following i.p. administration. Our earlier results [10] indicate that YFa degrades quite rapidly, hence the parent peptide was modified at its N-terminal. This synthetic stable analog [D-Ala2]YAGFMKKKFMRFamide ([D-Ala2]YFa) was administered centrally (i.c.v.) in mice to evaluate its effects on antinociception and its modulation. In the present study, i.c.v. administered [D-Ala2]YFa in doses of 1, 2, and 5 g/mouse (0.64, 1.28, and 3.22 nmol, respectively) displayed significant antinociceptive effect in the radiant-heat tailflick test in mice. However higher i.c.v. dose of the peptide—10 g/mouse—tended to produce respiratory depression. It has been previously reported that i.c.v. YGGFMRFamide produces no antinociception in rats [18]. In this study, it was demonstrated that although both YGGFMRF and YGGFMRFamide bind to opioid receptors, only YGGFMRF produces antinociception [18]. Because YGGFMRFamide also showed high binding potency at
NPFF receptors in comparison to YGGFMRF, it was proposed that both YGGFMRFamide and YGGFMRF may be acting as agonists at central opioid receptors, the additional anti-analgesic activity elicited by YGGFMRFamide at NPFF receptors may have masked its analgesic effect [18]. In the current experiments the reason for the difference in the antinociceptive effects of YGGFMRFamide and [D-Ala2]YFa is not clear. One possibility could be due to a difference in the animal model. In pilot studies with YGGFMRFamide, i.c.v. administration up to 40 g/mouse did not induce any antinociceptive effects in mice (data not shown). These results are in agreement with the earlier report by Payza et al. [18]. It should be noted, however, that it is not unprecedented for these types of chimeric sequences to show differing antinociceptive responses— [D-Ala2]metenkephalin-Arg6-Phe7amide is 130 times more potent analgesic than YGGFMRF and twice as potent than morphine when administered i.v. in mice [24]. One factor that may contribute to this variable response could be differential receptor binding of YGGFMRFamide and [D-Ala2]YFa. Our study does not exclude the possibility of [D-Ala2]YFa binding to receptors other than opioid and NPFF, such as MERF receptors [3]. The antinociceptive effect of [D-Ala2]YFa was completely reversed by i.c.v. administration of naloxone 1 g/mouse (4.12 nmol), administered 5 min prior, indicating opioid receptor mediation in the observed antinociception. FMRFa and NPFF have also been shown in one study to induce antinociception when administered by i.t. route in rats, and the mechanism ascribed is the indirect release of endogenous opioid peptides [8]. Because [D-Ala2]YFa contains the FMRFa sequence, the observed nalox-
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FIG. 3. Effect of [D-Ala2]YFa on the antinociceptive effect of morphine in the radiant-heat tail-flick test in mice. Morphine (2 g/mouse, intracerebroventricular [i.c.v.]) induced a significant antinociceptive effect. [D-Ala2]YFa (1 g/mouse, i.c.v.) when co-injected with morphine (2 g/mouse, i.c.v.) showed an additive antinociceptive effect. Antinociception (% maximum possible effect [MPE]) values for [D-Ala2]YFa (1 g/mouse, i.c.v.) alone are also represented (from Fig. 1) for comparison. Data expressed as mean ⫾ SEM. *Significant difference from the morphine-treated group (*p ⬍ 0.05). #p ⬍ 0.05 vs. saline-treated group.
