- Email: [email protected]
Characterization of intrathecally administered hemokinin-1-induced nociceptive behaviors in mice
Peptides 31 (2010) 1613–1616
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Characterization of intrathecally administered hemokinin-1-induced nociceptive behaviors in mice Chizuko Watanabe, Hirokazu Mizoguchi, Akihiko Yonezawa, Shinobu Sakurada ∗ Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 27 April 2010 Accepted 27 April 2010 Available online 6 May 2010 Keywords: Hemokinin-1 Tachykinin Nociceptive behavior Pain Spinal cord
a b s t r a c t Hemokinin-1 is a novel mammalian tachykinin cloned from mouse bone marrow. At present, pharmacological profile and physiological role of hemokinin-1 are still unclear. In the present study, we found that intrathecal (i.t.) administration of hemokinin-1 (0.00625–1.6 nmol) induced nociceptive responses consisting of scratching, biting and licking, which resemble substance P-induced behavioral responses in mice. The behaviors evoked by low-dose of hemokinin-1 (0.0125 nmol) were dose-dependently inhibited by i.t. co-administration of CP-99,994, a non-peptidic tachykinin NK1 receptor antagonist, whereas high-dose of hemokinin-1 (0.1 nmol)-induced behaviors were not affected. Moreover, sendide, a peptidic tachykinin NK1 receptor antagonist, failed to reduce the behavioral responses of both low- and high-dose of hemokinin-1. In contrast, substance P-induced behaviors were completely suppressed by both CP-99,994 and sendide. These results suggest that hemokinin-1 plays an important role in pain transmission at spinal cord. Moreover, the mechanism of hemokinin-1-induced nociceptive behaviors may be dose-dependent, and distinct from substance P-induced nociceptive behaviors. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The mammalian tachykinin family, substance P, neurokinin A and neurokinin B have been reported as endogenous ligands for tachykinin NK1 , NK2 and NK3 receptors, respectively. These tachykinin receptors, which are seven transmembrane G-proteincoupled receptors, are widely distributed in both central and peripheral nervous systems [10–12]. Hemokinin-1, a novel mammalian tachykinin, was cloned from mouse bone marrow on 2000 [15], and then identified from rat and named as rat/mouse (r/m) hemokinin-1 [8]. The sequence for r/m hemokinin-1 is RSRTRQFYGLM-NH2 . It has been found to resemble most closely to substance P and reported to have highest binding affinity for NK1 receptor than NK2 and NK3 receptors [1,9]. Tachykinin NK1 receptor is expressed in superficial layer of the spinal dorsal horn, an important site for pain transmission. Interestingly, hemokinin-1 mRNA, likewise substance P mRNA, is also expressed in the spinal cord and dorsal root ganglion [3]. The i.t. administration of substance P causes nociceptive responses consisting of scratching, biting and licking in mice [7,14]. Recently, it has been reported that i.t. administration of hemokinin-1 induced pain-related behavior such as scratching in rat [4]. Moreover, the intracerebroventricular administration of hemokinin-1 in
∗ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.04.025
mice or gerbils showed foot-tapping and scratching behaviors [3]. In the present study, the mechanism for the nociceptive response of hemokinin-1 at variable doses was investigated in the mouse spinal cord. 2. Materials and methods The experiments were performed according to a protocol approved by the Ethic Committee for Animal Experiments in Tohoku Pharmaceutical University. Every effort was made to minimize the number of animals used and their suffering. 2.1. Animals For the experiments, male ddY mice weighing 23–25 g (Japan SLC, Hamamatsu, Japan) were used. The animals were housed under controlled conditions for temperature (22–24 ◦ C), relative humidity (55 ± 5%), and a 12-h light-dark cycle (light on at 7:00 and light off at 19:00), and had free access to food and water. They were used only once. 2.2. Intrathecal administration Intrathecal administrations were made in conscious mice at the L5 and L6 intervertebral space in a volume at 5 l by the method of Hylden and Wilcox [6]. Drugs were administered i.t. with a 28-
