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Motilin increases food intake in mice
Peptides, Vol. 19, No. 6, pp. 987–990, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(97)00477-4
Motilin Increases Food Intake in Mice AKIHIRO ASAKAWA,* AKIO INUI,*1 KAZUHIRO MOMOSE,* NAOHIKO UENO,* MASAYUKI A. FUJINO† AND MASATO KASUGA* *Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650, Japan †First Department of Internal Medicine, Yamanashi Medical University, Tamaho, Nakakoma, Yamanashi 409-38, Japan Received 6 October 1997; Accepted 5 December 1997 ASAKAWA, A., A. INUI, K. MOMOSE, N. UENO, M. A. FUJINO AND M. KASUGA. Motilin increases food intake in mice. PEPTIDES 19(6) 987–990, 1998.—The effect of motilin on food intake was investigated in nonfood-deprived mice. A significant increase in food intake was observed 1 h after ICV administration of motilin (3 nmol/mouse) and continued for 2 h. This effect was attenuated markedly by the motilin receptor antagonist GM-109 (0.3–3 nmol/mouse) in a dose-related manner. GM-109 alone had no effect on food intake. These results indicate that motilin receptors are present in the brain and may have a role in the regulation of food intake. © 1998 Elsevier Science Inc. Motilin
ICV
Feeding
GM-109
Brain
regulated environment (22 6 2°C; 55 6 10% humidity; 12-h light– dark cycle with light on at 7:00 a.m.). Food and water were available ad libitum throughout the experiments. The mice were used only once each in the experiment, and the total number of mice was 240. Seven days before the experiments, the mice were anesthetized with sodium pentobarbital (80 – 85 mg/kg IP) and placed in a stereotaxic instrument (SR-6, Narishige, Tokyo, Japan). A hole was made in the skull by using a needle inserted 0.9-mm lateral to the central suture and 0.9-mm posterior to the bregma. A 24-gauge cannula beveled at one end over a distance of 3 mm (Safelet-Cas, Nipro, Osaka, Japan) was implanted into the third cerebral ventricle for ICV injection. The cannula was fixed to the skull with dental cement and capped with silicon without an obtruder (10,17). A 27-gauge injection insert was attached to a microsyringe by PE-20 tubing. Using tweezers, this was easily inserted into a fixed cannula without holding the mouse and thus without disturbing greatly the animal’s behavior. After completion of the experiment, the location of the cannula tip was confirmed by the injection of dye (Evans blue 0.5% and Zelatin 5%) and histological examination of frozen brain sections. More than 95% of the cannulas were confirmed to have been properly placed. Experiments were started at 10:00 a.m. Motilin and GM-
MOTILIN is a 22-amino acid polypeptide secreted from the endocrine cells of the mucosa of the upper part of the small intestine (28). Motilin stimulates gastrointestinal motor activity especially in the antrum and upper duodenum, and it is widely acknowledged that motilin is involved in the regulation of interdigestive motility (15,22). The existence of motilin in the brain has been debated. Earlier studies suggest that motilin immunoreactivity is present in the Purkinje cells of the cerebellum in mice, rats, pigs, monkeys, and humans (4,18), as well as in the cerebrum, pituitary body, hypothalamus, and epiphysis in mice, rats, dogs, pigs, cows, monkeys, and humans (1,2,18,20,29). Motilin is also reported to excite neurons in the cerebral cortex and spinal cord of rats (21). However, few studies have been performed to address the role of motilin in the brain. In this study, we investigated the effects of ICV administration of motilin on food intake in mice. METHOD Porcine motilin was purchased from the Peptide Research Institute (Osaka, Japan), and selective motilin receptor antagonist GM-109 (26) was generously supplied by Chugai Co.Ltd. (Tokyo, Japan). Male mice of the ddy strain (JAPAN SLC Inc. Japan), obtained at 7 weeks of age, were housed individually in a 1
Address all correspondence and requests to Akio Inui.
987
988
ASAKAWA ET AL.
2.782; p , 0.01] were significant. GM-109 is a cyclic peptide and a highly selective motilin receptor antagonist in the smooth muscle of the rabbit small intestine (26). IP injection of motilin (0.003–3 nmol/mouse), in contrast did not increase food intake in nonfood-deprived mice. GM-109 alone at doses of 0.003–3 nmol/mouse had no effect on food intake.
