Identification of the Neurotransmitter/Neuromodulator Functions of the Neuropeptide Gastrin-Releasing Peptide in the Porcine Antrum, Using the Antagonist ( Led3- q-CH NH-LeuI4)-B ombesin J. J . HOLST, H. HARLING, T. MESSELL & D. H. COY Institute of Medical Physiology C, The Panum Institute, and Dept. of Surgery C, Rigshospitalet. University of Copenhagen, Denmark, and Dept. of Medicine, Tulane University Medical Center, New Orleans, Louisiana, USA

Holst JJ , Harling H, Messell T, Coy DH. Identification of the neurotransmitter/ neuromodulator functions of the neuropeptide gastrin-releasing peptide in the porcine antrum, using the antagonist (Leu'3-~-CH,NH-Leu'4)-bombesin. Scand J Gastro- enterol 1990, 25, 89-96

We studied the effects of a new bombesin/gastrin-releasing peptide (GRP) receptor antagonist, Le~'~-~p-(CH~NH)-Leu'~-bornbesin, on the secretion of gastrin and somatostatin and on the motor activity of isolated perfused porcine antrum in response to infusions of GRP at lo-"' or lo-' mol/l and in response to electric stimulation of the vagus nerves. GRP significantly increased the secretion of gastrin and somatostatin and increased the frequency of antral contractions threefold. At 0.5 x mol/l the antagonist completely abolished the effects on motility and gastrin secretion and strongly inhibited the effect on somatostatin secretion. Vagus stimulation significantly increased gastrin and somatostatin secretion and increased the contraction frequency threefold. The antagonist strongly inhibited the somato- statin response, abolished the motility effects and reversed the stimulatory effect on gastrin secretion to a significant inhibition. Assuming that the antagonist interacts specifically with GRP receptors, we conclude that our data strongly support the concept that GRP-producing nerves are essential for vagally induced secretion of gastrin and somatostatin from the antrum. The GRP nerves may also play a role in the control of gastric motor activity.

Key words: Antrum; bombesin antagonist; gastrin; gastrin-releasing peptide; motor activity; nerve stimulation; pig; somatostatin

J . J . Holst, M. D., Institute of Medical Physiology C, The Panum lnstiture, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

The unequivocal identification of the neuro- transmitter/neuromodulator functions of a neuro- peptide requires fulfillment of a set of criteria that naturally resemble those used for the established neurotransmitters (1) but also differ in several respects. For example, t he traditional require- ment fo r the presence of a biosynthetic machinery for t he transmitter in the nerve terminal is obviously incompatible with the ribosomal syn- thesis of the neuropeptides. Ra the r , a require- ment for the demonstration of axonal transport

would seem relevant. We recently proposed for neuropeptides the set of criteria ( 2 ) shown in Table I. Whereas criteria 1-4 a re indispensable for identification of neurotransmitter function, they d o not provide proof. That this is true is evident when one considers the complex com- position of the contents of the vesicles of nerve terminals: a very large proportion of terminals release more than one transmitter substance (co- existence (3)) and other substances with potential or established regulatory functions (adenosine tri-

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Table 1. Identification of peptidergic control

I . The peptide must be there 2. The peptide must be active in that system (implying

that receptors must be present and that the peptide must he sufficiently potent)

3. The peptide must be released on appropriate stimu- lation

4. Release o f peptidc and response must parallel each iithcr (e.g. during pharmacologic blockade)

5 . Specific interference with peptide action/release should influence rcsponse:

a) desensitization b) antagonists c) antibody blockade (Fab fragments)

phosphatc, chromogranin A , Ca++). Thus, proof of the regulatory functions of a single neuro- peptide will require specific interference with the action o r release of the peptide in question. For this purpose specific and potent antagonists rep- resent valuable tools.

For the neuropeptide gastrin-releasing peptide 1,GRP) there is ample evidence of its transmitter role in the vaga.1 control of gastrin release from the gastric antrum of rats (4) and pigs ( S , 6 ) . The evidence includes inhibition/elimination of the gastrin response to electric vagus stimulation after desensitization of the gastrin cells for GRP, block- ade with antibodies against G R P , and the use of peptide antagonists. It may, however, be argued that the specificity of desensitization is not well established and that it is extremely difficult to produce a complete blockade in antibody per- fusion experiments. Finally, the antagonist used so far. a substance P analog (7), is not only weak (and must therefore be given in very high con- centrations. with subsequent risk of non-specific effects) but is also per definition unspecific. since it antagonizes both G R P and substance P actions. Thus, in the case of G R P there is a need for improved antagonists.

An antagonist that seems to be promising was introduced recently: (Le~'~-~pLeu'~)-bombesin (8). This antagonist effectively antagonizes bom- besin's effect on pancreatic acini with a 100-fold improvement in potency compared with pre- viously reported antagonists and showed no affin-

ity for substance P receptors. We therefore decided to repeat our studies of the effects of electric vagus stimulation on the endocrine secretion of the isolated perfused porcine antrum, this time including administration of the new antagonist. We also stildied the vagal effects on antral motility, since it became apparent that the antagonist strongly interfered with this as well.