one reversible antinociception could be due to direct opioid receptor stimulation and/or due to indirect release of endogenous opioid peptides. The interaction of this peptide [D-Ala2]YFa with anti-opioid peptides NPFF and FMRFa was also studied. The antinociceptive effect induced by [D-Ala2]YFa (1 g/mouse: 0.64 nmol, i.c.v.) was significantly attenuated by i.c.v. pretreatment (5 min) with either NPFF (1 g/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 g/ mouse:1.69 nmol, i.c.v.). Higher doses were avoided because both NPFF and FMRFa have been reported to induce antinociception [8,12]. These results again suggest that the observed antinociception may be due to multiple binding of [D-Ala2]YFa to opioid as well as NPFF/FMRFa receptor(s). Previous reports have described the antagonism of morphine antinociception by NPFF/FMRFa family in rats [17]. The present study also evaluated the opioid-modulating properties of [D-Ala2]YFa by concurrent i.c.v. administration of [D-Ala2]YFa (1 g/mouse) with morphine (2 g/mouse: 5.86 nmol). Interestingly, [D-Ala2]YFa does not antagonise morphine antinociception, rather an additive antinociceptive effect was seen. Taken together, our present findings suggest that [D-Ala2]YFa displays opioid-like effects following i.c.v. administration in mice and the antiopioid/ antianalgesic effects of the FMRFa portion of the peptide is not evident in these experiments. The lack of any antiopioid action could possibly be explained if the model proposing that NPFF- and FMRFa-induced effects— opioid agonist as well as antagonist like— can be mediated by different antiopioid receptor subtypes
through differential G-protein coupling—Gi and Gs—is taken into account [23]. Accordingly, [D-Ala2]YFa may be activating antiopioid receptor via Gi, thereby producing only opioid like effects. An alternate explanation could be [D-Ala2]YFa may be behaving as a putative antagonist at NPFF receptor/binding site(s)—then the observed antinociceptive effect and lack of attenuation of morphine antinociception could be due to opioid receptor(s) stimulation and/or by antagonism at antiopioid receptor(s). The effect of [D-Ala2]YFa on development of tolerance to morphine antinociception would provide further insight into its mechanism of action and is currently under investigation. In conclusion, [D-Ala2]YFa causes modest to good antinociception in mice following i.c.v. administration. This antinociceptive effect is completely reversed by opioid receptor antagonist— naloxone and also significantly attenuated by the antiopioid peptides NPFF and FMRFa, indicating these effects may be mediated by opioid and/or antiopioid mechanism(s). Coinjection of [D-Ala2]YFa with morphine showed an additive antinociceptive effect, suggesting that [D-Ala2]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation. ACKNOWLEDGEMENTS
This work was supported by Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC). We thank Rajbir Singh from Department of Biostatistics, All India Institute of Medical
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FIG. 4. Influence of antiopioid peptides neuropeptides FF (NPFF) and FMRFa on the antinociceptive effect of [D-Ala2]YFa in the radiant-heat tail-flick test in mice. NPFF (1 g/mouse, intracerebroventricular [i.c.v.]) (a) or FMRFa (1 g/mouse, i.c.v.) (b) was administered 5 min prior to [D-Ala2]YFa (1 g/mouse, i.c.v.). Both NPFF and FMRFa significantly attenuated antinociception induced by [D-Ala2]YFa. NPFF (1 g/mouse, i.c.v.) (a) or FMRFa (1 g/mouse, i.c.v.) (b) alone had no significant antinociceptive effects in the tail-flick test. Data expressed as mean ⫾ SEM. *Significant difference from NPFF or FMRFa-treated group (*p ⬍ 0.05). Abbreviation: MPE, maximum possible effect.
[D-ALA2]YFA IN ANTINOCICEPTION AND ITS MODULATION Sciences for statistical analysis of the data. We also thank Dr. Shyam Sunder Sharma, Department of Pharmacology, All India Institute of Medical Sciences for helping us with some of the pharmacological studies.
