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Anti-analgesic and anti-amnesic effect of complement C3a
Life Sciences 67 (2000) 2137Ð2143
Pharmacology letters Accelerated communication
Anti-analgesic and anti-amnesic effect of complement C3a Yunden Jinsmaaa, Masakatsu Takahashib, Masakazu Takahashic, Masaaki Yoshikawaa,* a
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8521, Japan c Faculty of Biotechnology, Fukui Prefectural University, Fukui 910-1195, Japan
b
(Submitted November 8, 1999; accepted November 29, 1999; received in Þnal form May 12, 2000)
Abstract In the present study, we found that complement C3a exerted central effects after intracerebroventricular administration in mice. At doses of 3 and 10 pmol/mouse, the peptide showed an antagonistic effect on analgesia induced by morphine and U-50488H, known to be m- and k-opioid receptor agonists, respectively. Moreover, complement C3a improved scopolamine- and ischemia-induced amnesia at a dose of 10 pmol/mouse. Anti-analgesia was not observed by C3a des-Arg at 10 pmol/mouse. The present Þndings suggest that complement C3a may act as a peptide with anti-opioid activity in the central nervous system. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Analgesia; Anti-opioid; Amnesia; Complement C3a; Morphine
Introduction Complement C3a is a 77-amino acid residue peptide of the immune system that is released from C3 by C3 convertase during activation of the complement system. The peptide loses its C3a activity following removal of its C-terminal arginine (C3a des-Arg) (1). C3a mediates a number of biological effects such as chemotaxis of neutrophils, degranulation of mast cells, smooth muscle contraction and increasing vascular permeability (2). Receptors for complement C3a were found in platelets, mast cells, neutrophils, eosinophilils and macrophages (2Ð4). Recently, it has been reported that receptors are also expressed in the lung, spleen, placenta, small intestine and throughout the brain (5); neurons are the predominant cell type expressing C3a receptor in the central nervous system (CNS), especially in cortical and hippocampal * Corresponding author. Tel.: 181-774-38-3725; fax: 181-774-38-3774. E-mail address: [email protected] (M. Yoshikawa) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 0 0 -6
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neurons (6). In our previous paper, we stated that complement C3a showed anti-opioid activity in guinea-pig ileum (GPI) assay by antagonizing opioid-induced inhibition of electricallystimulated contraction of a longitudinal muscle strip (7). In this study, we tested whether complement C3a exerts its anti-opioid effects in the CNS. Following i.c.v. injection, complement C3a showed an antagonistic effect on morphine-induced analgesia besides demonstrating improvement of scopolamine- and ischemia-induced amnesia in mice. Materials and methods Materials Human complement C3a, C3a des-Arg and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) were from Calbiochem-Novabiochem Co., San Diego, CA. Scopolamine, U-50488H (trans-(1)3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide) were from Sigma Chemical Co., St. Louis, MO. Morphine-HCl was from Takeda Chemical Industries Ltd., Osaka, Japan. Animals Male ddy mice (4 weeks old, 22Ð25 g) were purchased from Shimizu Laboratory Supplies, Kyoto, Japan. The animals were housed in standard plastic cages in a temperature-controlled room (238C) on a daily 12 h light : 12 h dark cycle with food and water freely available. Anti-analgesic test Anti-analgesic effect of C3a was measured on analgesia induced by m-, k- and d-opioid agonists in mice. The peptide solution in PBS was given intracerebroventricularly 5 min before morphine (5 mg/kg s.c.), U-50488H (30 mg/kg s.c.) and DTLET (3 nmole/mouse i.c.v.) administration, and analgesia was measured every 15 min for 1 hour after opioid injection by tail-pinch test or by hot plate test. The i.c.v. injection was made with a 4-mm-long needle attached to a 50 ml Hamilton microsyringe. Pharmacological tests and care of animals were in accordance with standard ethical guidelines (NIH, 1985). Passive avoidance test For the step-through type passive avoidance test, mice were trained in the apparatus, which consists of 2 compartments, illuminated and dark, with an electrode under the grid of the ßoor of the dark room. For the training, mice were placed in the illuminated room and the time taken to retreat to the dark room was measured. When the mouse stepped through the dark room, an electric shock (28Ð29 V, 5 sec delay) was delivered through the grid. The electric shock was continued until the mouse returned to the illuminated room. Twenty-four hours after the training, the mouse was placed again in illuminated room and the time taken to enter the dark room was measured. Amnesia was induced by i.p. administration of 0.1 mg/kg of scopolamine 30 min before training (scopolamine amnesia) or by interrupting the blood ßow of bilateral common carotid arteries for 10 min 3 days before training to induce ischemia in mice (ischemic amnesia). The peptide was given by i.c.v. administration just after the train-
