Biomedical Research 5 (3) 229-234, 1984 EFFECT OF NEUROMEDIN B ON GUT HORMONE SECRETION IN THE RAT - Mitsuyoshi NAMBA, Mohammad A. GHATEI, Thomas E. ADRIAN, Adolfo J. BACARESE-HAMILTON, Peter K. MULDERRY and Stephen R. BLOOM Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 OHS, U.K. ABSTRACT Neuromedin B is a novel decapeptide which has recently been isolated from porcine spi- nal cord and shows striking sequence homology with bombesin-like peptides at the C-ter- minal region. The effect of synthetic neuromedin B on the secretion of gastrointestinal and pancreatic regulatory peptides has been compared with bombesin in the rat. Insulin, glucagon, enteroglucagon, gastrin, cholecystokinin (CCK) and bombesin were measured in plasma by radioimmunoassays. Neuromedin B (1.0 nmol) had significant stimulatory effects on insulin, enteroglucagon, gastrin and CCK release, similar in pattern but slightly less potent than those ofbombesin (1.0 nmol). Neuromedin B had no significant effect on plasma concentrations of glucagon and bombesin. These results suggest the possibility that neuromedin B could be one of the neural factors which play a regulatory role in the control of the endocrine pancreas and gastrointestinal tract. A variety of putative peptide neurotransmitter with bombesin-like peptides at the C-terminal have been identified by radioimmunoassay and region (Table 1). immunocytochemistry in the gut and pancreas. Although the effect of neuromedin B has not Of these peptides, bombesin-like peptides which hitherto been investigated, it is apparent from was originally isolated from amphibian skin (4), the sequence homology that this peptide may exhibits biological activity on several mamma- well exhibit bombesin-like actions. The aim of lian digestive organs and in particular stimulation the present study was to compare the effect of of gut hormone secretion (5, 10, 14). Bombesin- neuromedin B with bombesin at a dose which like immunoreactivity has been found in the the latter peptide is effective in stimulatory secre- mammalian gastrointestinal tract (21, 29), brain tion, of gastrointestinal and pancreatic regulatory (25), lung (27) and plasma (7), but only porcine peptides in the rat. gastrin releasing peptide (GRP) has so far been isolated from pig gastric tissue (19). A smaller MATERIALS AND METHODS molecular form than that isolated, with a size similar to amphibian bombesin, has been Eighteen male Wistar rats, weighing 200-300 g, demonstrated chromatographically and may be were fasted for 18 h and anesthetized with diaze- the dominant form in some mammalian tissues pam (Valium, Roche Products, Welwyn Garden (22, 23). City, U.K.) and fentanyl citrate (Hypnorm, A novel decapeptide which was given the Crown. Chemical Comp., Kent, U.K.). A cannula name neuromedin B was recently isolated from was inserted into the jugular vein for blood porcine spinal cord (20). In structure-activity sampling, and another one into the femoral vein studies of bombesin-like peptides, the minimal for the injection of neuromedin B, bombesin or sequence with the full range of activity was the saline. Pure synthetic neuromedin B and bombe- C-terminal heptapeptide (6). Neuromedin B was sin (Peninsula Laboratories, San Carlos," CA, found to have a striking sequence homology U.S.A.) were dissolved in 150 mM saline contain- 230 M. NAMBA et al. Table 1 Amino Acid Sequences of Amplzfbian and Mariimafiai: Bombesfn-If/re Peptides Gly-Asn-Leu{Trp-Ala-Tin-Gly-His-k-Met-NH; g_l Neuromedin B - - - Ala-Lys-Met-Tyr-Pro-ArgGly-Asn—His{ Trp-Ala-Val-Gly-His-Leu-Met-NH2 , G R P pGlu-Gln-Arg-Leu-Gly-Asn-Gln-l Trp-A;a-Val-Gly-His-Leu-Met-NH; ,' Bombesin pGlu~Gly-Arg-Leu-Gly-Thr-Gln-I Trp-Ala-Val-Gly-His-Leu-Met-NH; I Alytesin pGlu-Gln~i' Trp-A;a-Val-Gly-His-Pg-Met-NH; I Litorin pGlu-Val-Pro-Gln-1' Trp-Ala-Val-Gly-His»-PIE-Met-NH; F Ranatensin Ser-Asn-Thr-Ala-Leu-Arg~Arg-Tyr-Asn~Gln4 Trp-Ala-1111;-oiy-His-m-Mei-NH, 1 Ranatensin-R l X-Glx-Thr—Pro-Gln-Lffrp-Ala I _ _ _ “ _ _ M{lfi-Gly-His-P_l_1e-Met-NH;_ _ _ _ _ _ _ _ _ _ _ _ W _ _ _ _ _ _ _ ____I Ranatensin-C ing 0.15 mM human serum albumin (Lister In- Plasma glucose showed a slight decrease at stitute, Elstree, Herts, U. K.). Rats were divided 10 min after neuromedin B and bombesin injec- into three groups, 6 rats in each group were ad- tion, but this did not reach statistical significance ministered the bolus injection of neuromedin B in either case (Fig. 1). The plasma levels of both (1.0 nmol), bombesin (1.0 nmol) or saline CCK and gastrin at 10 min after injection ofneu- respectively. The blood samples were taken from romedin B or bombesin were significantly higher the jugular vein at 10 min and 30 min after a than saline control (Fig. 2). bolus injection and transferred into heparinized Plasma enteroglucagon showed a significant tubes containing 400 KIU aprotinin (Trasylol, Bayer) per ml of blood. The sample was centri- pmol/l fuged as soon as possible and the plasma was 40$‘ INSULIN stored at —20°C until analyzed. *-l(- Gut hormone measurements were carried out II'II-‘ill- . 