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Blockade of the antinociceptive effect of spinally administered kyotorphin by naltrindole in mice
Neuroscience Letters 322 (2002) 95–98 www.elsevier.com/locate/neulet
Blockade of the antinociceptive effect of spinally administered kyotorphin by naltrindole in mice Takehiro Ochi*, Yoshitaka Ohkubo, Seitaro Mutoh Department of Immunology and Inflammation, Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan Received 18 October 2001; received in revised form 16 January 2002; accepted 16 January 2002
Abstract We investigated the role of spinal opioid receptors in the antinociceptive effect of kyotorphin (Tyr-Arg, KTP) by using an in vivo mice tail-pinch test and an in vitro opioid receptor binding assays. Intrathecal administration of KTP produced a dose-dependent antinociceptive effect with an ED50 value of 24 mg/mouse. This antinociception, which was reversed by the KTP antagonist Leu-Arg, was completely blocked by naltrindole but not by naloxonazine, b-funaltrexamine, or norbinaltorphimine. The results from the binding study in vitro indicated that KTP bound to spinal KTP receptors but not to any opioid receptors in the mouse spinal cord. These results suggest that KTP-induced antinociception is mediated by binding to KTP receptors followed by an indirect activation of the d-opioid receptors in the spinal cord. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kyotorphin; Antinociception; Tail-pinch test; d-Opioid receptor; Mice
Kyotorphin (KTP) is an endogenous neurodipeptide, ltyrosyl-l-arginine (Tyr-Arg), isolated from the bovine brain [9,11]. This neuropeptide elicits morphine-like and naloxone-reversible analgesic effects [5], possibly mediated through a release of Met-enkephalin [8], and is considered to be a neurotransmitter for KTP receptors or a neuromodulator for opioidergic neuron. KTP is found in the rat brain and spinal cord [12]. However, the physiological role of KTP in the spinal cord has never been examined in in vivo studies. The aim of this study was to clarify the mechanism of the antinociceptive action of KTP in the spinal cord. To assess the analgesic activity of KTP at the spinal level, here we examined the effect of intrathecally (i.t.) administered KTP in the tail-pinch test in mice. To determine possible mediation through opioid receptors in the mechanism of the antinociception of KTP, we examined the effects of selective antagonists of the m-, d- and k-opioid receptors on the antinociception produced by i.t. administered KTP and the effect of KTP on various opioid receptor binding in vitro, in comparison with those of various opioid receptor agonists. * Corresponding author. Tel.: 181-6-6390-1297; fax: 181-66304-5367. E-mail address: [email protected] (T. Ochi).
Ethical guidelines for the experimental use of animals were followed [13]. In addition, the experimental work was reviewed by the Fujisawa Pharmaceutical Animal Experiment Committee for Animal Experimentation. Male ddY mice (25–30 g, Japan SLC, Hamamatsu, Japan) at the age of 6 weeks were used in all studies. The animals were maintained in a group of ten animals for at least 5 days on a 12-h light–dark cycle (light on from 07:00 to 19:00 h) in a controlled temperature (23 ^ 1 8C) and humidity (55 ^ 5%) environment. The animals were given standard laboratory food and tap water ad libitum before the experiment. The nociceptive response in the tail-pinch test was measured according to the modified Haffner’s method as previously reported [10]. Briefly, mice were pretested by pinching their tail base with an artery clip (1.5 mm width, 500 g constant force), and only the mice that showed a nociceptive response such as biting the clip or vocalizing within 2 s were used for experiments. When the mice did not show the above-mentioned behaviors up to 6 s after pinching, the antinociceptive effect was regarded as positive. To prevent tissue damage, the pressure stimuli were not applied for more than 10 s. After drug treatments, the nociceptive responses in the tail-pinch test were measured at 15-min intervals for a period of 90 min. The maximal antinociceptive effects of drugs were obtained 30 min after treatment