1614
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
gauge needle attached to a 50 l Hamilton syringe. Puncture of the dura was sighed by a slight flick of the tail. 2.3. Behavioral observation Before the drug administration, mice were adapted to the plastic cage (22.0 cm × 15.0 cm × 12.5 cm) for 60 min, which was used as the observation chamber. Immediately following the i.t. administration, each mouse returned to their cage and the accumulated time for nociceptive responses, scratching, biting and licking, were measured at 5 min intervals. 2.4. Drugs The following drugs were used: hemokinin-1 (Tocris Cookson Inc, Bristol, UK), substance P (Peptide Institute, Osaka, Japan). CP-99,994 was obtained courtesy of Pfizer Pharmaceuticals. [Tyr6 ,D-Phe7 ,D-His9 ]substance P (6–11) (sendide) was synthesized by solid-phase peptide methodology in our laboratory. All drugs were dissolved in sterile artificial cerebrospinal fluid (CSF) containing: NaCl 126.6 mM, KCl 2.5 mM, MgCl2 2.0 mM and CaCl2 1.3 mM. NK1 receptor antagonists were co-administrated with hemokinin-1 or substance P. 2.5. Analyses of data The total time spent in scratching, biting and licking responses for 10 min is expressed as the mean ± SEM. The data were evaluated statistically for difference by Student’s t-test or one-way analysis of variance (ANOVA) followed by the Dunnett’s test. The dose–response curve with its ED50 value and 95% confidence interval was calculated with a computer-associated curve-fitting program (GraphPad Prism). 3. Results 3.1. Behavioral responses induced by i.t. administration of hemokinin-1 The i.t. administration of hemokinin-1 (0.00625–1.6 nmol) dose-dependently produced substance P-like behavioral responses consisting of scratching, biting and licking behaviors, which were peaked at 0–5 min and almost disappeared by 15 min after the injection (Fig. 1A). The nociceptive responses of hemokinin-1 were saturated at 0.1 nmol and no further increase of nociceptive behaviors were observed by higher doses of hemokinin-1 (0.4 and 1.6 nmol). The ED50 value with confidence interval for hemokini-1 was 0.02435 (0.01935–0.03064) nmol (Fig. 1B). 3.2. Effect of tachykinin NK1 receptor antagonists on the nociceptive behavior induced by i.t. administration of substance P and hemokinin-1 The nociceptive behaviors elicited by substance P (0.1 nmol) were dose-dependently attenuated by co-administration of CP-99,994 (0.125–2.0 nmol) and sendide (0.0625–2.0 pmol), and 2.0 nmol of CP-99,994 and 2.0 pmol of sendide completely suppressed substance P-induced nociceptive behaviors (Figs. 2A and 3A). The nociceptive behaviors elicited by low-dose of hemokinin-1 (0.00125 nmol) were also significantly inhibited by co-administration of CP-99,994 (Fig. 2B), whereas the nociceptive behaviors elicited by high-dose of hemokinin-1 (0.1 nmol) were not affected by CP-99,994 (Fig. 2C). On the other hand, co-administration of sendide (2.0 pmol) did not alter both lowand high-dose of hemokinin-1-induced behavioral responses (Fig. 3B and C).
Fig. 1. The behavioral responses induced by various doses of hemokinin-1 (0.00625–1.6 nmol) administered i.t. in mice. The duration of nociceptive behaviors induced by hemokinin-1 was measured over a 10-min period starting immediately after the injection. The data represent the mean ± SEM for 10 mice. (A) *P < 0.05, **P < 0.01, when compared with CSF treated group. (B) The dose–response curve was calculated with GraphPad Prism, a computer-associated curve-fitting program. ED50 = 0.02435 (0.01935–0.03064) nmol.