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57 enkephalin-Arg6-Phe7 in rat spinal cord. Brain Res. 264:336 –369; 1983. Malin, D. H.; Lake, J. R.; Hammond, M. V.; Fowler, D. E.; Rogillio, R. B.; Brown, S. L.; Sims, J. L.; Leecraft, B. M.; Yang, H.-Y. T. FMRFamide like mammalian octapeptide: Possible role in opiate dependence and abstinence. Peptides 11:969 –972; 1990. Panula, P.; Kalso, E.; Nieminen, M.-L.; Kontinen, V.; Brandt, A.; Pertovaara, A. Neuropeptide FF and modulation of pain. Brain Res. 848:191–196; 1999. Payza, K.; Akar, C. A.; Yang, H.-Y. T. Neuropeptide FF receptors: Structure activity relationship and effect of morphine. J. Pharmacol. Exp. Ther. 267:88 –94; 1993. Perry, S. J.; Huang, E. Y.-K.; Cronk, D.; Bagust, J.; Sharma, R.; Walker, R. J.; Wilson, S.; Burke, J. F. A human gene encoding morphine modulating peptides related to NPFF and FMRFamide. FEBS Lett. 409:426 – 430; 1997. Raffa, R. B.; Jacoby, H. I. Effect of [D-Met2]FMRFamide and other FMRFamide related peptides on mouse colonic propulsive motility: A structure-activity relationship study. J. Pharmacol. Exp. Ther. 254: 809 – 814; 1990. Raffa, R. B.; Kim, A.; Rice, K. C.; Costa, B. R.; Codd, E. E.; Rothman, R. B. Low affinity of FMRFa and four FaRPs (FMRFamide related peptides), including the mammalian-derived FaRPs-F8F (NPFF) and A18Fa, for opioid , ␦, 1 2a or 2b receptors. Peptides 15:401– 404; 1994. Raffa, R. B. The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals. Peptides 9:915–922; 1988. Raffa, R. B.; Stone, D. J. Could dual G-protein coupling explain [D-Met2] FMRFamide’s mixed action in vivo? Peptides 17:1261– 1265; 1996. Rossier, J.; Audigier, Y.; Ling, N.; Cros, J.; Udenfriend, S. Metenkephalin-Arg6-Phe7, present in high amounts in brain of rat, cattle and man, is an opioid agonist. Nature 288:88 –90; 1980. Rothman, R. B. A review of the role of anti-opioid peptides in morphine tolerance and dependence. Synapse 12:129 –138; 1992. Rothman, R. B.; Brady, L. S.; Xu, H.; Long, J. B. Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu- Phe-GlnPro-Gln-Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain. Peptides 14:1271–1277; 1993. Roumy, M.; Zajac, J.-M. Neuropeptide FF, pain and analgesia. Eur. J. Pharmacol. 345:1–11; 1998. Tang, J.; Yang, H.-Y. T.; Costa, E. Inhibition of spontaneous and opiate modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-NH2-like immunoreactivity. Proc. Natl. Acad. Sci. USA 81:5002–5005; 1984. Vilim, F. S.; Aarnisalo, A. A.; Nieminen, M.-L.; Lintunen, M.; Karlstedt, K.; Kontinen, V.; Kalso, E.; States, B.; Panula, P.; Ziff, E. Gene for pain modulatory neuropeptide NPFF: Induction in spinal cord by noxious stimuli. Mol. Pharmacol. 55:804 – 811; 1999. Vilim, F. S.; Ziff, E. B. Cloning of the neuropeptide NPFF and NPAF precursor from bovine, rat, mouse and human. Soc. Neurosci. Abstr. 21(pt. 1):760; 1995. Yang, H.-Y. T.; Fratta, W.; Majane, E. A.; Costa, E. Isolation, sequencing, synthesis and pharmacological characterisation of two brain neuropeptides that modulate the action of morphine. Proc. Natl. Acad. Sci. USA 82:7757–7761; 1985. Yang, H. Y.-T.; Panula, P.; Tang, J.; Costa, E. Characterisation and localization of met5-enkephalin-Arg6-Phe7 stored in various rat brain regions. J. Neurochem. 40:969 –976; 1983.
PII S0361-9230(01)00490-7
Effects of intracerebroventricularly administered chimeric peptide of Metenkephalin and FMRFa—[D-Ala2]YFa— on antinociception and its modulation in mice Suparna Gupta,1 Santosh Pasha,1* Y. K. Gupta2 and D. K. Bhardwaj3 1
Peptide Laboratory, Centre for Biochemical Technology (CSIR), Delhi, India; 2Department of Pharmacology, All India Institute of Medical Sciences, Delhi, India; and 3Department of Chemistry, University of Delhi, Delhi, India [Received 18 September 2000; Revised 6 February 2001; Accepted 10 March 2001]
ABSTRACT: An enzymatically stable analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of metenkephalin and FMRFa, was synthesised. The antinociceptive effects of intracerebroventricular injections of this analog—[D-Ala2]YAGFMKKKFMRFamide ([D-Ala2]YFa)—was then investigated using the mouse radiantheat tail-flick test. [D-Ala2]YFa produced modest to good antinociception at 1, 2, and 5 g/mouse (0.64, 1.28, and 3.22 nmol, respectively). This antinociceptive effect was completely reversed by the opioid receptor antagonist naloxone (1.5 g/mouse: 4.12 nmol, intracerebroventricular [i.c.v.]), administered 5 min prior. Pretreatment (5 min) with either neuropeptides FF (1 g/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 g/mouse: 1.69 nmol, i.c.v.) significantly attenuated the antinociceptive effects induced by [D-Ala2]YFa (1 g/mouse, i.c.v.). Intracerebroventricular administration of [D-Ala2]YFa at 1 g/mouse dose with morphine (2 g/ mouse: 5.86 nmol, i.c.v.) produced an additive antinociceptive effect, suggesting that [D-Ala2]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation. © 2001 Elsevier Science Inc.