3'-'§IP-'. using previously described radioimmunoassays H ‘ M i iii.‘ for insulin (2), glucagon (3), enteroglucagon (12), l Iilii ‘I M;"% Iii 200-1-Q u l "'- gastrin (9), cholecystokinin (CCK) (1) and bom- 1 I--il- 7'1?‘ II-I-il-I-I|—I- - besin (18). None of these assays showed any -q-pn-Fibr- I I-I—-I- in-II-""- cross-reaction, except the enteroglucagon assay I-ix-III nu‘-||——l|-III-I ulna‘.-InIn-|_ '' with glucagon and the CCK assay with gastrin. III-|II— Illnxlll "-" --.—g Enteroglucagon was calculated by subtracting Cl- |:l— III-III l:l~ specifically measured pancreatic glucagon (C-ter- minal immunoreactivity measured with anti- pmol/I serum RCS5) from total N~terminal glucagon 20 PANCREATIC GLUCAGON immunoreactivity (measured with antiserum R59) (12). The concentration of CCK was derived by subtracting gastrin (determined by antiserum Gas-179) (9), from the total chole- cystokinin and gastrin, as determined by the ewe ti CCK assay (1). "W01/I GLUCOSE Plasma glucose estimations were carried out 10 ‘Z’-2 using standard glucose oxidase methodology as Xx _1I_ adapted for the Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA, U.S.A.). 5 ;._llllllllllllllll Statistical significance was determined using E Student’s non-paired t test, and all values are 0 W—— i given as the meaniSEM. sarme BOMBESIN NEUROMEDIN B Fig. 1 Effect of 1.0 nmol bolus of neuromedin B or RESULTS bombesin injected into a femoral vein on plasma insu- Plasma insulin level at 30 min after neuromedin lin, pancreatic glucagon and glucose in anesthetized B (1.0 nmol) injection was significantly higher rats. n=6, meant SEM. Open columns represent the level at 10 min after injection, and shaded columns than saline control (228i30 vs. 93137 pmol/l, at 30 min after injection respectively. *P<0.05, P<0.05, n=6) and was similar to the response to **P<0.0l for comparison between the groups of pep» equimolar bombesin (319142 pmol/l) (Fig. 1). tide injection and saline control. GUT HORMONE RELEASE BY NEUROMEDIN B 231 pmol/l injection were 47.51125 and 34.li4.1pmol/l 50_ GASTRIN respectively, these were similar to the levels 1 =i= =i= observed in the saline injection group. ‘ =i= DISCUSSION The results described in this study indicate that 21- tie lg Ba neuromedin B, the novel mammalian bombesin- ___,J pmol/1 like decapeptide, stimulates the secretion of gas- 50- CCK trointestinal and pancreatic regulatory peptides. =i= l ** =i=*=|= * Neuromedin B injection produces a rise in plasma insulin which is not secondary to a rise in glucose, since plasma glucose actually decreases. llllll We have already found bombesin-like immuno- reactivity to be present locally in the pancreas, 25-1OJ | 1 1:: ‘El Fl especially in bombesin-immunoreactive nerve fibres in the peri-insular spaces (13). Although it P"‘°'/1 ENTEROGLUCAGON is to date unknown whether neuromedin B plays 60-i * * a physiological role as a neurotransmitter in such IlK# I nerve fibres, the peptide seems to exert at least a bombesin-like insulinotropic effect. 1 =i= 30-i A stimulatory effect of bombesin on glucagon secretion in dogs (17, 18, 24) and in man (8, 11, 14) has been reported. These data would OJI U U suggest that bombesin might act on the A-cells of SALINE BOMBESlN NEUROMEDIN B the islets of Langerhans as well. In the present study, however, neither bombesin nor neuro- Fig. 2 Effect of 1.0 nmol bolus of neuromedin B or medin B has shown the effect on glucagon secre- bombesin injected into a femoral vein on plasma gas- tion in contrast to insulin. A similar negative trin, cholecystokinin (CCK) and enteroglucagon in effect on glucagon release in dogs receiving bom- anesthetized rats. n=6, meaniSEM. Open columns represent the level at 10 min after injection, and besin infusion intrapancreatically has been shaded columns at 30 min after injection respectively. reported by Kaneto er al. (16). The discrepancy *P<0.05, **P<0.01, ***P<0.001 for comparison be- between these findings is difficult to explain. tween the groups of peptide injection and saline con- Species, situation and/or dose differences in each trol experiment may explain the discrepancy. In contrast, neuromedin B stimulates the CCK, gastrin and enteroglucagon release signifi- rise at 30 min after neuromedin B injection, and cantly.