FIG. 1. Effects of motilin (0.003–3 nmol/mouse) injected ICV on cumulative food intake in nonfood-deprived mice. The number of mice used are 10 –14 per each group. **p , 0.01 compared to the ACSF control group by Bonferroni’s t-test.
109 were dissolved in 4 ml of ACSF (NaCl, 138.9 mmol; KCl, 3.4 mmol; CaCl2, 1.26 mmol; NaHCO3, 4 mmol; NaH2PO4 z 2H2O, 0.6 mmol; and glucose, 5.6 mmol.) and injected at doses of 0.003–3 nmol. Food intake was measured by placing the preweighed pellets in the cage and weighing the uneaten pellets at 20 min, 1 and 2 h after drug administration. Results were expressed as mean value 6 SE. Data were evaluated by ANOVA with repeated measures. If the F ratio for any of the tests was significant, Bonferroni’s t-test was made between the control and experimental groups; p , 0.05 was considered to be statistically significant. RESULTS Administration of motilin increased significantly the cumulative food intake at 1 and 2 h after ICV injection of 3 nmol/mouse compared to ACSF-treated controls (p , 0.01; Bonferroni’s t-test; Fig. 1). Analysis by ANOVA indicated that the main effect of time at which food intake was measured [F(2, 116) 5 77.973; p , 0.0001], and the interaction of dose 3 time [F(8, 174) 5 2.750; p , 0.01] were significant. This effect of motilin (3 nmol/mouse) was totally antagonized by the simultaneous ICV administration of the motilin receptor antagonist GM-109 (0.003-3 nmol/ mouse) in a dose-related manner (p , 0.05 at 1 h by a dose of 3 nmol; p , 0.01 at 2 h by doses of 0.3 and 3 nmol; Bonferroni’s t-test; Fig. 2). Analysis by ANOVA indicated that the main effect of dose of motilin and GM-109 [F(5, 42) 5 3.816; p , 0.01], the main effect of time at which food intake was measured [F(2, 84) 5 107.977; p , 0.0001], and the interaction of dose 3 time [F(10, 126) 5
DISCUSSION In this study, we showed that ICV injection of motilin increased food intake in non-food deprived mice. This is the first report about the effects of ICV injection of motilin and motilin antagonist on feeding in mice. It is generally accepted that motilin is involved in the regulation of interdigestive motility (15,22). Motilin induces Phase III-like contractions in the lower esophageal sphincter (12), stomach, and duodenum (13,25), and the motilin-induced contractions migrate along the small intestine to sweep the accumulated intraluminal contents in a caudad direction toward the terminal ileum (14). Therefore, motilin might stimulate food intake by decreasing the volume of gastric contents (6,24), although we (in the present study) and others (19) have failed to observe feeding stimulation by motilin administerd peripherally. Until now, only a few peptides were thought to increase food intake, including opioid peptides (16), neuropeptide Y (NPY; 5,11), galanin (27), and growth hormone-releasing factor (GRF; 30). The results presented here and reported by Rosenfeld et al. in rats (23) suggest
FIG. 2. Effects of motilin (3 nmol/mouse) or motilin (3 nmol/ mouse) 1 GM109 (0.003–3 nmol/mouse) injected ICV on cumulative food intake in nonfood-deprived mice. The number of mice used are eight per each group. #p , 0.05; ##p , 0.01 compared to the ACSF control group by Bonferroni’s t-test. *p , 0.05; **p , 0.01 compared to the motilin (3 nmol/mouse) alone group by Bonferroni’s t-test.
MOTILIN INCREASES FOOD INTAKE IN MICE
989
that motilin may be one of the orexigenic peptides, at least when administered into the brain. Motilin gene expression in the brain has been suggested previously on the basis of immunohistochemical data (1,2,4,18,20,29) but has been partially excluded in northern assays containing polyA1 from monkey pituitary and cerebellum; human cerebellum (7) and porcine pituitary, cerebellum, cortex, hypothalamus, and hippocampus (3). However, a recent study demonstrates the presence of positive signals from human brain polyA1 (9). This is in agreement with the detection of the motilin-like peptide in the brain of rodent, canine, porcine, ovine, and primate species reported by several groups using independently derived antisera.