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and after that their effects gradually decreased. The antinociceptive effect was therefore measured 30 min after drug administration. Mouse spinal membranes were prepared as previously described [1]. The receptor sites were characterized by utilizing 5 nM [ 3H][d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin ([ 3H]DAMGO), 5 nM [ 3H][d-Phe 2,5]-enkephalin ([ 3H]DPDPE), 5 nM [ 3H](5a,7a,8a)-(1)-N-methyl-N-[7(1-pyrrolidinyl)-1-oxaspiro (4,5) dec-8-yl] benzeneacetamide ([ 3H]U-69,593) or 5 nM [ 3H]KTP. The obtained membranes were suspended in 30 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA, 0.025 units/ml aprotinin, 5 mM leupeptin and 0.5 mM pepstatin A. Drugs were dissolved in the above-mentioned buffer. The system was incubated for 1 h at 25 8C. Non-specific binding was determined in the presence of 100 mM or 1 mM of the unlabeled ligand for opioid receptor binding or KTP receptor binding, respectively. Free ligand was separated from bound ligand by vacuum filtration onto Whatman GF/B glass filters which had been preincubated in 0.5% polyethylimine for 2 h to reduce non-specific binding. Radioactivity was determined in the presence of 3 ml aqueous scintillant. The following drugs: KTP acetate salt; Leu-Arg acetate salt; DAMGO; DPDPE; U-69,593; naltrindole HCl; and nor-binaltorphimine diHCl, were obtained from Sigma (St. Louis, MO). Naloxonazine diHCl and b-funaltrexamine (bFNA) HCl were obtained from Research Biochemical International (Natick, MA). [ 3H]DAMGO, [ 3H]DPDPE and [ 3H]U-69,593 were from New England Nuclear (Wilmington, DE). [ 3H]KTP was from Amersham (Buckinghamshire, UK). For in vivo assay, drugs were dissolved and diluted in saline. Drug solutions were prepared just before starting experiments. Subcutaneous (s.c.) injection was performed in a volume of 10 ml/kg of animal weight, and i.t. injection was done in a volume of 5 ml/mouse. Intrathecal injection was performed according to the method of Hylden and Wilcox [2]. Ten animals were used for each of 4–5 doses to determine the ED50 value of a drug. The ED50 and IC50 values and 95% confidence limits (95% CL) were calculated from the dose– percentage inhibition relations by computer log-linear analysis [6]. The antinociceptive effect of KTP after i.t. administration was measured in the tail-pinch test in mice. As shown in Fig. 1A, the antinociceptive effect of KTP (8–64 mg/mouse) in a dose-dependent manner, which had an ED50 value (95% CL) of 24 (16–38) mg/mouse, was completely blocked by coadministration of naltrindole, a d-opioid receptor antagonist, at a dose of 10 mg/mouse. Moreover, the antinociceptive effect of KTP was also abolished by systemic (0.2 mg/kg, s.c.) administration of naltrindole (Fig. 1B). However, the antinociceptive effect of KTP was not attenuated by i.t. injection of 10 mg/mouse naloxonazine or 10 mg/mouse b-FNA, both m-opioid receptor antagonists, or 10 mg/ mouse nor-binaltorphimine, a k-opioid receptor antagonist
(Table 1). The antinociceptive effect of various opioid receptor agonists, DAMGO (m-), DPDPE (d-) and U69,593 (k-), was reversed by the respective antagonists.
Fig. 1. Effect of naltrindole on the antinociceptive effect of i.t. KTP in the tail-pinch test in mice. After measuring the normal nociceptive responses, KTP was administered i.t. (A) Naltrindole (closed circles) at 10 mg/mouse i.t. was co-administered with KTP (control: open circles). (B) Naltrindole (closed circles) at 0.2 mg/kg s.c. was injected immediately before i.t. administration of KTP (control: open circles). The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
T. Ochi et al. / Neuroscience Letters 322 (2002) 95–98
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Table 1 Effect of i.t. co-administered various opioid receptor antagonists on the antinociceptive effect of i.t. KTP in the tail-pinch test in mice a Antagonist (mg/mouse; i.t.)