brain mechanisms [17]. NPFF/FMRFa has been found to decrease the tail-flick latency in rats and attenuate the prolongation of the tail-flick latency induced by morphine [28,31], indicating these peptides may be acting as endogenous anti-opioid peptides in rats and mice. Pretreatment with immunoglobulin G from a NPFF antiserum prevents the naloxone-precipitated abstinence syndrome in morphine-dependent rats [16], and it restores the analgesic response to morphine in morphine-tolerant rats [14]. Moreover, the concentration of NPFF in the cerebrospinal fluid of morphinedependent rats is higher than in naı¨ve rats [16]. Though the detailed mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that NPFF can regulate opioid receptor number [6]. Several studies have shown that chronic infusion of NPFF into the lateral ventricle down regulates opioid receptors [26]. Conversely, administration of an antibody to NPFF increases opioid receptor binding, suggesting tonic regulation of opioid receptor number by NPFF [7]. Evidence from binding studies suggests that NPFF does not bind significantly to opioid receptors [21]. NPFF/FMRFa binding sites, distinct from opioid receptor sites have been demonstrated [16] suggesting that opioid modulating effects of NPFF/FMRFa are most likely mediated through their own set of receptors/ binding sites [1]. Interestingly, NPFF/FMRFa can also display opioid like effects—intrathecal (i.t.) infusion of NPFF/FMRFa produces longlasting analgesia [8]. Similarly, NPFF analogs inhibit intestinal transit after intracerebroventricular (i.c.v.) injection and potentiate opioid effects [20]. Taken together, these experiments reveal the complex interplay existing between NPFF/FMRFa and opioid systems in pain regulatory mechanisms [11,22,25,27]. Methionine-enkephalin-Arg6-Phe7 (MERF) is widely distributed in the central nervous system of different mammals [15,24, 32] and is considered largely to belong to the opioid family [13]. However the most noticeable feature of MERF is the presence of the FMRF sequence within it [9], indicating that MERF and related
KEY WORDS: Chimeric peptide, MERF, Metenkephalin, FMRFa, Antinociception, Morphine, Mice, i.c.v.
INTRODUCTION Neuropeptides FF (NPFF) and AF (NPAF) are homologous amidated peptides that were originally identified on the basis of their similarity to the molluscan neuropeptide FMRFamide [5,31]. Recent cloning of the NPFF gene revealed that the gene encodes for both NPFF and related neuropeptide NPAF [17,19,29,30]. There is considerable pharmacological data which suggest NPFF/FMRFa family of neuropeptides has a significant role in pain modulation as well as in opioid tolerance and dependence in the mammalian central nervous system [11,17,27]. NPFF has been shown to modulate pain sensation and morphine analgesia under both normal and pathological conditions, apparently through spinal as well as
* Address for correspondence: Dr. Santosh Pasha, Peptide Laboratory, Centre for Biochemical Technology (CSIR), Mall Road, Delhi-110007, India. Fax: ⫹ 91-11-766-7471; E-mail: [email protected]
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peptides may not be merely opioid-like peptides. Based on the various reports of NPFF/FMRFa family in pain modulation, it is reasonable to hypothesise that these amphiactive peptides may have a possible role in antinociception and its modulation. Therefore in our earlier study, a chimeric peptide of met-enkephalin and FMRFa was designed as a useful probe [10]. It was demonstrated that intraperitoneal (i.p.) administration of this chimeric peptide, YGGFMKKKFMRFamide (YFa), induced a dose-dependent increase in tail-flick latency in mice, indicating an antinociceptive action. This effect was naloxone reversible, suggesting that opioid receptors are probably involved in the mediation of the antinociceptive effect. Further, YFa potentiated morphine-induced antinociception and attenuated development of tolerance to the antinociceptive action of morphine. These preliminary results indicate a possible role of these types of amphiactive sequences in pain modulation [10]. Following these studies on effects of YFa on antinociception and tolerance development via the peripheral (i.p.) route, an analog of this chimeric peptide [D-Ala2]YAGFMKKKFMRFamide ([DAla2]YFa)—was designed to further investigate the central (i.c.v.) effects of this peptide. This analog was synthesised with N-terminal modification—substitution of Gly2 of parent peptide with D-Ala2—to make it more resistant to degradation by peptidases. The present study evaluated the possible antinociceptive effects of [D-Ala2]YFa via the i.c.v. route, using the tail-flick nociceptive test in mice. Experiments were also carried out to estimate the modulatory effect of this peptide on morphine antinociception. The interaction of [D-Ala2]YFa—with opioid antagonist naloxone as well as the antiopioid peptides- NPFF and FMRFa- was also investigated. MATERIALS AND METHODS Animals Male albino mice weighing 25–30 g were used. They were housed six per cage maintained at room temperature (25–30°C). Food and water was allowed ad libitum. All animals were used only once in the study and were treated in accordance to Indian National Science Academy guidelines for usage of small animals in scientific research. Peptide Synthesis All peptides were synthesised as described earlier [10] by the solid phase method on a mechanical shaker, using the standard chemistry of Fmoc amino acids and HOBt/DCC activation method on Rink amide—MBHA resin. The peptides were purified by semi-preparative reverse phase high-performance liquid chromatography with a 40-min linear gradient from 10 to 50% acetonitrile containing 0.05% tri-fluoroacetic acid in water. The correct peptide sequences synthesised were confirmed through automated peptide sequencing (490 Applied Biosystems) and by MALDI. Chemicals Naloxone hydrochloride and bestatin were purchased from Sigma (St. Louis, MO, USA) and morphine hydrochloride was obtained from Department of Pharmacology, AIIMS. All peptides/ drugs were dissolved in physiological saline and administered i.c.v. [4] in a volume of 5 l. Peptidase inhibitor bestatin (1 M) was included in saline in an attempt to minimise the enzymatic inactivation of the injected neuropeptide(s). A preliminary experiment indicated that i.c.v. injection of bestatin (5 l) did not induce any significant antinociceptive effects.
