Gut: first published as 10.1136/gut.21.2.94 on 1 February 1980. Downloaded from
Gut, 1980, 21, 94-97
Effects of [Asu'7]-eel calcitonin on gastric somatostatin and gastrin release
T CHIBA, T TAMINATO. S KADOWAKI, Y GOTO, K MORI, Y SEINO, H ABE, K CHIHARA, S MATSUKURA, T FUJITA, AND T KONDO From the Third Division, Department of Medicine, Kobe University School of Medicine, Kobe, and Uwajima City Hospital, Ehime, Japan
SUMMARY Effects of [Asul17]-eel calcitonin on gastric somatostatin and gastrin secretion were studied by using the isolated perfused rat stomach. [Asul 7]-eel calcitonin (10-9 -10-7M) caused a simultaneous dose-dependent increase of gastric somatostatin release and decrease of gastrin secretion, with a significant correlation between these two. The demonstration of calcitonin stimula- tion of gastric somatostatin release raises the possibility of somatostatin-mediated suppression of gastrin secretion by calcitonin.
In addition to its effects on bone and kidney as a of Lefebvre et al.'s with minor modifications.'4 hypocalcaemic agent, calcitonin was found to Animals were anaesthetised with pentobarbital suppress various gastrointestinal functions,1-5 in- (40 mg/kg) after an overnight fast. After a midline cluding secretion of gastrin and gastric acid.6,8 laparotomy, polyethylene cannulae were inserted Recently, significant amounts of somatostatin, a into the left gastric artery and gastric vein. All other potent inhibitor of gastric acid and gastrin secre- vessels and pancreas were carefully excluded by http://gut.bmj.com/ tion," 10 were demonstrated in the stomach by radio- ligation. After cardial ligation, a catheter was in- immunoassayll 12 as well as by immunocyto- serted into the stomach through the pyloric ring to chemical technique.'3 More recently, we demon- drain gastric juice. Infusion was then started with strated somatostatin release from the stomach.14 4-6% dextran (mean mol.wt.70,000) Krebs-Ringer In view of the location of somatostatin-producing bicarbonate buffer containing 5.5 mM glucose D cells in the mucosa of the fundus and the antrum (DKRBG) into the left gastric artery at a flow rate in the of the cells and of 2 ml/min without recirculation. The perfusate
vicinity parietal gastrin- on September 25, 2021 by guest. Protected copyright. producing G cells,15 16 an influence of gastric somato- was equilibrated with a gas phase of 95% 02/5% statin on gastric acid and gastrin secretion was CO2 immediately before use and maintained at pH suggested. It therefore appeared worth while to 7-4 and PaO2 350 mmHg. The entire perfusion investigate the effect of calcitonin on gastric somato- system including the perfusate was kept at 37°C statin release with reference to gastrin secretion. throughout the experiment. After the pre-stimula- In the present study, we studied the effects of tion period (20 minutes) perfused with DKRBG [Asul"7]-eel calcitonin, an analogue of eel calcitonin,17 alone, the perfusate with or without [Asu"7]-eel on gastric somatostatin and gastrin secretion from calcitonin (Toyo Jozo Research Laboratories, the isolated perfused rat stomach. Shizuoka) was infused for 15 minutes at the con- centrations of 10-9, 10-8, and 10-7 M. The number Methods of animals was six for each dose of [Asul 7]-eel calcitonin, and each stomach was exposed to only Male Wistar rats weighing 300-350 g were main- one perfusion. The venous effluents were collected atined in a temperature-controlled and air-con- every minute into chilled tubes containing Bacitracin ditioned room under light-dark cycle, fed Oriental (Sigma Chemical Company, St. Louis)-Trasylol laboratory chow containing 0-71 % calcium and (Bayer Leverkusen, Germany) mixture (2x 10-5 M 2 IU/g vitamin D (Oriental Yeast Company, and 1000 KIU/ml, respectively), frozen immediately, Tokyo) and given tap water ad libitum. The per- and stored at -20°C until the assay. fusion of rat stomach was performed by the method Immunoreactive somatostatin levels were measured by a specific radioimmunoassay using Received for publication 20 August 1979 antiserum RA-823 described previously.'4 Anti- 94 Gut: first published as 10.1136/gut.21.2.94 on 1 February 1980. Downloaded from