KTP ED50: mg/mouse (95% CL)
Vehicle (saline) b-FNA 10 Naloxonazine 10 Naltrindole 10 Nor-binaltorphimine 10
24 (16–38) 19 (11–31) 25 (14–45) .128 23 (15–34)
a After measuring the normal nociceptive responses, KTP was administered i.t. Opioid receptor antagonists at 10 mg/mouse i.t. were co-administered with KTP. The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
Vehicle (saline) i.t. and various opioid antagonists i.t. or s.c. alone did not produce any antinociceptive effect. The antinociceptive effect caused by i.t. administration of KTP was blocked by co-administration of Leu-Arg, a KTP receptor antagonist, at a dose of 10 mg/mouse (Table 2). However, Leu-Arg at a dose of 100 mg/mouse i.t. failed to block the antinociceptive effect of DAMGO, DPDPE and U69,593. Leu-Arg at doses of 10 and 100 mg/mouse i.t. alone did not produce any antinociceptive effect. Binding studies were also carried out with membranes prepared from mouse spinal cord. KTP at concentrations of 0.01–100 mM did not bind to the various opioid receptors (data not shown). On the other hand, KTP and Leu-Arg showed potent displacement of the [ 3H]KTP binding in mouse spinal cord membranes with IC50 values of 1.9 ^ 0.47 and 0.45 ^ 0.082 mM, respectively (Fig. 2). The IC50 values of various opioid receptor agonists, DAMGO, DPDPE and U-59,693, in the displacement of [ 3H]KTP binding were over 100 mM. We previously reported that KTP (i.t.)-induced antinociception is antagonized by the KTP receptor antagonist LeuArg [7]. In this report, antinociception of i.t. injected KTP in mice was completely reversed by naltrindole. This finding suggests the involvement of d-opioid receptors in the spinal antinociception of KTP. Certainly, KTP depolarizes the
Fig. 2. Effect of various compounds on specific [ 3H]KTP binding to mouse spinal KTP receptors. Mouse spinal membranes were characterized by utilizing the following specific radiolabeled ligand [ 3H]KTP (5 nM) after incubation for 1 h at 25 8C. The binding of [ 3H]KTP to the KTP receptors was tested by the indicated concentrations of KTP (open circles), Leu-Arg (closed circles), DAMGO (open triangles), DPDPE (closed triangles) or U-69,593 (open squares; n ¼ 3).
Met-enkephalinergic neuron and releases endogenous opioid peptide, especially Met-enkephalin, from guinea pig striatum and spinal cord [8], resulting in naloxone-reversible antinociception [9]. l-Arg, a constituent amino acid of KTP, is considered to be an effective KTP precursor, and the antinociception of l-Arg after intracerebroventricular injection is antagonized by naltrindole and Leu-Arg [3,4]. In this study, i.t. co-administered Leu-Arg failed to reverse the antinociception of the d-opioid receptor agonist DPDPE, which was antagonized by naltrindole. Additionally, KTP itself bound to KTP receptors but not d-opioid receptors in the spinal cord. These results suggest the presence of a functional link between the KTP receptors, d-opioid peptide containing neuron and the d-opioid receptor system in the spinal cord as well as in the brain. Thus, KTP attenuates
Table 2 Effect of i.t. co-administered Leu-Arg on the antinociceptive effect of i.t. KTP and various opioid receptor agonists in the tail-pinch test in mice a Drug
KTP DAMGO DPDPE U-69,693
ED50: mg/mouse (95% CL) 1Vehicle (saline, i.t.)
1Leu-Arg (10 mg/mouse, i.t.)
1Leu-Arg (100 mg/mouse, i.t.)