Very recently, using autoradiography, specific- and highaffinity motilin-binding sites were observed in the molecular layer of the rabbit cerebellar cortex (8). Our findings that the motilin receptor antagonist GM-109 (26) potently inhibited the feeding-stimulatory effect of motilin also suggest motilin receptors are present in the brains of mice, to which porcine motilin could act as a ligand. ACKNOWLEDGEMENTS The authors acknowledge with gratitude the generous gifts of GM-109 from Chugai Co. Ltd. (Tokyo, Japan). This work was supported in part by Grants-in-Aid for Scientific Research (A) 08559012 and (C) 09671057 from The Ministry of Education, Science and Culture of Japan (to AI).
REFERENCES 1. Beinfeld, M. C.; Bailey, G. J. The distribution of motilin-like peptides in rhesus monkey brain as determined by radioimmunoassay. Neurosci. Lett. 54:345–350; 1985. 2. Beinfeld, M. C.; Korchak, D. M. The regional distribution and the chemical, chromatographic, and immunologic characterization of motilin brain peptides: the evidence for a difference between brain and intestinal motilin-immunoreactive peptides. J. Neurosci. 5:2502–2509; 1985. 3. Bond, C. T.; Nilaver, G.; Godfrey, B.; Zimmerman, E. A.; Adelman, J. P. Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin. Mol. Endocrinol. 2:175–180; 1988. 4. Chan-Palay, V.; Nilaver, G.; Palay, S. L.; Beinfeld, M. C.; Zimmerman, E. A.; Wu, J.-Y.; O’Donohue, T. L. Chemical heterogeneity in cerebellar Purkinje cells: Existence and coexistence of glutamic acid decarboxylase-like and motilin-like immunoreactivities. Proc. Natl. Acad. Sci. USA. 78:7787– 7791; 1981. 5. Clark, J. T.; Kalra, P. S.; Crowley, W. R.; Kalra, S. P. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behaviour in rats. Endocrinology. 115:427– 429; 1984. 6. Code, C. F.; Marlett, J. A. The interdigestive myoelectric complex of the stomach and small bowel of dogs. J. Physiol. 246:289 –309; 1975. 7. Daikh, D. I.; Douglass, J. O.; Adelman, J. P. Structure and expression of the human motilin gene. DNA (NY). 8:615– 621; 1989. 8. Depoortere, I.; Peeters, T. L. Demonstration and characterization of motilin-binding sites in the rabbit cerebellum. Am. J. Physiol. 272:G994 –G999; 1997. 9. Gasparini, P.; Grifa, A.; Savasta, S.; Merlo, I.; Bisceglia, L.; Totaro, A.; Zelante, L. The motilin gene: subregional localisation, tissue expression, DNA polymorphisms and exclusion as a candidate gene for the HLA-associated immotile cilia syndrome. Hum. Genet. 94:671– 674; 1994. 10. Hirosue, Y.; Inui, A.; Miura, M.; Nakajima, M.; Okita, M.; Himori, N.; Baba, S.; Kasuga, M. Effects of CCK antagonists on CCK-induced suppression of locomotor activity in mice. Peptides. 13:155–157; 1992. 11. Inui, A.; Okita, M.; Nakajima, M.; Inoue, T.; Sakatani, K.; Oya, M.; Morioka, H.; Okimura, Y.; Chihara, K.; Baba, S. Neuropeptide regulation of feeding in dogs. Am. J. Physiol. 261:R588 –R594; 1991. 12. Itoh, Z.; Aizawa, I.; Honda, R.; Hiwatashi, K.; Couch, E. F. Control of lower-esophageal–sphincter contractile activity by motilin in conscious dogs. Am. J. Dig. Dis. 23:341–345; 1978.