4. Discussion In the present study, we found that i.t. administration of hemokinin-1 elicited pain-related behaviors consisting of scratching, biting and licking, which are very similar with nociceptive behaviors induced by i.t. administration of substance P. It has been reported that hemokinin-1, as well as substance P, have a very high affinity and selectivity to the tachykinin NK1 receptor as compared with NK2 and NK3 receptors, and works as a NK1 receptor agonist. In the present study, a non-peptidic NK1 receptor antagonist, CP-99,994, suppressed low-dose of hemokinin-1-induced nociceptive behaviors as well as substance P-induced nociceptive behaviors. However, sendide, a peptidic NK1 receptor antagonist, failed to affect low-dose of hemokinin-1-elicited nociceptive behaviors, although substance P-induced nociceptive behaviors were completely inhibited by sendide. These results suggest that unlike substance P, low-dose of hemokinin-1-induced nociceptive responses may be mediated by the spinal NK1 receptor, which is insensitive to sendide. The tachykinin receptors are seven transmembrane G-proteincoupled receptors. Three types of NK receptors, NK1 , NK2 and NK3 receptors, have been described for substance P, neurokinin A and neurokinin B, respectively. The NK1 receptor is expressed in the superficial layers of spinal dorsal horn associated with pain pathway. It was recently reported that NK1 receptor is
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
1615
Fig. 2. Effect of CP-99,994 on the behavioral responses evoked by i.t.-administered substance P (A), low-dose (0.0125 nmol) of hemokinin-1 (B) and high-dose (0.1 nmol) of hemokinin-1 (C) in mice. The duration of nociceptive behaviors induced by substance P, low-dose of hemokinin-1 and high-dose of hemokinin-1 was measured over a 10-min period starting immediately after the injection. CP-99,994 was co-administered i.t. with each agonist. The data represent the mean ± SEM for 10 mice. **P < 0.01, when compared to each agonist alone-treated group.
Fig. 3. Effect of sendide on the behavioral responses evoked by i.t.-administered substance P (A), low-dose (0.0125 nmol) of hemokinin-1 (B) and high-dose (0.1 nmol) of hemokinin-1 (C) in mice. The duration of nociceptive behaviors induced by substance P, low-dose of hemokinin-1 and high-dose of hemokinin-1 was measured over a 10-min period starting immediately after the injection. Sendide was co-administered i.t. with each agonist. The data represent the mean ± SEM for 10 mice. **P < 0.01, when compared to each agonist alone-treated group.
divided into two receptor isoforms by different lengths of the C-terminal, long or short isoform. Long isoform is more responsive to substance P than the short isoform [5]. Moreover, the long isoform is abundantly expressed in the central nervous system including the spinal cord [2]. In the present study, NK1 receptor antagonist CP-99,994 completely suppressed substance P and low-dose of hemokinin-1-induced nociceptive responses,
whereas peptidic NK1 receptor antagonist sendide only suppresses substance P-induced nociceptive behaviors, but not low-dose of hemokinin-1-induced nociceptive behaviors. These results suggest that at low-dose hemokinin-1 binds to a different type of NK1 receptor with substance P. Sendide is a C-terminal analog of substance P and shows highest sensitivity to substance P as compared with other NK1 receptor agonists [13]. These evidences reveal the
1616
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
possibility that sendide may bind to the long isoform of NK1 receptor, which is more sensitive to substance P as compared with the short isoform, and low-dose of hemokinin-1 may preferentially bind to short isoform of NK1 receptor. In the present study, high-dose of hemokinin-1-induced nociceptive responses were not affected by the both NK1 receptor antagonists CP-99,994 and sendide. These data clearly suggest that high-dose of hemokinin-1-induced nociceptive responses are not mediated through NK1 receptors. At this point, the mechanism for high-dose of hemokinin-1-induced nociceptive responses is unknown. Extensive research is required to investigate the mechanism. In conclusion, i.t. administration of hemokinin-1 induced nociceptive behaviors consisting of scratching, biting and licking in mice. These effects were elicited through the NK1 receptor by low doses of hemikinin-1 and appeared to be insensitive to sendide, whereas high doses of hemikinin-1 induced nociceptive behaviors through mechanisms not implicating the NK1 receptor. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) (KAKENHI 18613015) from the Japan Society for the Promotion of Science, and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Bellucci F, Carni F, Catalani C, Cucchi P, Lecci A, Meini S, et al. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br J Pharmacol 2002;135:266–74.