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ing. For scopolamine-induced amnesia, statistical analysis was performed to test for difference between the treated and non-treated groups, while in the ischemia-induced amnesia, statistical comparison was made between the treated group and the sham-operated group, in which mice were operated upon just as in the experimental groups without pinching the cervical artery. Statistical analysis Statistical analyses for anti-analgesic test and passive avoidance experiment were performed by StudentÕs and Mann-WhitneyÕs U-tests, respectively. Results Anti-analgesic effect of complement C3a The anti-analgesic effect of complement C3a was measured by tail-pinch test in mice. Morphine, U-50488H and DTLET, m- , k- and d-opioid receptor agonists, respectively, induced analgesia after s.c. or i.c.v. administration. Intracerebroventricularly injected complement C3a dose-dependently inhibited morphine- and U-50488H-induced analgesia, whereas C3a failed to show any antagonistic effect on analgesia induced by DTLET (data not shown). As shown in Fig. 1A, morphine-induced analgesia was signiÞcantly inhibited by C3a at doses of 3 and 10 pmol/mouse, whereas U-50488H-induced analgesia only by 10 pmol/ mouse of C3a (Fig. 1B). The antagonistic effect of C3a was also observed against morphineinduced analgesia by the hot plate test (Fig. 1C). C3a show neither hyperalgesic nor analgesic activity by itself (data not shown). As reported previously, C3a was evidenced to exert strong anti-opioid activity in GPI assay via complement C3a receptor (11). Arginine at the carboxyl terminus of C3a is an impor-
Fig. 1. Antagonistic effect of C3a on morphine- (A), U-50488H-induced (B) analgesia by tail pinch test and on morphine-induced analgesia by hot plate test in mice (C). C3a was given intracerebroventricularly. Physiological saline was given to control mice. Each point represents the mean 6 s.e.m. ** p , 0.02, * p , 0.05 vs control (n 5 6).
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tant residue for binding to C3a receptor. Removal of this residue by carboxypeptidase N leads to disappearance of C3a activity (1). Morphine-induced analgesia was not blocked by C3a des-Arg at a dose of 10 pmol/mouse (data not shown). Anti-amnesic effect of complement C3a It is known that anti-opioids such as naloxone, cholecystokinin and thyrotropin-releasing hormone have learning- and memory-improving effects (8Ð11). These Þndings prompted us to address the possible effect of complement C3a on learning by a step-through passive avoidance task in mice. Despite the lack of improvement of learning ability in normal mice (data not shown), complement C3a was found to improve learning in scopolamine- and ischemia-amnesic mice. Scopolamine-induced amnesia Step-through passive avoidance experiment in mice was performed to verify the possible effect of C3a on scopolamine-induced amnesia. In the preceding training trials, achievement of the control mice was consistent with that of the mice treated with scopolamine (data not shown). However, scopolamine reduced the latency in the test trial, indicating the onset of amnesia. C3a reduced the amnesia at a dose of 10 pmol/mouse after i.c.v. administration to the level of the control group (Fig. 2). Ischemia-induced amnesia The effect of complement C3a on ischemia-induced amnesia was studied in mice. Interruption of the blood ßow of cervical arteries for 10 min established deÞnite amnesia in mice 3 days later. C3a improved amnesia also in this model system at a dose of 10 pmol/mouse (Fig. 3).
Fig. 2. Anti-amnesic effect of C3a on scopolamine (scop)-induced amnesia. Peptide (i.c.v.) was given after the training. Data are expressed as medians 6 quarter point. * p , 0.01 vs control, # p , 0.01 vs scop. group (n 5 9).
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Fig. 3. Anti-amnesic effect of C3a on ischemia-induced amnesia. Peptide (i.c.v.) was given after the training. Data are expressed as medians 6 quarter point. * p , 0.05 vs control, # p , 0.05 vs ischemia (n 5 8).