13. Itoh, Z.; Honda, R.; Hiwatashi, K.; Takeuchi, S.; Aizawa, I.; Takayanagi, R.; Couch, E. F. Motilin-induced mechanical activity in the canine alimentary tract. Scand. J. Gastroenterol. 11(Suppl. 39):93–110; 1976. 14. Itoh, Z. Hormones, peptides, opioids and prostaglandins in normal gastric contractions. In: Akkermans, L. M. A.; Johnson, A. G.; Read, N. W., Eds. Gastric and gastroduodenal motility. East Sussex, UK: Praeger Publishers; 1984:41–59. 15. Lee, K. Y.; Chang, T.; Chey, W. Y. Effect of rabbit antimotilin serum on myoelectric activity and plasma motilin concentration in fasting dog. Am. J. Physiol. 245:G547–G553; 1983. 16. Morley, J. E.; Levine, A. S.; Yim, G. K.; Lowy, M. T. Opioid modulation of appetite. Neurosci. Biobehav. Rev. 7:281–305; 1983. 17. Nakajima, M.; Inui, A.; Teranishi, A.; Miura, M.; Hirosue, Y.; Okita, M.; Himori, N.; Baba, S.; Kasuga, M. Effects of pancreatic polypeptide family peptides on feeding and learning behavior in mice. J. Pharmacol. Exp. Ther. 268:1010 –1014; 1994. 18. Nilaver, G.; Beinfeld, M. C.; Bond, C. T.; Daikh, D.; Godfrey, B.; Adelman, J. P. Heterogeneity of motilin immunoreactivity in mammalian tissue. Synapse. 2:266 –275; 1988. 19. Olson, R. D.; Kastin, A. J.; von-Almen, T. K.; Coy, D. H.; Olson, G. A. Systemic injections of gastro-intestinal peptides alter behavior in rats. Peptides. 1:383–385; 1980. 20. O’Donohue, T. L.; Beinfeld, M. C.; Chey, W. Y.; Chang, T.; Nilaver, G.; Zimmerman, E. A.; Yajima, H.; Adachi, H.; Poth, M.; Mcdevitt, R. P.; Jacobowitz, D. M. Identification, characterization and distribution of motilin immunoreactivity in the rat central nervous system. Peptides. 2:467– 477; 1981. 21. Phillis, J. W.; Kirkpatrick, J. R. Motilin excites neurons in the cerebral cortex and spinal cord. Eur. J. Pharmacol. 58:469 – 472; 1979. 22. Poitras, P. Motilin is a digestive hormone in the dog. Gastroenterology. 87:909 –913; 1984. 23. Rosenfeld, D. J.; Garthwaite T. L. Central administration of motilin stimulates feeding in rats. Physol. Behav. 39:753–756; 1987. 24. Szurszewski, J. H. A migrating electric complex of the canine small intestine. Am. J. Physiol. 217:1757–1763; 1969. 25. Takahashi, I.; Honda, R.; Dodds, W. J.; Sarna, S.; Itoh, Z.; Chey, W. Y.; Hogan, W. J.; Greiff, D.; Baker, K. Effect of motilin on opossum upper gastrointestinal tract and sphincter of Oddi. Am. J. Physiol. 245:G476 –G481; 1983. 26. Takanashi, H.; Yogo, K.; Ozaki, K.; Ikuta, M.; Akima, M.; Koga, H.; Nabata, H. GM-109: a novel, selective motilin
990 receptor antagonist in the smooth muscle of the rabbit small intestine. J. Pharmacol. Exp. Ther. 273:624 – 628; 1995. 27. Tempel, D. L.; Leibowitz, K. J.; Leibowitz, S. F. Effects of PVN galanin on macronutrient selection. Peptides. 9:309 – 314; 1988. 28. Usellin, L.; Buchan, A. M. J.; Polak, J. M.; Capella, C.; Cornaggia, M.; Solcia, E. Ultrastructural localization of motilin in endocrine cells of human and dog intestine by the immunogold technique. Histochemistry. 81:363–368; 1984.
ASAKAWA ET AL. 29. Yanaihara, C.; Sato, H.; Yanaihara, N.; Naruse, S.; Forssmann, W. G.; Helmstaedter, V.; Fujita, T.; Yamaguchi, K.; Abe, K. Motilin-, substance P- and somatostatin-like immunoreactivities in extracts from dog, tupaia and monkey brain and GI tract. Adv. Exp. Med. Biol. 106:269 –283; 1978. 30. Vaccarino, F. J.; Bloom, F. E.; Rivier, J.; Vale, W.; Koob, G. F. Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor. Nature (London). 314:167–168; 1985.