24 (16–38) 0.015 (0.0083–0.023) 0.030 (0.019–0.044) 0.013 (0.0058–0.022)
.128 – – –
– 0.017 (0.0099–0.027) 0.029 (0.017–0.045) 0.016 (0.010–0.023)
a After measuring the normal nociceptive responses, drugs were administered i.t. Leu-Arg at 1 or 10 mg/mouse i.t. was co-administered with drugs. The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
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pain transmission through an indirect activation of spinal dopioid receptors. By contrast, KTP-induced antinociception was not antagonized by naloxonazine, b-FNA or nor-binaltorphimine. In addition to this, the antinociception of DAMGO and U69,593 was not attenuated by Leu-Arg. Our results imply that the antinociceptive mechanism of KTP is independent of the m- and k-opioid receptor systems, differing from those of m- and k-opioid agonists. In conclusion, spinal KTP is antinociceptive via the KTP–Met-enkephalin pathway followed by indirect activation of the d-opioid receptors in the spinal cord. [1] Chen, J., Smith, E.R., Cahill, M., Cohen, R. and Fishman, J.B., The opioid receptor binding of dezocine, morphine, fentanyl, butorphanol and nalbuphine, Life Sci., 52 (1993) 389–396. [2] Hylden, J.L.K. and Wilcox, G.L., Intrathecal morphine in mice: a new technique, Eur. J. Pharmacol., 67 (1980) 313– 316. [3] Kawabata, A., Nishimura, Y. and Takagi, H., l-Leucyl-l-arginine, naltrindole and d-arginine block antinociception elicited by l-arginine in mice with carrageenin-induced hyperalgesia, Br. J. Pharmacol., 107 (1992) 1096–1101. [4] Kawabata, A., Umeda, N. and Takagi, H., l-Arginine exerts a dual role in nociceptive processing in the brain: involvement of the kyotorphin-Met-enkephalin pathway and NOcyclic GMP pathway, Br. J. Pharmacol., 109 (1993) 73–79. [5] Kitabatake, S., Tsurutani, R., Nakajima, H., Tomita, K.,
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Yoshihara, Y., Ueda, H., Takagi, H. and Imahori, K., A novel method for the synthesis of kyotorphin, Tyr-Arg, and 3H-Tyr-Arg, catalyzed by tyrosyl-tRNA synthetase from Bacillus stearothermophilus, Pharmaceut. Res., 4 (1987) 154–157. Litchfield Jr., J.T. and Wilcoxon, F., A simplified method of evaluating dose–effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99–113. Ochi, T., Motoyama, Y. and Goto, T., The spinal antinociceptive effect of FR140423 is mediated through kyotorphin receptors, Life Sci., 66 (2000) 2239–2245. Shiomi, H., Kuraishi, Y., Ueda, H., Harada, Y., Amano, H. and Takagi, H., Mechanism of kyotorphin-induced release of Met-enkephalin from guinea pig striatum and spinal cord, Brain Res., 221 (1981) 161–169. Shiomi, H., Ueda, H. and Takagi, H., Isolation and identification of an analgesic opioid dipeptide kyotorphin (TyrArg) from bovine brain, Neuropharmacology, 20 (1981) 633–638. Takagi, H., Inukai, T. and Nakama, M., A modification of Haffner’s method for testing analgesics, Jpn. J. Pharmacol., 16 (1966) 287–294. Takagi, H., Shiomi, H., Ueda, H. and Amano, H., A novel analgesic dipeptide from bovine brain is a possible Metenkephalin releaser, Nature, 282 (1979) 410–412. Ueda, H., Shiomi, H. and Takagi, H., Regional distribution of a novel analgesic dipeptide kyotorphin (Tyr-Arg) in the rat brain and spinal cord, Brain Res., 198 (1980) 460–464. Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 (1983) 109–110.