Measurement of the Antinociceptive Response The antinociceptive response was measured by the radiant-heat tail-flick test as described previously [10]. Briefly, at the beginning of the study, the intensity of heat stimulus in the tail-flick apparatus was so adjusted to elicit a response in control or untreated animals within 3–5 s. To minimize tail skin tissue damage, the cut-off time was set at 10 s. Mice were given three baseline trials, each separated by 10 min, followed by an i.c.v. injection of either physiological saline (5 l) or the peptide/drug and tested 5, 10, 15, 30, and 60 min later. The basal tail-flick latencies were subtracted from the effect induced by the peptide/drug for each mouse. Percent maximum possible effect (% MPE) was calculated for each mouse using the following formula: %MPE ⫽ 100 ⫻ [(test latency ⫺ control latency)/(10 ⫺ control latency)]. Eight mice were used for each treatment group. Intracerebroventricular Injections in Mice The i.c.v. injection procedure was adapted from the method of Zhao and Bhargava [4]. Briefly, the i.c.v. injections were given as follows: under light ether anesthesia, bregma was exposed. An injection volume of 5 l was delivered over a 60-s period, 2 mm lateral and caudal to bregma at a depth of 3 mm by using a 10-ml Hamilton syringe with a 27-gauge needle. To facilitate diffusion into the tissue, the syringe was left in place an additional 15 s [2]. Proper placement was verified in pilot experiments by injection and localization of methylene blue dye. Statistical Analysis All data are expressed as % MPE (mean ⫾ SEM) and analyzed using two-way analysis of variance (ANOVA) with treatment (saline vs. drug/peptide) as a between-subjects variable and time as a within-subject variable. Subsequent post-hoc comparison by Newman-Keul’s test was carried out. Significance level was set at p ⬍ 0.05. RESULTS 2
[D-Ala ]YFa-induced Antinociception The i.c.v. injection of physiological saline had no measurable effect on baseline tail-flick latency. [D-Ala2]YFa produced a significant dose related increase in response latency of mice in the tail-flick test compared to saline-treated mice, with peak effect from 15–30 min after i.c.v. administration (Fig. 1) (two-way ANOVA results for 1 g/mouse (0.64 nmol) dose—Ftreatment (1,84) ⫽ 47.13, p ⬍ 0.0001; Ftime(5,84) ⫽ 4.20, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 3.818, p ⬍ 0.001, 2 g/mouse (1.28 nmol) dose—Ftreatment(1,84) ⫽ 96.52, p ⬍ 0.0001; Ftime(5,84) ⫽ 9.18, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 8.36, p ⬍ 0.001, 5 g/mouse dose (3.22 nmol)—Ftreatment(1,84) ⫽ 117.17, p ⬍ 0.0001; Ftime (5,84) ⫽ 12.29, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 10.94, p ⬍ 0.001). i.c.v. administration of bestatin alone (5 l) showed no significant difference in response latency from saline-treated group (Fig. 1) [Ftreatment(1,84) ⫽ 2.76, n.s.; Ftime(5,84) ⫽ 1.75, n.s.; Ftreatment ⫻ time (5,84) ⫽ 2.29, n.s.]. Naloxone Antagonism of [D-Ala2]YFa-induced Antinociception The i.c.v. administration of the opioid receptor antagonist naloxone (1.5 g/mouse: 4.12 nmol), 5 minutes prior to i.c.v. injection of [D-Ala2]YFa (1 g/mouse: 0.64 nmol) significantly reversed the antinociceptive effect produced by the peptide alone (Fig. 2) [two-way ANOVA showed—Ftreatment(1,84) ⫽ 39.75, p ⬍ 0.0001; Ftime(5,84) ⫽ 5.62, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 2.35, p ⬍ 0.001]. Naloxone at this dose (1.5 g/mouse) did not
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FIG. 1. Effect of the intracerebroventricular (i.c.v.) administration of [D-Ala2]YFa (1, 2, and 5 g/mouse/5 l) or saline (5 l/mouse) in mice in the radiant-heat tail-flick test. [D-Ala2]YFa produced dose-related antinociception which lasted from 60 –90 min (data up to 60 min shown), with peak effect from 15 to 30 min after i.c.v. injection. Effect of bestatin (5 l, i.c.v.) alone on tail-flick values in mice is also represented. Data expressed as mean ⫾ SEM. *Significant difference from the saline-treated group (*p ⬍ 0.05). Abbreviation: MPE, maximum possible effect.