Efects of [Asul7-eel calcitonin on gastric somatostatin and gastrin release 95
serum RA-823 was found not to cross-react with [Asu'17]-eel calcitonin and synthetic human gastrin I (Imperial Chemical Industries, England). The minimal detectable quantity of somatostatin was 10 pg/ml in this system. The coefficients of variation within and between assays were 5A4% and 8.5o', respectively. Immunoreactive gastrin was measured by the dextran-coated charcoal radioimmunoassay re- ported previously.19 The antisera raised in rabbits .against synthetic human gastrin I was used for this assay. Synthetic human gastrin I was used as the standard and also as the labelled tracer prepared by 250 a modification of the chloramine T method.20 The z minimal detectable quantity by this assay was were - 200 20 pg/ml. Intra- and interassay variations 6.5% z and 888%, respectively. In our radioimmunoassay system [Asul 7]-eel calcitonin and somatostatin ur
(Protein Research Institute, Osaka) did not cross- < 150 react with the antigastrin antisera. The serial dilu- 0 tion curve of gastrin-like immunoreactivity in the perfusate was completely superimposable on the lQuu -5 0 5 10 15 20 25 standard curve. MIN U T ES Statistical analysis was performed by Duncan's new multiple range test. Fig. 1 Gastric somatostatin and gastrin responses to [Asul17]-eel calcitonin, used in this study, has been the 10-9M ( x-x ), 10-8M (-), and 10-7M (-*) of shown to have an equal hypocalcaemic potency to [ASU '7,-eel calcitonin for 15 minutes. The light solid line ( ) also indicates the control group. Each that of natural eel calcitonin,21 with a high physico-
value represents the mean SEM. http://gut.bmj.com/ chemical stability.22
Results 10 ng/l (P<005), and 340±21 ng/l (P<001) were
obtained by 10-9, 10-8 , and 10-7 M [Asul'71-eel In the control experiments perfused only with calcitonin, respectively. In most experiments, the DKRBG, somatostatin levels ranged from 130 ng/l peak values were attained at 1 1-13 minutes after the to 218 ng/l (mean ±SEM: 176±5 ng/l), and gastrin start of [Asul"7]-eel calcitonin infusion (Fig. 1). levels from 165 ng/l to 250 ng/l (mean ±SEM: 223 ± Conversely,[Asu' 7]-eel calcitonin elicited a significant on September 25, 2021 by guest. Protected copyright. 7 ng/), neither of which changed significantly decrease ofgastrin secretion also in a dose-dependent throughout the perfusion period (Fig. 1). fashion (Table, Fig. 1). The nadir of gastrin release The infusion of [Asul"7]-eel calcitonin caused a caused by 10-9, 10-, and 10-7 M eel calcitonin was significant and dose-related increase of gastric 184±11 ng/l (P<005 vs. control), 180±10 ng/1 somatostatin secretion (Table, Fig. 1). The peak (p<0.05), and 152 ±6 ng/l (p<0.05), respectively. values of 246± 14 ng/l (p<0 05 vs. control), 254± The most prominent suppression of gastrin release
Table Total amounts ofgastric somatostatin andgastrin release during infusion of iA su",7 -eel calcitonin for 15 minutes Dose ofpeptide Gastric somatostatin* Relative amounts oft Gastrin release* Relative amountst of (M) release (pg/15 min) somatostatin release (pg/ 1S min) gastrin release (%)
Control 0 5330± 120 100 0 6660± 70 100 0 Calcitonin 10-s 6350±2308 1191 6080± 608 91-3 10-8 7220±220b. c 135-4 5560±260b. c 83-8 10-7 9110±450b, d 170.9 4780±210b, d 71-8 *AIl values are the mean ± SEM (six animals). tThe relative amounts of somatostatin and gastrin release are expressed as a percentage of the amounts released by the control. a: P<0-05 compared with the control. b: P<0 01 compared with the control. c: P<005 compared with 10-9M. d: P<0-05 compared with 10-8M. Gut: first published as 10.1136/gut.21.2.94 on 1 February 1980. Downloaded from