Blockade of the antinociceptive effect of spinally administered kyotorphin by naltrindole in mice Takehiro Ochi*, Yoshitaka Ohkubo, Seitaro Mutoh Department of Immunology and Inflammation, Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan Received 18 October 2001; received in revised form 16 January 2002; accepted 16 January 2002
Abstract We investigated the role of spinal opioid receptors in the antinociceptive effect of kyotorphin (Tyr-Arg, KTP) by using an in vivo mice tail-pinch test and an in vitro opioid receptor binding assays. Intrathecal administration of KTP produced a dose-dependent antinociceptive effect with an ED50 value of 24 mg/mouse. This antinociception, which was reversed by the KTP antagonist Leu-Arg, was completely blocked by naltrindole but not by naloxonazine, b-funaltrexamine, or norbinaltorphimine. The results from the binding study in vitro indicated that KTP bound to spinal KTP receptors but not to any opioid receptors in the mouse spinal cord. These results suggest that KTP-induced antinociception is mediated by binding to KTP receptors followed by an indirect activation of the d-opioid receptors in the spinal cord. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kyotorphin; Antinociception; Tail-pinch test; d-Opioid receptor; Mice
Kyotorphin (KTP) is an endogenous neurodipeptide, ltyrosyl-l-arginine (Tyr-Arg), isolated from the bovine brain [9,11]. This neuropeptide elicits morphine-like and naloxone-reversible analgesic effects [5], possibly mediated through a release of Met-enkephalin [8], and is considered to be a neurotransmitter for KTP receptors or a neuromodulator for opioidergic neuron. KTP is found in the rat brain and spinal cord [12]. However, the physiological role of KTP in the spinal cord has never been examined in in vivo studies. The aim of this study was to clarify the mechanism of the antinociceptive action of KTP in the spinal cord. To assess the analgesic activity of KTP at the spinal level, here we examined the effect of intrathecally (i.t.) administered KTP in the tail-pinch test in mice. To determine possible mediation through opioid receptors in the mechanism of the antinociception of KTP, we examined the effects of selective antagonists of the m-, d- and k-opioid receptors on the antinociception produced by i.t. administered KTP and the effect of KTP on various opioid receptor binding in vitro, in comparison with those of various opioid receptor agonists. * Corresponding author. Tel.: 181-6-6390-1297; fax: 181-66304-5367. E-mail address: [email protected] (T. Ochi).
Ethical guidelines for the experimental use of animals were followed [13]. In addition, the experimental work was reviewed by the Fujisawa Pharmaceutical Animal Experiment Committee for Animal Experimentation. Male ddY mice (25–30 g, Japan SLC, Hamamatsu, Japan) at the age of 6 weeks were used in all studies. The animals were maintained in a group of ten animals for at least 5 days on a 12-h light–dark cycle (light on from 07:00 to 19:00 h) in a controlled temperature (23 ^ 1 8C) and humidity (55 ^ 5%) environment. The animals were given standard laboratory food and tap water ad libitum before the experiment. The nociceptive response in the tail-pinch test was measured according to the modified Haffner’s method as previously reported [10]. Briefly, mice were pretested by pinching their tail base with an artery clip (1.5 mm width, 500 g constant force), and only the mice that showed a nociceptive response such as biting the clip or vocalizing within 2 s were used for experiments. When the mice did not show the above-mentioned behaviors up to 6 s after pinching, the antinociceptive effect was regarded as positive. To prevent tissue damage, the pressure stimuli were not applied for more than 10 s. After drug treatments, the nociceptive responses in the tail-pinch test were measured at 15-min intervals for a period of 90 min. The maximal antinociceptive effects of drugs were obtained 30 min after treatment