[2] Caberlotto L, Hurd YL, Murdock P, Wahlin JP, Melotto S, Corsi M, et al. Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. Eur J Neurosci 2003;17:1736– 46. [3] Dufy RA, Hedrick JA, Randolph G, Morgan CA, Cohen-williams ME, Vassileva G, et al. Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance P-like behavioral effects in mice and gerbils. Neuropharmacology 2003;45:242–50. [4] Endo D, Ikeda T, Ishida Y, Yoshioka D, Nishimori T. Effects of intrathecal administration of hemokinin-1 on the withdrawal response to noxious thermal stimulation of the rat hind paw. Neurosci Lett 2006;392:114–7. [5] Fong TM, Anderson SA, Yu H, Huang RR, Strader CD. Differential action of intracellular effector by two isoforms of human neurokinin-1 receptor. Mol Pharmacol 1992;41:24–30. [6] Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–6. [7] Hylden JL, Wilcox GL. Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice. Brain Res 1981;217:212–5. [8] Kurtz MM, Wang R, Clements MK, Cascieri MA, Austin CP, Cunningham BR, et al. Identification, localization and receptor characterization of novel mammalian substance-like peptide. Gene 2002;296:205–12. [9] Morteau O, Lu B, Gerard C, Geraed NP. Hemokinin-1 is a full agonist at the substance P receptor. Nat Immunol 2001;2:1088. [10] Quartara L, Maggi CA. The tachykinin NK1 receptor: Part I. Ligands and mechanisms of cellular activation. Neuropeptides 1997;31:537– 63. [11] Quartara L, Maggi CA. The tachykinin NK1 receptor: Part II. Distribution and pathophysiological roles. Neuropeptides 1998;32:1–49. [12] Regoli D, Boudon A, Fauchere JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994;46:551–99. [13] Sakurada T, Manome Y, Tan-No K, Sakurada S, Kisara K, Ohba M, et al. A selective and extremely potent antagonist of the neurokinin-1 receptor. Brain Res 1992;593:319–22. [14] Takahasi K, Sakurada T, Sakurada S, Kuwahara H, Yonezawa A, Ando R, et al. Behavioural characterization of substance P-induced nociceptive response in mice. Neuropharmacology 1987;26:1289–93. [15] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoieticspecific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392–7.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Characterization of intrathecally administered hemokinin-1-induced nociceptive behaviors in mice Chizuko Watanabe, Hirokazu Mizoguchi, Akihiko Yonezawa, Shinobu Sakurada ∗ Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 27 April 2010 Accepted 27 April 2010 Available online 6 May 2010 Keywords: Hemokinin-1 Tachykinin Nociceptive behavior Pain Spinal cord
a b s t r a c t Hemokinin-1 is a novel mammalian tachykinin cloned from mouse bone marrow. At present, pharmacological profile and physiological role of hemokinin-1 are still unclear. In the present study, we found that intrathecal (i.t.) administration of hemokinin-1 (0.00625–1.6 nmol) induced nociceptive responses consisting of scratching, biting and licking, which resemble substance P-induced behavioral responses in mice. The behaviors evoked by low-dose of hemokinin-1 (0.0125 nmol) were dose-dependently inhibited by i.t. co-administration of CP-99,994, a non-peptidic tachykinin NK1 receptor antagonist, whereas high-dose of hemokinin-1 (0.1 nmol)-induced behaviors were not affected. Moreover, sendide, a peptidic tachykinin NK1 receptor antagonist, failed to reduce the behavioral responses of both low- and high-dose of hemokinin-1. In contrast, substance P-induced behaviors were completely suppressed by both CP-99,994 and sendide. These results suggest that hemokinin-1 plays an important role in pain transmission at spinal cord. Moreover, the mechanism of hemokinin-1-induced nociceptive behaviors may be dose-dependent, and distinct from substance P-induced nociceptive behaviors. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The mammalian tachykinin family, substance P, neurokinin A and neurokinin B have been reported as endogenous ligands for tachykinin NK1 , NK2 and NK3 receptors, respectively. These tachykinin receptors, which are seven transmembrane G-proteincoupled receptors, are widely distributed in both central and peripheral nervous systems [10–12]. Hemokinin-1, a novel mammalian tachykinin, was cloned from mouse bone marrow on 2000 [15], and then identified from rat and named as rat/mouse (r/m) hemokinin-1 [8]. The sequence for r/m hemokinin-1 is RSRTRQFYGLM-NH2 . It has been found to resemble most closely to substance P and reported to have highest binding affinity for NK1 receptor than NK2 and NK3 receptors [1,9]. Tachykinin NK1 receptor is expressed in superficial layer of the spinal dorsal horn, an important site for pain transmission. Interestingly, hemokinin-1 mRNA, likewise substance P mRNA, is also expressed in the spinal cord and dorsal root ganglion [3]. The i.t. administration of substance P causes nociceptive responses consisting of scratching, biting and licking in mice [7,14]. Recently, it has been reported that i.t. administration of hemokinin-1 induced pain-related behavior such as scratching in rat [4]. Moreover, the intracerebroventricular administration of hemokinin-1 in