PII S0196-9781(97)00477-4
Motilin Increases Food Intake in Mice AKIHIRO ASAKAWA,* AKIO INUI,*1 KAZUHIRO MOMOSE,* NAOHIKO UENO,* MASAYUKI A. FUJINO† AND MASATO KASUGA* *Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650, Japan †First Department of Internal Medicine, Yamanashi Medical University, Tamaho, Nakakoma, Yamanashi 409-38, Japan Received 6 October 1997; Accepted 5 December 1997 ASAKAWA, A., A. INUI, K. MOMOSE, N. UENO, M. A. FUJINO AND M. KASUGA. Motilin increases food intake in mice. PEPTIDES 19(6) 987–990, 1998.—The effect of motilin on food intake was investigated in nonfood-deprived mice. A significant increase in food intake was observed 1 h after ICV administration of motilin (3 nmol/mouse) and continued for 2 h. This effect was attenuated markedly by the motilin receptor antagonist GM-109 (0.3–3 nmol/mouse) in a dose-related manner. GM-109 alone had no effect on food intake. These results indicate that motilin receptors are present in the brain and may have a role in the regulation of food intake. © 1998 Elsevier Science Inc. Motilin
ICV
Feeding
GM-109
Brain
regulated environment (22 6 2°C; 55 6 10% humidity; 12-h light– dark cycle with light on at 7:00 a.m.). Food and water were available ad libitum throughout the experiments. The mice were used only once each in the experiment, and the total number of mice was 240. Seven days before the experiments, the mice were anesthetized with sodium pentobarbital (80 – 85 mg/kg IP) and placed in a stereotaxic instrument (SR-6, Narishige, Tokyo, Japan). A hole was made in the skull by using a needle inserted 0.9-mm lateral to the central suture and 0.9-mm posterior to the bregma. A 24-gauge cannula beveled at one end over a distance of 3 mm (Safelet-Cas, Nipro, Osaka, Japan) was implanted into the third cerebral ventricle for ICV injection. The cannula was fixed to the skull with dental cement and capped with silicon without an obtruder (10,17). A 27-gauge injection insert was attached to a microsyringe by PE-20 tubing. Using tweezers, this was easily inserted into a fixed cannula without holding the mouse and thus without disturbing greatly the animal’s behavior. After completion of the experiment, the location of the cannula tip was confirmed by the injection of dye (Evans blue 0.5% and Zelatin 5%) and histological examination of frozen brain sections. More than 95% of the cannulas were confirmed to have been properly placed. Experiments were started at 10:00 a.m. Motilin and GM-
MOTILIN is a 22-amino acid polypeptide secreted from the endocrine cells of the mucosa of the upper part of the small intestine (28). Motilin stimulates gastrointestinal motor activity especially in the antrum and upper duodenum, and it is widely acknowledged that motilin is involved in the regulation of interdigestive motility (15,22). The existence of motilin in the brain has been debated. Earlier studies suggest that motilin immunoreactivity is present in the Purkinje cells of the cerebellum in mice, rats, pigs, monkeys, and humans (4,18), as well as in the cerebrum, pituitary body, hypothalamus, and epiphysis in mice, rats, dogs, pigs, cows, monkeys, and humans (1,2,18,20,29). Motilin is also reported to excite neurons in the cerebral cortex and spinal cord of rats (21). However, few studies have been performed to address the role of motilin in the brain. In this study, we investigated the effects of ICV administration of motilin on food intake in mice. METHOD Porcine motilin was purchased from the Peptide Research Institute (Osaka, Japan), and selective motilin receptor antagonist GM-109 (26) was generously supplied by Chugai Co.Ltd. (Tokyo, Japan). Male mice of the ddy strain (JAPAN SLC Inc. Japan), obtained at 7 weeks of age, were housed individually in a 1
Address all correspondence and requests to Akio Inui.
987
988
ASAKAWA ET AL.