induce any effect on tail-flick latency. Two-way ANOVA did not reveal a significant difference in response latency between the groups receiving naloxone and the saline-treated group [Ftreatment (1,84) ⫽ 0.97, n.s.; Ftime(5,84) ⫽ 0.31, n.s.; Ftreatment ⫻ time(5,84) ⫽ 0.75, n.s.]. [D-Ala2]YFa Potentiation of Morphine-induced Antinociception The i.c.v. administration of morphine (2 g/mouse: 5.86 nmol) alone produced a clear antinociceptive effect in the tail-flick test (Fig. 3) (Ftreatment(1,84) ⫽ 50.29, p ⬍ 0.0001; Ftime(5,84) ⫽ 3.08, p ⬍ 0.01; Ftreatment ⫻ time(5,84) ⫽ 3.77, p ⬍ 0.001). [D-Ala2]YFa (1 g/mouse: 0.64 nmol) coinjected i.c.v. with morphine (2 g/ mouse) demonstrated an additive antinociceptive effect [two-way ANOVA: Ftreatment(1,84) ⫽ 16.95, p ⬍ 0.0001; Ftime(5,84) ⫽ 8.13, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 4.88, p ⬍ 0.001]. Effects of Antiopioid Peptides NPFF and FMRFa on [DAla2]YFa-induced Antinociception The effects of i.c.v. NPFF and FMRFa on [D-Ala2]YFa-induced antinociception are shown in Figs. 4a and 4b, respectively. NPFF (1 g/mouse: 0.92 nmol) or FMRFa (1 g/mouse: 1.69 nmol) alone produced no significant change in the tail-flick values
in mice [Two-way ANOVA results for NPFF—Ftreatment(1,84) ⫽ 0.22, n.s.; Ftime(5,84) ⫽ 1.02, n.s.; Ftreatment ⫻ time(5,84) ⫽ 0.24, n.s., FMRFa—Ftreatment(1,84) ⫽ 1.18, n.s.; Ftime(5,84) ⫽ 1.82, n.s.; Ftreatment ⫻ time(5,84) ⫽ 1.68, n.s.]. However, pretreatment (5 min) with NPFF (1 g/mouse) or FMRFa (1 g/mouse) significantly attenuated [D-Ala2]YFa (1 g/mouse: 0.64 nmol) antinociception from 63.78 ⫾ 11.72 (% MPE, peak effect) to 39.33 ⫾ 13.07 and 31.19 ⫾ 10.87, respectively [two-way ANOVA showed—NPFF (1 g/mouse) Ftreatment(1,84) ⫽ 15.43, p ⬍ 0.0001; Ftime(5,84) ⫽ 4.11, p ⬍ 0.001; Ftreatment ⫻ time(5,84) ⫽ 3.71, p ⬍ 0.001, FMRFa (1 g/mouse) Ftreatment(1,84) ⫽ 7.08, p ⬍ 0.001; Ftime(5,84) ⫽ 3.99, p ⬍ 0.0001; Ftreatment ⫻ time (5,84) ⫽ 2.5, p ⬍ 0.01]. DISCUSSION In our earlier study [10], a chimeric peptide of met-enkephalin and FMRFa—YGGFMKKKFMRFa (YFa) was designed, based on the endogenous amphiactive peptide MERF and various reports on the role of NPFF/FMRFa peptides in pain modulation [11,22, 25,27]. The rationale of the study was to explore the possibility of a role of these sequences in antinociception and its modulation. Briefly, i.p. YFa in mice produced naloxone reversible, dose-
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FIG. 2. Influence of naloxone on the antinociceptive effect of [D-Ala2]YFa in the radiant-heat tail-flick test in mice. Naloxone (1.5 g/mouse, intracerebroventricular [i.c.v.]) administered 5 min prior reversed the antinociceptive effect of [D-Ala2]YFa. Naloxone (1.5 g/mouse, i.c.v.) alone had no significant antinociceptive effect. Antinociception (% maximum possible effect [MPE]) values for [D-Ala2]YFa (1 g/mouse, i.c.v.) are also represented (from Fig. 1). Data expressed as mean ⫾ SEM. *Significant difference from [D-Ala2]YFa-treated group (*p ⬍ 0.05).