∗ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.04.025
mice or gerbils showed foot-tapping and scratching behaviors [3]. In the present study, the mechanism for the nociceptive response of hemokinin-1 at variable doses was investigated in the mouse spinal cord. 2. Materials and methods The experiments were performed according to a protocol approved by the Ethic Committee for Animal Experiments in Tohoku Pharmaceutical University. Every effort was made to minimize the number of animals used and their suffering. 2.1. Animals For the experiments, male ddY mice weighing 23–25 g (Japan SLC, Hamamatsu, Japan) were used. The animals were housed under controlled conditions for temperature (22–24 ◦ C), relative humidity (55 ± 5%), and a 12-h light-dark cycle (light on at 7:00 and light off at 19:00), and had free access to food and water. They were used only once. 2.2. Intrathecal administration Intrathecal administrations were made in conscious mice at the L5 and L6 intervertebral space in a volume at 5 l by the method of Hylden and Wilcox [6]. Drugs were administered i.t. with a 28-
1614
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
gauge needle attached to a 50 l Hamilton syringe. Puncture of the dura was sighed by a slight flick of the tail. 2.3. Behavioral observation Before the drug administration, mice were adapted to the plastic cage (22.0 cm × 15.0 cm × 12.5 cm) for 60 min, which was used as the observation chamber. Immediately following the i.t. administration, each mouse returned to their cage and the accumulated time for nociceptive responses, scratching, biting and licking, were measured at 5 min intervals. 2.4. Drugs The following drugs were used: hemokinin-1 (Tocris Cookson Inc, Bristol, UK), substance P (Peptide Institute, Osaka, Japan). CP-99,994 was obtained courtesy of Pfizer Pharmaceuticals. [Tyr6 ,D-Phe7 ,D-His9 ]substance P (6–11) (sendide) was synthesized by solid-phase peptide methodology in our laboratory. All drugs were dissolved in sterile artificial cerebrospinal fluid (CSF) containing: NaCl 126.6 mM, KCl 2.5 mM, MgCl2 2.0 mM and CaCl2 1.3 mM. NK1 receptor antagonists were co-administrated with hemokinin-1 or substance P. 2.5. Analyses of data The total time spent in scratching, biting and licking responses for 10 min is expressed as the mean ± SEM. The data were evaluated statistically for difference by Student’s t-test or one-way analysis of variance (ANOVA) followed by the Dunnett’s test. The dose–response curve with its ED50 value and 95% confidence interval was calculated with a computer-associated curve-fitting program (GraphPad Prism). 3. Results 3.1. Behavioral responses induced by i.t. administration of hemokinin-1 The i.t. administration of hemokinin-1 (0.00625–1.6 nmol) dose-dependently produced substance P-like behavioral responses consisting of scratching, biting and licking behaviors, which were peaked at 0–5 min and almost disappeared by 15 min after the injection (Fig. 1A). The nociceptive responses of hemokinin-1 were saturated at 0.1 nmol and no further increase of nociceptive behaviors were observed by higher doses of hemokinin-1 (0.4 and 1.6 nmol). The ED50 value with confidence interval for hemokini-1 was 0.02435 (0.01935–0.03064) nmol (Fig. 1B). 3.2. Effect of tachykinin NK1 receptor antagonists on the nociceptive behavior induced by i.t. administration of substance P and hemokinin-1 The nociceptive behaviors elicited by substance P (0.1 nmol) were dose-dependently attenuated by co-administration of CP-99,994 (0.125–2.0 nmol) and sendide (0.0625–2.0 pmol), and 2.0 nmol of CP-99,994 and 2.0 pmol of sendide completely suppressed substance P-induced nociceptive behaviors (Figs. 2A and 3A). The nociceptive behaviors elicited by low-dose of hemokinin-1 (0.00125 nmol) were also significantly inhibited by co-administration of CP-99,994 (Fig. 2B), whereas the nociceptive behaviors elicited by high-dose of hemokinin-1 (0.1 nmol) were not affected by CP-99,994 (Fig. 2C). On the other hand, co-administration of sendide (2.0 pmol) did not alter both lowand high-dose of hemokinin-1-induced behavioral responses (Fig. 3B and C).