96 Chiba et al.
3000 suppressive effect of calcitonin on gastrin secretion, confirming previous reports.7 As a constant level X0. (2.5 mM) of calcium was supplied to the isolated W 2500 perfused stomach in the present study, the suppressive W effect of calcitonin on gastrin secretion was ap- W parently not mediated by the change in the calcium 2000 concentration, though the subtle intra- and extra- z cellular movement of calcium ions in the secretory q 1500 cells cannot be excluded. The most striking observation in the present LL study is the dose-related increase of gastric somato- ° 1000 statin release in response to [Asul 7]-eel calcitonin. Somatostatin-producing D cells have been demon- z r =0.60 within mucosa in the vicinity of 2 a P <001 strated the antral W soo0500 the G cells.'516 Furthermore, significant amounts of somatostatin have been found to be released from the stomach of dogs23 and rats in response to various 1000 2000 3000 4000 5000 6000 stimuli. These findings'4 raise the possibility that INCREMENT OF SOMATOSTATIN RELEASE(p9) gastrin secretion may be regulated by gastric somato- mucosa. In Fig. 2 Correlation between the increment of total statin within the local area of the antral amounts ofsomatostatin release and the decrement of view of the simultaneous increase of somatostatin total amounts ofgastrin secretion during the infusion release and decrease of gastrin secretion with a of 109M (x ), 1 O-8I ((), and 1O-7M (0) of[Asul,7'-eel significant mutual correlation, as well as the known calcitonin compared with the amounts released by the potent inhibiting action of somatostatin on gastrin control. secretion, the suppression of gastrin secretion by calcitonin may, at least in part, be mediated by coincided with the peak of gastric somatostatin endogenous gastric somatostatin, though other release (Fig. 1). possibilities independent of these two phenomena The total amounts of gastric somatostatin release cannot be completely excluded. http://gut.bmj.com/ and gastrin secretion during the infusion of [Asu-7]_ The fact that both calcitonin and somatostatin eel calcitonin (15 min) are summarised in the Table. inhibit histamine- and pentagastrin-induced gastric The increment in total somatostatin release and acid secretion610 may suggest a similar mechanism decrement in total gastrin secretion during the of action of these two substances in inhibiting infusion of 10-9, 10-8, and 1O-7 M[Asul7]-eel gastric acid secretion. While somatostatin-secreting calcitonin were plotted in Fig. 2. A significant cor- D cells are distributed both in the fundic and antral relation (r=0.60, P
Effects of[Asul 7]-eel calcitonin on gastric somatostatin and gastrin release 97 thank Toyo Jozo Research Laboratories, Shizuoka, 13Polak JM, Pearse AGE, Grimelius L, Bloom SR, for the generous gift of [Asul"7]-eel calcitonin. We Arimura A. Growth-hormone release-inhibiting hor- also thank Mr. T. Ichii and Miss C. Shiba for mone in gastrointestinal and pancreatic D cells. Lancet technical assistance and Miss M. Usui and Miss S. 1975; 1: 1220-2. Yanagihara for typing the manuscript. '4Chiba T, Seino Y, Goto Y, et al. Somatostatin release from isolated perfused rat stomach. Biochem Biophys References Res Commun 1978; 82: 731-7. '5Rufener C, Dubois MP, Malaisse-Lagae F, Orci L. 'Hufner M, Hesch RD, Schmidt H, et al. The gastro- Immunofluorescent reactivity to anti-somatostatin in intestinal effects of calcitonin. (Abstract.) Acta Endo- the gastrointestinal mucosa of the dog. Diabetologia crinol 1972; 159 (suppl): 65. 1975; 11: 321-4. ?Schmidt H, Hesch RD, Hufner M, Paschen K, Creutz- '6Polak JM, Bloom SR, McCrossan M, Timson CM, feldt W. Hemmung der exokrinen Pankreassekretion Arimura A, Pearse AGE. Studies in gastric D cell des Menschen durch Calcitonin. Dtsch Med Wochenschr pathology. Gut 1976; 17 (abstr): 400-1. 