Discussion There are many reports showing effects of neuropeptides on the immune system (12Ð14), while a certain report deals with the effect of immunopeptides on the nervous system (15). To our best knowledge, certain effects of an immunopeptide complement C3a are considered to be a new example in this Þeld. It has been reported that C3a increased the eating response to norepinephrine and the drinking response to carbamyl choline after hypothalamic injection in rats (16). Furthermore, C3a receptor mRNA was found in the brain (17), hippocampus (5), astrocytes, astrocyte cell lines (18) and in neurons (6) together with C3 mRNA in the brain (19). All these Þndings suggest that complement C3a might play an important role in the CNS. In our previous paper, we reported that C3a showed strong anti-opioid activity against m-opioid agonist DAMGO in GPI assay and caused ileum contraction by release of histamine from mast cells as well as prostaglandin E2 (PGE2) and acetylcholine from neuronal cells (7). In this study, we showed that complement C3a injected intracerebroventricularly in mice acts on CNS as a peptide having anti-analgesic and anti-amnesic activities. An anti-analgesic effect was not observed by C3a des-Arg, which suggests that these effects are speciÞc for C3a. It is conceivable that anti-analgesic and anti-amnesic effects of exogenously injected C3a might be mediated by release of histamine, PGE2 and/or acetylcholine in the brain, because all these substances act on pain perception and on memory function after i.c.v. administration in experimental animals (20Ð23). C3a antagonized m- and k-opioid analgesia, whereas it failed to show any effect on d-opioid analgesia, which might be explained by differences in neurotransmitters released by opioids. In fact, C3a did not antagonize d-opioid activity of DTLET in a mouse vas deferens assay (data not shown). Further study on the mechanisms of action of complement C3a in the CNS would contribute to the treatment of memory deÞcits.
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Thus, complement C3a exerts effects on impaired systems such as scopolamine- and ischemia-induced amnesia, though it had no effect on learning ability in normal mice. Acknowledgments This work was supported in part by grants to M.Y. from the Ministry of Education, Science, Sports and Culture of Japan, and PROBRAIN grant from Bio-oriented Technology Research Advancement Institution. References 1. Wilken HC, Gotze O, Werfel T. Zwirner J. C3a(desArg) does not bind to and signall through the human C3a receptor. Immunol Lett. 1999; 67(2): 141Ð5. 2. Bitter-Suermann D. The Complement System. In: Rother K, Till G, editors. Springer-Verlag, Heidelberg: 1988. pp. 367Ð395. 3. Fukuoka Y, Hugli TE. Demonstration of a speciÞc C3a receptor on guinea pig platelets. J Immunol. 1988; 140(10): 3496Ð501. 4. Legler DF, Loetscher M, Jones SA, Dahinden CA, Arock M, Moser B. Expression of high- and low-afÞnity receptors for C3a on the human mast cell line, HMC-1. Eur J Immunol. 1996; 26(4): 753Ð8. 5. Ames RS, Li Y, Sarau HM, Nuthulaganti P, Folley JJ, Ellis C, Zeng Z, Su K, Jurewicz AJ, Hertzberg RP, Bergsma DJ, Kumar C. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J Biol Chem. 1996; 271(34): 20231Ð4. 6. Davoust N, Jones J, Stahel PF, Ames RS, Barnum SR. Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells. Glia. 1999; 26(3): 201Ð11. 7. Takahashi T, Moriguchi S, Tani F, Shiota A, Suganuma H, Kono S, Usui H, Kurahashi K, Sasaki R, Yoshikawa M. IdentiÞcation of casoxin C, an ileum-contracting peptide derived from bovine kappa-casein, as an agonist for C3a receptors. Peptides. 1997; 8(3): 329Ð36. 8. Rush DK. Reversal of scopolamine-induced amnesia of passive avoidance by pre- and post-training naloxone. Psychopharmacology (Berl). 1986; 89(3): 296Ð300. 9. Ilyutchenok RY, Dubrovina NI. Memory retrieval enhancement by kappa opioid agonist and mu, delta antagonists. Pharmacol Biochem Behav. 1995; 52(4): 683Ð7. 10. Maurice T, Hiramatsu M, Kameyama T, Hasegawa T, Nabeshima T. Cholecystokinin-related peptides, after systemic or central administration, prevent carbon monoxide-induced amnesia in mice. J Pharmacol Exp Ther. 1994; 269(2): 665Ð73. 11. Ogasawara T, Itoh Y, Tamura M, Ukai Y, Yoshikuni Y, Kimura K. NS-3, a TRH-analog, reverses memory disruption by stimulating cholinergic and noradrenergic systems. Pharmacol Biochem Behav. 1996; 53(2): 391Ð9. 12. Cheido MA, Idova GV. Effect of opioid peptides on immunomodulation. Ross Fiziol Zh Im I M Sechenova. 1998; 84(4): 385Ð90. 