Fig. 1. The behavioral responses induced by various doses of hemokinin-1 (0.00625–1.6 nmol) administered i.t. in mice. The duration of nociceptive behaviors induced by hemokinin-1 was measured over a 10-min period starting immediately after the injection. The data represent the mean ± SEM for 10 mice. (A) *P < 0.05, **P < 0.01, when compared with CSF treated group. (B) The dose–response curve was calculated with GraphPad Prism, a computer-associated curve-fitting program. ED50 = 0.02435 (0.01935–0.03064) nmol.
4. Discussion In the present study, we found that i.t. administration of hemokinin-1 elicited pain-related behaviors consisting of scratching, biting and licking, which are very similar with nociceptive behaviors induced by i.t. administration of substance P. It has been reported that hemokinin-1, as well as substance P, have a very high affinity and selectivity to the tachykinin NK1 receptor as compared with NK2 and NK3 receptors, and works as a NK1 receptor agonist. In the present study, a non-peptidic NK1 receptor antagonist, CP-99,994, suppressed low-dose of hemokinin-1-induced nociceptive behaviors as well as substance P-induced nociceptive behaviors. However, sendide, a peptidic NK1 receptor antagonist, failed to affect low-dose of hemokinin-1-elicited nociceptive behaviors, although substance P-induced nociceptive behaviors were completely inhibited by sendide. These results suggest that unlike substance P, low-dose of hemokinin-1-induced nociceptive responses may be mediated by the spinal NK1 receptor, which is insensitive to sendide. The tachykinin receptors are seven transmembrane G-proteincoupled receptors. Three types of NK receptors, NK1 , NK2 and NK3 receptors, have been described for substance P, neurokinin A and neurokinin B, respectively. The NK1 receptor is expressed in the superficial layers of spinal dorsal horn associated with pain pathway. It was recently reported that NK1 receptor is
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
1615
Fig. 2. Effect of CP-99,994 on the behavioral responses evoked by i.t.-administered substance P (A), low-dose (0.0125 nmol) of hemokinin-1 (B) and high-dose (0.1 nmol) of hemokinin-1 (C) in mice. The duration of nociceptive behaviors induced by substance P, low-dose of hemokinin-1 and high-dose of hemokinin-1 was measured over a 10-min period starting immediately after the injection. CP-99,994 was co-administered i.t. with each agonist. The data represent the mean ± SEM for 10 mice. **P < 0.01, when compared to each agonist alone-treated group.
Fig. 3. Effect of sendide on the behavioral responses evoked by i.t.-administered substance P (A), low-dose (0.0125 nmol) of hemokinin-1 (B) and high-dose (0.1 nmol) of hemokinin-1 (C) in mice. The duration of nociceptive behaviors induced by substance P, low-dose of hemokinin-1 and high-dose of hemokinin-1 was measured over a 10-min period starting immediately after the injection. Sendide was co-administered i.t. with each agonist. The data represent the mean ± SEM for 10 mice. **P < 0.01, when compared to each agonist alone-treated group.