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 10 9- X
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and after that their effects gradually decreased. The antinociceptive effect was therefore measured 30 min after drug administration. Mouse spinal membranes were prepared as previously described [1]. The receptor sites were characterized by utilizing 5 nM [ 3H][d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin ([ 3H]DAMGO), 5 nM [ 3H][d-Phe 2,5]-enkephalin ([ 3H]DPDPE), 5 nM [ 3H](5a,7a,8a)-(1)-N-methyl-N-[7(1-pyrrolidinyl)-1-oxaspiro (4,5) dec-8-yl] benzeneacetamide ([ 3H]U-69,593) or 5 nM [ 3H]KTP. The obtained membranes were suspended in 30 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA, 0.025 units/ml aprotinin, 5 mM leupeptin and 0.5 mM pepstatin A. Drugs were dissolved in the above-mentioned buffer. The system was incubated for 1 h at 25 8C. Non-specific binding was determined in the presence of 100 mM or 1 mM of the unlabeled ligand for opioid receptor binding or KTP receptor binding, respectively. Free ligand was separated from bound ligand by vacuum filtration onto Whatman GF/B glass filters which had been preincubated in 0.5% polyethylimine for 2 h to reduce non-specific binding. Radioactivity was determined in the presence of 3 ml aqueous scintillant. The following drugs: KTP acetate salt; Leu-Arg acetate salt; DAMGO; DPDPE; U-69,593; naltrindole HCl; and nor-binaltorphimine diHCl, were obtained from Sigma (St. Louis, MO). Naloxonazine diHCl and b-funaltrexamine (bFNA) HCl were obtained from Research Biochemical International (Natick, MA). [ 3H]DAMGO, [ 3H]DPDPE and [ 3H]U-69,593 were from New England Nuclear (Wilmington, DE). [ 3H]KTP was from Amersham (Buckinghamshire, UK). For in vivo assay, drugs were dissolved and diluted in saline. Drug solutions were prepared just before starting experiments. Subcutaneous (s.c.) injection was performed in a volume of 10 ml/kg of animal weight, and i.t. injection was done in a volume of 5 ml/mouse. Intrathecal injection was performed according to the method of Hylden and Wilcox [2]. Ten animals were used for each of 4–5 doses to determine the ED50 value of a drug. The ED50 and IC50 values and 95% confidence limits (95% CL) were calculated from the dose– percentage inhibition relations by computer log-linear analysis [6]. The antinociceptive effect of KTP after i.t. administration was measured in the tail-pinch test in mice. As shown in Fig. 1A, the antinociceptive effect of KTP (8–64 mg/mouse) in a dose-dependent manner, which had an ED50 value (95% CL) of 24 (16–38) mg/mouse, was completely blocked by coadministration of naltrindole, a d-opioid receptor antagonist, at a dose of 10 mg/mouse. Moreover, the antinociceptive effect of KTP was also abolished by systemic (0.2 mg/kg, s.c.) administration of naltrindole (Fig. 1B). However, the antinociceptive effect of KTP was not attenuated by i.t. injection of 10 mg/mouse naloxonazine or 10 mg/mouse b-FNA, both m-opioid receptor antagonists, or 10 mg/ mouse nor-binaltorphimine, a k-opioid receptor antagonist
(Table 1). The antinociceptive effect of various opioid receptor agonists, DAMGO (m-), DPDPE (d-) and U69,593 (k-), was reversed by the respective antagonists.
Fig. 1. Effect of naltrindole on the antinociceptive effect of i.t. KTP in the tail-pinch test in mice. After measuring the normal nociceptive responses, KTP was administered i.t. (A) Naltrindole (closed circles) at 10 mg/mouse i.t. was co-administered with KTP (control: open circles). (B) Naltrindole (closed circles) at 0.2 mg/kg s.c. was injected immediately before i.t. administration of KTP (control: open circles). The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
T. Ochi et al. / Neuroscience Letters 322 (2002) 95–98
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Table 1 Effect of i.t. co-administered various opioid receptor antagonists on the antinociceptive effect of i.t. KTP in the tail-pinch test in mice a Antagonist (mg/mouse; i.t.)