2.782; p , 0.01] were significant. GM-109 is a cyclic peptide and a highly selective motilin receptor antagonist in the smooth muscle of the rabbit small intestine (26). IP injection of motilin (0.003–3 nmol/mouse), in contrast did not increase food intake in nonfood-deprived mice. GM-109 alone at doses of 0.003–3 nmol/mouse had no effect on food intake.
FIG. 1. Effects of motilin (0.003–3 nmol/mouse) injected ICV on cumulative food intake in nonfood-deprived mice. The number of mice used are 10 –14 per each group. **p , 0.01 compared to the ACSF control group by Bonferroni’s t-test.
109 were dissolved in 4 ml of ACSF (NaCl, 138.9 mmol; KCl, 3.4 mmol; CaCl2, 1.26 mmol; NaHCO3, 4 mmol; NaH2PO4 z 2H2O, 0.6 mmol; and glucose, 5.6 mmol.) and injected at doses of 0.003–3 nmol. Food intake was measured by placing the preweighed pellets in the cage and weighing the uneaten pellets at 20 min, 1 and 2 h after drug administration. Results were expressed as mean value 6 SE. Data were evaluated by ANOVA with repeated measures. If the F ratio for any of the tests was significant, Bonferroni’s t-test was made between the control and experimental groups; p , 0.05 was considered to be statistically significant. RESULTS Administration of motilin increased significantly the cumulative food intake at 1 and 2 h after ICV injection of 3 nmol/mouse compared to ACSF-treated controls (p , 0.01; Bonferroni’s t-test; Fig. 1). Analysis by ANOVA indicated that the main effect of time at which food intake was measured [F(2, 116) 5 77.973; p , 0.0001], and the interaction of dose 3 time [F(8, 174) 5 2.750; p , 0.01] were significant. This effect of motilin (3 nmol/mouse) was totally antagonized by the simultaneous ICV administration of the motilin receptor antagonist GM-109 (0.003-3 nmol/ mouse) in a dose-related manner (p , 0.05 at 1 h by a dose of 3 nmol; p , 0.01 at 2 h by doses of 0.3 and 3 nmol; Bonferroni’s t-test; Fig. 2). Analysis by ANOVA indicated that the main effect of dose of motilin and GM-109 [F(5, 42) 5 3.816; p , 0.01], the main effect of time at which food intake was measured [F(2, 84) 5 107.977; p , 0.0001], and the interaction of dose 3 time [F(10, 126) 5
DISCUSSION In this study, we showed that ICV injection of motilin increased food intake in non-food deprived mice. This is the first report about the effects of ICV injection of motilin and motilin antagonist on feeding in mice. It is generally accepted that motilin is involved in the regulation of interdigestive motility (15,22). Motilin induces Phase III-like contractions in the lower esophageal sphincter (12), stomach, and duodenum (13,25), and the motilin-induced contractions migrate along the small intestine to sweep the accumulated intraluminal contents in a caudad direction toward the terminal ileum (14). Therefore, motilin might stimulate food intake by decreasing the volume of gastric contents (6,24), although we (in the present study) and others (19) have failed to observe feeding stimulation by motilin administerd peripherally. Until now, only a few peptides were thought to increase food intake, including opioid peptides (16), neuropeptide Y (NPY; 5,11), galanin (27), and growth hormone-releasing factor (GRF; 30). The results presented here and reported by Rosenfeld et al. in rats (23) suggest
FIG. 2. Effects of motilin (3 nmol/mouse) or motilin (3 nmol/ mouse) 1 GM109 (0.003–3 nmol/mouse) injected ICV on cumulative food intake in nonfood-deprived mice. The number of mice used are eight per each group. #p , 0.05; ##p , 0.01 compared to the ACSF control group by Bonferroni’s t-test. *p , 0.05; **p , 0.01 compared to the motilin (3 nmol/mouse) alone group by Bonferroni’s t-test.