dependent antinociception; potentiated morphine-induced antinociception and attenuated tolerance development—indicating a possible role of these amphiactive sequences in pain modulation [10]. Further, several reports indicate that NPFF, FMRFa and related peptides display either analgesia or blockade of opiate analgesia depending on such experimental conditions as route of administration. For example, intraventricular (i.v.) NPFF attenuates the antinociceptive effect of morphine administered i.c.v. [28,31], whereas NPFF administered i.t. produces a dose-dependent, longlasting antinociceptive effect that is partially naloxone reversible [8]. Because YFa contains FMRFa sequence [10], it would be interesting to examine if YFa administered i.v. also displays effects distinct from those following i.p. administration. Our earlier results [10] indicate that YFa degrades quite rapidly, hence the parent peptide was modified at its N-terminal. This synthetic stable analog [D-Ala2]YAGFMKKKFMRFamide ([D-Ala2]YFa) was administered centrally (i.c.v.) in mice to evaluate its effects on antinociception and its modulation. In the present study, i.c.v. administered [D-Ala2]YFa in doses of 1, 2, and 5 g/mouse (0.64, 1.28, and 3.22 nmol, respectively) displayed significant antinociceptive effect in the radiant-heat tailflick test in mice. However higher i.c.v. dose of the peptide—10 g/mouse—tended to produce respiratory depression. It has been previously reported that i.c.v. YGGFMRFamide produces no antinociception in rats [18]. In this study, it was demonstrated that although both YGGFMRF and YGGFMRFamide bind to opioid receptors, only YGGFMRF produces antinociception [18]. Because YGGFMRFamide also showed high binding potency at
NPFF receptors in comparison to YGGFMRF, it was proposed that both YGGFMRFamide and YGGFMRF may be acting as agonists at central opioid receptors, the additional anti-analgesic activity elicited by YGGFMRFamide at NPFF receptors may have masked its analgesic effect [18]. In the current experiments the reason for the difference in the antinociceptive effects of YGGFMRFamide and [D-Ala2]YFa is not clear. One possibility could be due to a difference in the animal model. In pilot studies with YGGFMRFamide, i.c.v. administration up to 40 g/mouse did not induce any antinociceptive effects in mice (data not shown). These results are in agreement with the earlier report by Payza et al. [18]. It should be noted, however, that it is not unprecedented for these types of chimeric sequences to show differing antinociceptive responses— [D-Ala2]metenkephalin-Arg6-Phe7amide is 130 times more potent analgesic than YGGFMRF and twice as potent than morphine when administered i.v. in mice [24]. One factor that may contribute to this variable response could be differential receptor binding of YGGFMRFamide and [D-Ala2]YFa. Our study does not exclude the possibility of [D-Ala2]YFa binding to receptors other than opioid and NPFF, such as MERF receptors [3]. The antinociceptive effect of [D-Ala2]YFa was completely reversed by i.c.v. administration of naloxone 1 g/mouse (4.12 nmol), administered 5 min prior, indicating opioid receptor mediation in the observed antinociception. FMRFa and NPFF have also been shown in one study to induce antinociception when administered by i.t. route in rats, and the mechanism ascribed is the indirect release of endogenous opioid peptides [8]. Because [D-Ala2]YFa contains the FMRFa sequence, the observed nalox-
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FIG. 3. Effect of [D-Ala2]YFa on the antinociceptive effect of morphine in the radiant-heat tail-flick test in mice. Morphine (2 g/mouse, intracerebroventricular [i.c.v.]) induced a significant antinociceptive effect. [D-Ala2]YFa (1 g/mouse, i.c.v.) when co-injected with morphine (2 g/mouse, i.c.v.) showed an additive antinociceptive effect. Antinociception (% maximum possible effect [MPE]) values for [D-Ala2]YFa (1 g/mouse, i.c.v.) alone are also represented (from Fig. 1) for comparison. Data expressed as mean ⫾ SEM. *Significant difference from the morphine-treated group (*p ⬍ 0.05). #p ⬍ 0.05 vs. saline-treated group.