KTP ED50: mg/mouse (95% CL)
Vehicle (saline) b-FNA 10 Naloxonazine 10 Naltrindole 10 Nor-binaltorphimine 10
24 (16–38) 19 (11–31) 25 (14–45) .128 23 (15–34)
a After measuring the normal nociceptive responses, KTP was administered i.t. Opioid receptor antagonists at 10 mg/mouse i.t. were co-administered with KTP. The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
Vehicle (saline) i.t. and various opioid antagonists i.t. or s.c. alone did not produce any antinociceptive effect. The antinociceptive effect caused by i.t. administration of KTP was blocked by co-administration of Leu-Arg, a KTP receptor antagonist, at a dose of 10 mg/mouse (Table 2). However, Leu-Arg at a dose of 100 mg/mouse i.t. failed to block the antinociceptive effect of DAMGO, DPDPE and U69,593. Leu-Arg at doses of 10 and 100 mg/mouse i.t. alone did not produce any antinociceptive effect. Binding studies were also carried out with membranes prepared from mouse spinal cord. KTP at concentrations of 0.01–100 mM did not bind to the various opioid receptors (data not shown). On the other hand, KTP and Leu-Arg showed potent displacement of the [ 3H]KTP binding in mouse spinal cord membranes with IC50 values of 1.9 ^ 0.47 and 0.45 ^ 0.082 mM, respectively (Fig. 2). The IC50 values of various opioid receptor agonists, DAMGO, DPDPE and U-59,693, in the displacement of [ 3H]KTP binding were over 100 mM. We previously reported that KTP (i.t.)-induced antinociception is antagonized by the KTP receptor antagonist LeuArg [7]. In this report, antinociception of i.t. injected KTP in mice was completely reversed by naltrindole. This finding suggests the involvement of d-opioid receptors in the spinal antinociception of KTP. Certainly, KTP depolarizes the
Fig. 2. Effect of various compounds on specific [ 3H]KTP binding to mouse spinal KTP receptors. Mouse spinal membranes were characterized by utilizing the following specific radiolabeled ligand [ 3H]KTP (5 nM) after incubation for 1 h at 25 8C. The binding of [ 3H]KTP to the KTP receptors was tested by the indicated concentrations of KTP (open circles), Leu-Arg (closed circles), DAMGO (open triangles), DPDPE (closed triangles) or U-69,593 (open squares; n ¼ 3).
Met-enkephalinergic neuron and releases endogenous opioid peptide, especially Met-enkephalin, from guinea pig striatum and spinal cord [8], resulting in naloxone-reversible antinociception [9]. l-Arg, a constituent amino acid of KTP, is considered to be an effective KTP precursor, and the antinociception of l-Arg after intracerebroventricular injection is antagonized by naltrindole and Leu-Arg [3,4]. In this study, i.t. co-administered Leu-Arg failed to reverse the antinociception of the d-opioid receptor agonist DPDPE, which was antagonized by naltrindole. Additionally, KTP itself bound to KTP receptors but not d-opioid receptors in the spinal cord. These results suggest the presence of a functional link between the KTP receptors, d-opioid peptide containing neuron and the d-opioid receptor system in the spinal cord as well as in the brain. Thus, KTP attenuates
Table 2 Effect of i.t. co-administered Leu-Arg on the antinociceptive effect of i.t. KTP and various opioid receptor agonists in the tail-pinch test in mice a Drug
KTP DAMGO DPDPE U-69,693
ED50: mg/mouse (95% CL) 1Vehicle (saline, i.t.)
1Leu-Arg (10 mg/mouse, i.t.)
1Leu-Arg (100 mg/mouse, i.t.)
24 (16–38) 0.015 (0.0083–0.023) 0.030 (0.019–0.044) 0.013 (0.0058–0.022)
.128 – – –
– 0.017 (0.0099–0.027) 0.029 (0.017–0.045) 0.016 (0.010–0.023)
a After measuring the normal nociceptive responses, drugs were administered i.t. Leu-Arg at 1 or 10 mg/mouse i.t. was co-administered with drugs. The antinociceptive effect was determined by the modified Haffner’s method 30 min after drug injection in mice (n ¼ 10).
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