divided into two receptor isoforms by different lengths of the C-terminal, long or short isoform. Long isoform is more responsive to substance P than the short isoform [5]. Moreover, the long isoform is abundantly expressed in the central nervous system including the spinal cord [2]. In the present study, NK1 receptor antagonist CP-99,994 completely suppressed substance P and low-dose of hemokinin-1-induced nociceptive responses,
whereas peptidic NK1 receptor antagonist sendide only suppresses substance P-induced nociceptive behaviors, but not low-dose of hemokinin-1-induced nociceptive behaviors. These results suggest that at low-dose hemokinin-1 binds to a different type of NK1 receptor with substance P. Sendide is a C-terminal analog of substance P and shows highest sensitivity to substance P as compared with other NK1 receptor agonists [13]. These evidences reveal the
1616
C. Watanabe et al. / Peptides 31 (2010) 1613–1616
possibility that sendide may bind to the long isoform of NK1 receptor, which is more sensitive to substance P as compared with the short isoform, and low-dose of hemokinin-1 may preferentially bind to short isoform of NK1 receptor. In the present study, high-dose of hemokinin-1-induced nociceptive responses were not affected by the both NK1 receptor antagonists CP-99,994 and sendide. These data clearly suggest that high-dose of hemokinin-1-induced nociceptive responses are not mediated through NK1 receptors. At this point, the mechanism for high-dose of hemokinin-1-induced nociceptive responses is unknown. Extensive research is required to investigate the mechanism. In conclusion, i.t. administration of hemokinin-1 induced nociceptive behaviors consisting of scratching, biting and licking in mice. These effects were elicited through the NK1 receptor by low doses of hemikinin-1 and appeared to be insensitive to sendide, whereas high doses of hemikinin-1 induced nociceptive behaviors through mechanisms not implicating the NK1 receptor. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) (KAKENHI 18613015) from the Japan Society for the Promotion of Science, and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] Bellucci F, Carni F, Catalani C, Cucchi P, Lecci A, Meini S, et al. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br J Pharmacol 2002;135:266–74.
[2] Caberlotto L, Hurd YL, Murdock P, Wahlin JP, Melotto S, Corsi M, et al. Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. Eur J Neurosci 2003;17:1736– 46. [3] Dufy RA, Hedrick JA, Randolph G, Morgan CA, Cohen-williams ME, Vassileva G, et al. Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance P-like behavioral effects in mice and gerbils. Neuropharmacology 2003;45:242–50. [4] Endo D, Ikeda T, Ishida Y, Yoshioka D, Nishimori T. Effects of intrathecal administration of hemokinin-1 on the withdrawal response to noxious thermal stimulation of the rat hind paw. Neurosci Lett 2006;392:114–7. [5] Fong TM, Anderson SA, Yu H, Huang RR, Strader CD. Differential action of intracellular effector by two isoforms of human neurokinin-1 receptor. Mol Pharmacol 1992;41:24–30. [6] Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–6. [7] Hylden JL, Wilcox GL. Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice. Brain Res 1981;217:212–5. [8] Kurtz MM, Wang R, Clements MK, Cascieri MA, Austin CP, Cunningham BR, et al. Identification, localization and receptor characterization of novel mammalian substance-like peptide. Gene 2002;296:205–12. [9] Morteau O, Lu B, Gerard C, Geraed NP. Hemokinin-1 is a full agonist at the substance P receptor. Nat Immunol 2001;2:1088. [10] Quartara L, Maggi CA. The tachykinin NK1 receptor: Part I. Ligands and mechanisms of cellular activation. Neuropeptides 1997;31:537– 63. [11] Quartara L, Maggi CA. The tachykinin NK1 receptor: Part II. Distribution and pathophysiological roles. Neuropeptides 1998;32:1–49. [12] Regoli D, Boudon A, Fauchere JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994;46:551–99. [13] Sakurada T, Manome Y, Tan-No K, Sakurada S, Kisara K, Ohba M, et al. A selective and extremely potent antagonist of the neurokinin-1 receptor. Brain Res 1992;593:319–22. [14] Takahasi K, Sakurada T, Sakurada S, Kuwahara H, Yonezawa A, Ando R, et al. Behavioural characterization of substance P-induced nociceptive response in mice. Neuropharmacology 1987;26:1289–93. [15] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoieticspecific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392–7.