13. Goldman R, Bar-Shavit Z. On the mechanism of the augmentation of the phagocytic capability of phagocytic cells by Tuftsin, substance P, neurotensin, and kentsin and the interrelationship between their receptors. Ann N Y Acad Sci. 1983; 419: 143Ð55. 14. von Horsten S, Ballof J, Helfritz F, Meyer D, Schnmidt RE, Stalp M, Klemm A, Tscening T, Pabst R. Modulation of innate immune functions by intracerebrovent ricularly applied neuropeptide Y: dose and time dependent effects. Life Sci. 1998; 63(11): 909Ð22. 15. Ma TC, Zhu XZ. Intrahippocampal infusion of interleukin-6 impairs avoidance learning in rats. Chung Kuo Yao Li Hsueh Pao. 1997; 18(2): 121Ð3. 16. Schupf N, Williams CA, Hugli TE, Cox J. Psychopharmacological activity of anaphylatoxin C3a in rat hypothalamus. J Neuroimmunol. 1983; 5(3): 305Ð16. 17. Hsu MN, Eember JA, Wang M, Prossnitz ER, Hugli TE, Ye RD. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene. Immunogenetics. 1997; 47(1): 64Ð72.
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18. Gasque P, Singhrao SK, Neal JW, Wang P, Sayah S, Fontaine M, Morgan BP. The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inßamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. J Immunol. 1998; 160(7): 3543Ð54. 19. Haga S, Ikeda K, Sato M, Ishii T. Synthetic Alzheimer amyloid beta/A4 peptides enhance production of complement C3 component by cultured microglial cells. Brain Res. 1993; 601(1Ð2): 88Ð94. 20. Khasar SG, Ho T, Green PG, Levine JD. Comparison of prostaglandin E1- and prostaglandin E2-induced hyperalgesia in the rat. Neuroscience. 1994; 62(2): 345Ð50. 21. Onodera K, Miyazaki S, Imaizumi M, Stark H, Schunack W. Improvement by FUB 181, a novel histamine H3-receptor antagonist, of learning and memory in the elevated plus-maze test in mice. Naunyn Schmiedebergs Arch Pharmacol. 1998; 357(5): 508Ð13. 22. Yamamuro Y, Iwano H, Sensui N, Hori K, Nomura M. Acetylcholine in the hippocampus during the discrimination learning performance in a rat model of chronic cerebral ischaemia. Neuroreport. 1996; 7(11): 1837Ð40. 23. Matsuda S, Wen TC, Karasawa Y, Araki H, Otsuka H, Ishihara K, Sakanaka M. Protective effect of a prostaglandin I2 analog, TEI-7165, on ischemic neuronal damage in gerbils. Brain Res. 1997; 769(2): 321Ð8.
Pharmacology letters Accelerated communication
Anti-analgesic and anti-amnesic effect of complement C3a Yunden Jinsmaaa, Masakatsu Takahashib, Masakazu Takahashic, Masaaki Yoshikawaa,* a
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8521, Japan c Faculty of Biotechnology, Fukui Prefectural University, Fukui 910-1195, Japan
b
(Submitted November 8, 1999; accepted November 29, 1999; received in Þnal form May 12, 2000)
Abstract In the present study, we found that complement C3a exerted central effects after intracerebroventricular administration in mice. At doses of 3 and 10 pmol/mouse, the peptide showed an antagonistic effect on analgesia induced by morphine and U-50488H, known to be m- and k-opioid receptor agonists, respectively. Moreover, complement C3a improved scopolamine- and ischemia-induced amnesia at a dose of 10 pmol/mouse. Anti-analgesia was not observed by C3a des-Arg at 10 pmol/mouse. The present Þndings suggest that complement C3a may act as a peptide with anti-opioid activity in the central nervous system. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Analgesia; Anti-opioid; Amnesia; Complement C3a; Morphine
Introduction Complement C3a is a 77-amino acid residue peptide of the immune system that is released from C3 by C3 convertase during activation of the complement system. The peptide loses its C3a activity following removal of its C-terminal arginine (C3a des-Arg) (1). C3a mediates a number of biological effects such as chemotaxis of neutrophils, degranulation of mast cells, smooth muscle contraction and increasing vascular permeability (2). Receptors for complement C3a were found in platelets, mast cells, neutrophils, eosinophilils and macrophages (2Ð4). Recently, it has been reported that receptors are also expressed in the lung, spleen, placenta, small intestine and throughout the brain (5); neurons are the predominant cell type expressing C3a receptor in the central nervous system (CNS), especially in cortical and hippocampal * Corresponding author. Tel.: 181-774-38-3725; fax: 181-774-38-3774. E-mail address: [email protected] (M. Yoshikawa) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 0 0 -6