one reversible antinociception could be due to direct opioid receptor stimulation and/or due to indirect release of endogenous opioid peptides. The interaction of this peptide [D-Ala2]YFa with anti-opioid peptides NPFF and FMRFa was also studied. The antinociceptive effect induced by [D-Ala2]YFa (1 g/mouse: 0.64 nmol, i.c.v.) was significantly attenuated by i.c.v. pretreatment (5 min) with either NPFF (1 g/mouse: 0.92 nmol, i.c.v.) or FMRFa (1 g/ mouse:1.69 nmol, i.c.v.). Higher doses were avoided because both NPFF and FMRFa have been reported to induce antinociception [8,12]. These results again suggest that the observed antinociception may be due to multiple binding of [D-Ala2]YFa to opioid as well as NPFF/FMRFa receptor(s). Previous reports have described the antagonism of morphine antinociception by NPFF/FMRFa family in rats [17]. The present study also evaluated the opioid-modulating properties of [D-Ala2]YFa by concurrent i.c.v. administration of [D-Ala2]YFa (1 g/mouse) with morphine (2 g/mouse: 5.86 nmol). Interestingly, [D-Ala2]YFa does not antagonise morphine antinociception, rather an additive antinociceptive effect was seen. Taken together, our present findings suggest that [D-Ala2]YFa displays opioid-like effects following i.c.v. administration in mice and the antiopioid/ antianalgesic effects of the FMRFa portion of the peptide is not evident in these experiments. The lack of any antiopioid action could possibly be explained if the model proposing that NPFF- and FMRFa-induced effects— opioid agonist as well as antagonist like— can be mediated by different antiopioid receptor subtypes
through differential G-protein coupling—Gi and Gs—is taken into account [23]. Accordingly, [D-Ala2]YFa may be activating antiopioid receptor via Gi, thereby producing only opioid like effects. An alternate explanation could be [D-Ala2]YFa may be behaving as a putative antagonist at NPFF receptor/binding site(s)—then the observed antinociceptive effect and lack of attenuation of morphine antinociception could be due to opioid receptor(s) stimulation and/or by antagonism at antiopioid receptor(s). The effect of [D-Ala2]YFa on development of tolerance to morphine antinociception would provide further insight into its mechanism of action and is currently under investigation. In conclusion, [D-Ala2]YFa causes modest to good antinociception in mice following i.c.v. administration. This antinociceptive effect is completely reversed by opioid receptor antagonist— naloxone and also significantly attenuated by the antiopioid peptides NPFF and FMRFa, indicating these effects may be mediated by opioid and/or antiopioid mechanism(s). Coinjection of [D-Ala2]YFa with morphine showed an additive antinociceptive effect, suggesting that [D-Ala2]YFa may have a modulatory effect on opioid (morphine) analgesia. These results provide further support for a role of such amphiactive sequences in antinociception and its modulation. ACKNOWLEDGEMENTS
This work was supported by Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC). We thank Rajbir Singh from Department of Biostatistics, All India Institute of Medical
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FIG. 4. Influence of antiopioid peptides neuropeptides FF (NPFF) and FMRFa on the antinociceptive effect of [D-Ala2]YFa in the radiant-heat tail-flick test in mice. NPFF (1 g/mouse, intracerebroventricular [i.c.v.]) (a) or FMRFa (1 g/mouse, i.c.v.) (b) was administered 5 min prior to [D-Ala2]YFa (1 g/mouse, i.c.v.). Both NPFF and FMRFa significantly attenuated antinociception induced by [D-Ala2]YFa. NPFF (1 g/mouse, i.c.v.) (a) or FMRFa (1 g/mouse, i.c.v.) (b) alone had no significant antinociceptive effects in the tail-flick test. Data expressed as mean ⫾ SEM. *Significant difference from NPFF or FMRFa-treated group (*p ⬍ 0.05). Abbreviation: MPE, maximum possible effect.
[D-ALA2]YFA IN ANTINOCICEPTION AND ITS MODULATION Sciences for statistical analysis of the data. We also thank Dr. Shyam Sunder Sharma, Department of Pharmacology, All India Institute of Medical Sciences for helping us with some of the pharmacological studies.
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