1971; 96: 1773-5. "Morikawa T, Munekata E, Sakakibara S, Noda T, 3Waldeck F, Siewert R, Jennewein HM, Weiser F. Das Otani M. Synthesis of eel-calcitonin and [Asul7]-eel Druckprofil im unteren Osophagussphinkter beim calcitonin: contribution of the disulfide bond to the Menschen und seine Beeinflussung durch Gastrin, hormonal activity. Experientia 1976; 32: 104-6. Calcitonin und Glucagon. Dtsch Med Wochenschr 1973; 18Lefebvre PJ, Luyckx AS, Brassinne AH. Vagal 98: 1059-63. stimulation and its role in eliciting gastrin but not 'Winckler K, Hesch RD, Schmidt H. Hemmung der glucagon release from the isolated perfused dog sto- Gallenblasenkontraktion durch Calcitonin. Dtsch Med mach. Gut 1978; 19: 185-8. Wochenschr 1973; 98: 957-9. '9Seino Y, Matsukura S, Miyamoto Y, Goto Y, Taminato 5Hotz J, Goebell H, Ziegler R. Calcitonin and exocrine T, Imura H. Hypergastrinemia in hyperthyroidism. pancreatic secretion in man: inhibition of enzymes J Clin Endrocrinol Metab 1976; 43: 852-5. stimulated by CCK-pancreoeymin, cazrulein, or calcium 20Hunter WM, Greenwood FC. Preparation of iodine-1 31 no response to vagal stimulation. Gut 1977; 18: 615-22. labelled human growth hormone of high specific '6Becker HD, Konturek SJ, Reeder DD, Thompson JC. activity. Nature (Lond) 1962; 194: 495-6. Effect of calcium and calcitonin on gastrin and gastric 21Noda T, Sakurada S, Yamauchi H, et al. [Asu1,7]-eel secretion in cats. Am J Physiol 1973; 225: 277-80. calcitonin, a new analogue of eel calcitonin. In: 7Fahrenkrug J, Hornum 1, Rehfeld JF. Effect of calci- Copp DH, Talmage RV eds. Endocrinology of http://gut.bmj.com/ tonin on serum gastrin concentration and component calcium metabolism. Amsterdam-Oxford: Excerpta pattern in man. J Clin Endocrinol Metab 1975; 41: Medica 1977: 383. 149-52. 22Yamauchi H, Shiraki M, Otani M, Matsuo M, Orimo "Ito H, Sakurada T, Orimo H. Role of endogenous H. Stability of [Asu1l7]-eel calcitonin and eel calcitonin calcitonin in the secretion of gastrin and gastric acid in in vitro and in vivo. Endocrinol Jap 1977; 24: 281-5. rats. Horm Metab Res 1976; 9: 89-92. 23Schusdziarra V, Harris V, Conlon JM, Arimura A, "Bloom SR, Mortimer CH, Thorner MO, et al. Inhibi- Unger R. Pancreatic and gastric somatostatin release in tion of gastrin and gastric-acid secretion by growth- response to intragastric and intraduodenal nutrients on September 25, 2021 by guest. Protected copyright. hormone release-inhibiting hormone. Lancet 1974; 2: and HC1 in the dog. Clin Invest 1978; 62: 509-18. 1106-9. 24Van Noorden S, Polak JM, Pearse AGE. Single cellular 10Barros D'Sa AAJ, Bloom SR, Baron JH. Inhibition by origin of somatostatin and calcitonin in the rat thyroid somatostatin (growth-hormone release-inhibiting hor- gland. Histochemistry 1977; 53: 243-7. mone, GH-RIH) of gastric acid and pepsin and G-cell 25Barlet JP, Garel JM. Influence de la somatostatine sur release of gastrin. Gut 1978; 19: 315-20. le taux plasmatique de calcitonine chez le veau et chez '1Arimura A, Sato H, Dupont A, Nishi N, Schaily AV. le porc. C R Soc Biol (Paris) 1975; 169: 1476-82. Somatostatin: abundance of immunoreactive hormone 26Metz SA, Deftos LJ, Baylink DJ, Robertson RP. in rat stomach and pancreas. Science (Wash D.C.) Neuroendocrine modulation of calcitonin and para- 1975; 189: 1007-9. thyroid hormone in man. J Clin Endocrinol Metab 1978; 'McIntosh C, Arnold R, Bothe E, Becker H, Kobberling 47: 151-9. J, Creutzfeldt W. Gastrointestinal somatostatin: 27Hargis GK, Williams GA, Reynolds WA, et al. Effect extraction and radioimmunoassay in different species. of somatostatin on parathyroid hormone and calcitonin Gut 1978; 19: 655-63. secretion. Endocrinology 1978; 102: 745-50.