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neurons (6). In our previous paper, we stated that complement C3a showed anti-opioid activity in guinea-pig ileum (GPI) assay by antagonizing opioid-induced inhibition of electricallystimulated contraction of a longitudinal muscle strip (7). In this study, we tested whether complement C3a exerts its anti-opioid effects in the CNS. Following i.c.v. injection, complement C3a showed an antagonistic effect on morphine-induced analgesia besides demonstrating improvement of scopolamine- and ischemia-induced amnesia in mice. Materials and methods Materials Human complement C3a, C3a des-Arg and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) were from Calbiochem-Novabiochem Co., San Diego, CA. Scopolamine, U-50488H (trans-(1)3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide) were from Sigma Chemical Co., St. Louis, MO. Morphine-HCl was from Takeda Chemical Industries Ltd., Osaka, Japan. Animals Male ddy mice (4 weeks old, 22Ð25 g) were purchased from Shimizu Laboratory Supplies, Kyoto, Japan. The animals were housed in standard plastic cages in a temperature-controlled room (238C) on a daily 12 h light : 12 h dark cycle with food and water freely available. Anti-analgesic test Anti-analgesic effect of C3a was measured on analgesia induced by m-, k- and d-opioid agonists in mice. The peptide solution in PBS was given intracerebroventricularly 5 min before morphine (5 mg/kg s.c.), U-50488H (30 mg/kg s.c.) and DTLET (3 nmole/mouse i.c.v.) administration, and analgesia was measured every 15 min for 1 hour after opioid injection by tail-pinch test or by hot plate test. The i.c.v. injection was made with a 4-mm-long needle attached to a 50 ml Hamilton microsyringe. Pharmacological tests and care of animals were in accordance with standard ethical guidelines (NIH, 1985). Passive avoidance test For the step-through type passive avoidance test, mice were trained in the apparatus, which consists of 2 compartments, illuminated and dark, with an electrode under the grid of the ßoor of the dark room. For the training, mice were placed in the illuminated room and the time taken to retreat to the dark room was measured. When the mouse stepped through the dark room, an electric shock (28Ð29 V, 5 sec delay) was delivered through the grid. The electric shock was continued until the mouse returned to the illuminated room. Twenty-four hours after the training, the mouse was placed again in illuminated room and the time taken to enter the dark room was measured. Amnesia was induced by i.p. administration of 0.1 mg/kg of scopolamine 30 min before training (scopolamine amnesia) or by interrupting the blood ßow of bilateral common carotid arteries for 10 min 3 days before training to induce ischemia in mice (ischemic amnesia). The peptide was given by i.c.v. administration just after the train-
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ing. For scopolamine-induced amnesia, statistical analysis was performed to test for difference between the treated and non-treated groups, while in the ischemia-induced amnesia, statistical comparison was made between the treated group and the sham-operated group, in which mice were operated upon just as in the experimental groups without pinching the cervical artery. Statistical analysis Statistical analyses for anti-analgesic test and passive avoidance experiment were performed by StudentÕs and Mann-WhitneyÕs U-tests, respectively. Results Anti-analgesic effect of complement C3a The anti-analgesic effect of complement C3a was measured by tail-pinch test in mice. Morphine, U-50488H and DTLET, m- , k- and d-opioid receptor agonists, respectively, induced analgesia after s.c. or i.c.v. administration. Intracerebroventricularly injected complement C3a dose-dependently inhibited morphine- and U-50488H-induced analgesia, whereas C3a failed to show any antagonistic effect on analgesia induced by DTLET (data not shown). As shown in Fig. 1A, morphine-induced analgesia was signiÞcantly inhibited by C3a at doses of 3 and 10 pmol/mouse, whereas U-50488H-induced analgesia only by 10 pmol/ mouse of C3a (Fig. 1B). The antagonistic effect of C3a was also observed against morphineinduced analgesia by the hot plate test (Fig. 1C). C3a show neither hyperalgesic nor analgesic activity by itself (data not shown). As reported previously, C3a was evidenced to exert strong anti-opioid activity in GPI assay via complement C3a receptor (11). Arginine at the carboxyl terminus of C3a is an impor-
Fig. 1. Antagonistic effect of C3a on morphine- (A), U-50488H-induced (B) analgesia by tail pinch test and on morphine-induced analgesia by hot plate test in mice (C). C3a was given intracerebroventricularly. Physiological saline was given to control mice. Each point represents the mean 6 s.e.m. ** p , 0.02, * p , 0.05 vs control (n 5 6).