MOTILIN INCREASES FOOD INTAKE IN MICE
989
that motilin may be one of the orexigenic peptides, at least when administered into the brain. Motilin gene expression in the brain has been suggested previously on the basis of immunohistochemical data (1,2,4,18,20,29) but has been partially excluded in northern assays containing polyA1 from monkey pituitary and cerebellum; human cerebellum (7) and porcine pituitary, cerebellum, cortex, hypothalamus, and hippocampus (3). However, a recent study demonstrates the presence of positive signals from human brain polyA1 (9). This is in agreement with the detection of the motilin-like peptide in the brain of rodent, canine, porcine, ovine, and primate species reported by several groups using independently derived antisera.
Very recently, using autoradiography, specific- and highaffinity motilin-binding sites were observed in the molecular layer of the rabbit cerebellar cortex (8). Our findings that the motilin receptor antagonist GM-109 (26) potently inhibited the feeding-stimulatory effect of motilin also suggest motilin receptors are present in the brains of mice, to which porcine motilin could act as a ligand. ACKNOWLEDGEMENTS The authors acknowledge with gratitude the generous gifts of GM-109 from Chugai Co. Ltd. (Tokyo, Japan). This work was supported in part by Grants-in-Aid for Scientific Research (A) 08559012 and (C) 09671057 from The Ministry of Education, Science and Culture of Japan (to AI).
REFERENCES 1. Beinfeld, M. C.; Bailey, G. J. The distribution of motilin-like peptides in rhesus monkey brain as determined by radioimmunoassay. Neurosci. Lett. 54:345–350; 1985. 2. Beinfeld, M. C.; Korchak, D. M. The regional distribution and the chemical, chromatographic, and immunologic characterization of motilin brain peptides: the evidence for a difference between brain and intestinal motilin-immunoreactive peptides. J. Neurosci. 5:2502–2509; 1985. 3. Bond, C. T.; Nilaver, G.; Godfrey, B.; Zimmerman, E. A.; Adelman, J. P. Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin. Mol. Endocrinol. 2:175–180; 1988. 4. Chan-Palay, V.; Nilaver, G.; Palay, S. L.; Beinfeld, M. C.; Zimmerman, E. A.; Wu, J.-Y.; O’Donohue, T. L. Chemical heterogeneity in cerebellar Purkinje cells: Existence and coexistence of glutamic acid decarboxylase-like and motilin-like immunoreactivities. Proc. Natl. Acad. Sci. USA. 78:7787– 7791; 1981. 5. Clark, J. T.; Kalra, P. S.; Crowley, W. R.; Kalra, S. P. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behaviour in rats. Endocrinology. 115:427– 429; 1984. 6. Code, C. F.; Marlett, J. A. The interdigestive myoelectric complex of the stomach and small bowel of dogs. J. Physiol. 246:289 –309; 1975. 7. Daikh, D. I.; Douglass, J. O.; Adelman, J. P. Structure and expression of the human motilin gene. DNA (NY). 8:615– 621; 1989. 8. Depoortere, I.; Peeters, T. L. Demonstration and characterization of motilin-binding sites in the rabbit cerebellum. Am. J. Physiol. 272:G994 –G999; 1997. 9. Gasparini, P.; Grifa, A.; Savasta, S.; Merlo, I.; Bisceglia, L.; Totaro, A.; Zelante, L. The motilin gene: subregional localisation, tissue expression, DNA polymorphisms and exclusion as a candidate gene for the HLA-associated immotile cilia syndrome. Hum. Genet. 94:671– 674; 1994. 10. Hirosue, Y.; Inui, A.; Miura, M.; Nakajima, M.; Okita, M.; Himori, N.; Baba, S.; Kasuga, M. Effects of CCK antagonists on CCK-induced suppression of locomotor activity in mice. Peptides. 13:155–157; 1992. 11. Inui, A.; Okita, M.; Nakajima, M.; Inoue, T.; Sakatani, K.; Oya, M.; Morioka, H.; Okimura, Y.; Chihara, K.; Baba, S. Neuropeptide regulation of feeding in dogs. Am. J. Physiol. 261:R588 –R594; 1991. 12. Itoh, Z.; Aizawa, I.; Honda, R.; Hiwatashi, K.; Couch, E. F. Control of lower-esophageal–sphincter contractile activity by motilin in conscious dogs. Am. J. Dig. Dis. 23:341–345; 1978.
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