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tant residue for binding to C3a receptor. Removal of this residue by carboxypeptidase N leads to disappearance of C3a activity (1). Morphine-induced analgesia was not blocked by C3a des-Arg at a dose of 10 pmol/mouse (data not shown). Anti-amnesic effect of complement C3a It is known that anti-opioids such as naloxone, cholecystokinin and thyrotropin-releasing hormone have learning- and memory-improving effects (8Ð11). These Þndings prompted us to address the possible effect of complement C3a on learning by a step-through passive avoidance task in mice. Despite the lack of improvement of learning ability in normal mice (data not shown), complement C3a was found to improve learning in scopolamine- and ischemia-amnesic mice. Scopolamine-induced amnesia Step-through passive avoidance experiment in mice was performed to verify the possible effect of C3a on scopolamine-induced amnesia. In the preceding training trials, achievement of the control mice was consistent with that of the mice treated with scopolamine (data not shown). However, scopolamine reduced the latency in the test trial, indicating the onset of amnesia. C3a reduced the amnesia at a dose of 10 pmol/mouse after i.c.v. administration to the level of the control group (Fig. 2). Ischemia-induced amnesia The effect of complement C3a on ischemia-induced amnesia was studied in mice. Interruption of the blood ßow of cervical arteries for 10 min established deÞnite amnesia in mice 3 days later. C3a improved amnesia also in this model system at a dose of 10 pmol/mouse (Fig. 3).
Fig. 2. Anti-amnesic effect of C3a on scopolamine (scop)-induced amnesia. Peptide (i.c.v.) was given after the training. Data are expressed as medians 6 quarter point. * p , 0.01 vs control, # p , 0.01 vs scop. group (n 5 9).
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Fig. 3. Anti-amnesic effect of C3a on ischemia-induced amnesia. Peptide (i.c.v.) was given after the training. Data are expressed as medians 6 quarter point. * p , 0.05 vs control, # p , 0.05 vs ischemia (n 5 8).
Discussion There are many reports showing effects of neuropeptides on the immune system (12Ð14), while a certain report deals with the effect of immunopeptides on the nervous system (15). To our best knowledge, certain effects of an immunopeptide complement C3a are considered to be a new example in this Þeld. It has been reported that C3a increased the eating response to norepinephrine and the drinking response to carbamyl choline after hypothalamic injection in rats (16). Furthermore, C3a receptor mRNA was found in the brain (17), hippocampus (5), astrocytes, astrocyte cell lines (18) and in neurons (6) together with C3 mRNA in the brain (19). All these Þndings suggest that complement C3a might play an important role in the CNS. In our previous paper, we reported that C3a showed strong anti-opioid activity against m-opioid agonist DAMGO in GPI assay and caused ileum contraction by release of histamine from mast cells as well as prostaglandin E2 (PGE2) and acetylcholine from neuronal cells (7). In this study, we showed that complement C3a injected intracerebroventricularly in mice acts on CNS as a peptide having anti-analgesic and anti-amnesic activities. An anti-analgesic effect was not observed by C3a des-Arg, which suggests that these effects are speciÞc for C3a. It is conceivable that anti-analgesic and anti-amnesic effects of exogenously injected C3a might be mediated by release of histamine, PGE2 and/or acetylcholine in the brain, because all these substances act on pain perception and on memory function after i.c.v. administration in experimental animals (20Ð23). C3a antagonized m- and k-opioid analgesia, whereas it failed to show any effect on d-opioid analgesia, which might be explained by differences in neurotransmitters released by opioids. In fact, C3a did not antagonize d-opioid activity of DTLET in a mouse vas deferens assay (data not shown). Further study on the mechanisms of action of complement C3a in the CNS would contribute to the treatment of memory deÞcits.
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