MATERIALS A N D METHODS

Twelve pigs of the LYY strain, weighing 12- 16 kg, served as donors. Pig antrum with intact vagal innervation was isolated and perfused as described (5,9). In short, the antrum and the pancreas were isolated together and perfused through a segment of the aorta that included the celiac trunk and the superior mesenteric artery. The vagal innervation was preserved by including the lesser omentum in the preparation. The venous effluent from the preparation was drained from the portal vein, draining most of the prep- aration, and from a catheter inserted in the right gastroeploic vein, previously demonstrated to drain exclusively the antrum (9). The vagus nerves were threaded through a tunnel electrode and stimulated electrically with square-wave impulses of 4 msec and 10 m A at 4-8 Hz. The preparation was perfused in a single-pass system with a Krebs-- Ringer bicarbonate solution containing 0.1 9+ human serum albumin, S% dextran T70,7 nimoI/l glucose, aprotinin (100,000 KIU/I; Trasylol@. Bayer, Leverkusen, FRG), and a mixture of amino acids (17 amino acids, both essential and non-essential; Amodex Asa, Pharmacia. Sweden) added to give a final concentration of Smmol amino acids/l perfusate. The medium was sup- plemented with 20%: fresh, washed bovine erythrocytes and perfused at a rate of 0.4 ml' g.min, whereby an oxygen consumption for the whole preparation of 9-12 yllmin x g was ensured (5,9). Because of the presence of the erythrocytes we also added indomethacin (Con- fortid@, Dumex, Copenhagen. Denmark) to a concentration of 5 mg/I perfusate, to prevent for- mation of prostaglandins in the perfusate.

The flow of perfusate from the antral vein

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remained constant in the basal state and amounted to 2-6ml/min in the various prep- arations. The effluent was collected for 1-min periods in graded tubes (Minisorp, Technunc, Roskilde, Denmark), measured, chilled in ice, and centrifuged within a few minutes at 4°C. During vagus stimulation the antral flow often decreased synchronously with antral contractions. The average flow, however, showed no consistent changes. The supernatant was frozen immedi- ately, until assayed for gastrin (10) and somato- statin (12). The gastrin antiserum was directed against the C-terminus of gastrin-17. It binds all four gastrin components in the circulation (12). The somatostatin antiserum (1758) binds to the mid-portion of the somatostatin molecule and thus binds all molecular forms that possess this sequence (13, 11). For both assays, recovery of the respective peptides added to perfusate devi- ated less than 10% from expected values.

50 -

c .- 5 25- 0

5

0 -

Neuropeptide GRP 91

Hormone outputs were calculated by multi- plication of flow and hormone concentration in perfusate.

GRP-(1-27) was purchased from Peninsula (code 7188; St. Helens, Merseyside, U.K.). The antagonist, (Le~'~-+-Leu'~)-bombesin, was pre- pared by one of us (D. H. Coy (8)). Before use the peptides were dissolved in small amounts of 0.5 mol/l acetic acid and diluted further with per- fusion medium.

As previously described (9), this preparation shows annular, propulsive contractions with reg- ular intervals. To record these contractions, a suture was tied to the serosal surface of either the ventral or the dorsal face of the antrum and connected to a force-displacement transducer that was connected to a Mingograph recorder. Motility was recorded continuously.

Statistical evaluation of the data consisted of paired comparisons of accumulated hormone out-

=

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3 0 t

1z1 c .- E - 25 \

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E

0 - 1 10 20 1 10

min

Fig. 1. The secretion of gastrin from isolated perfused antrum in response to GRP infused to reach a concentration of lo-'" mol/l or in response to electric stimulation of the vagus nerves at a stimulation frequency of 8 Hz (marked 'Va') with or without the concomitant infusion of a bombesin-receptor antagonist. Mean ?SEM of nine perfusion experiments.

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92 J . J. Holst el al.

puts or contraction frequencies obtained during nerve stimulations or GRP infusions with or with- out administration of the antagonist o r by com- parison of values obtained during nerve stimulation and in the immediately preceding 5- min periods. The significance of the differences was evaluated by the t test for paired data. The level of significance was chosen as 5%.

Erperirnental protocol After a 30-min equilibration period the vagus

nerves were stimulated electrically for 5 min at 4- 8 Hz (in nine perfusion experiments). After a t least a 10-min rest period GRP was infused by means of a precision pump via a side arm into the arterial line to reach calculated, final perfusate concentrations of IO-"' ( n = 9) or lo-' mol/l ( n = 7). In similar, previous experiments ( 5 , 6 ) the final concentration deviated less than 10% from the expected values. After another 10 min of rest the antagonist was added to the perfusate, to reach a concentration of 0.5 x mol/l. After 15 min of perfusion with the antagonist, the vagus stimulation and G R P infusions were repeated. In previous control experiments it has been demon- strated that the secretory responses to both repeated vagus stimulations and G R P infusions (reaching concentrations of 10-"' mol/l) remain unimpaired for up to 180 min of perfusion ( 5 , 6 ) . In three perfusion experiments the antagonist was given alone with no other stimulation. Antral motility was recorded in seven perfusion experi- ments.

RESULTS

Fig. I shows the secretion of gastrin from the isolated perfused antrum in response to infusion o f G R P to a final concentration of 10-"' mol/l and in response to electric stimulation of the vagus nerves at 8IHz both with and without perfusion with the bombesin antagonist. G R P infusion Iiicreascd gastrin secretion significantly and approximately twofold. Administration of the antagonist completely abolished this response. Administration of the antagonist alone had no significant effect on gastrin secretion (not shown). Vagus stimulation increased gastrin secretion sig-

nificantly from a basal level of 18.5 t 3.1 pmol/ min to a peak value of 35.1 * 10pmol/min. By contrast, during antagonist administration vagus stimulation caused a significant inhibition of gastrin secretion from a basal level of 27.2 t 3.0 to a minimum of 14.9 t 2.5 pmol/min. Because of GRP-induced desensitization, the gastrin responses to mol/l G R P were irregular and generally smaller than the response to lo-"' mo l l 1 ( 5 ) . Administration of the antagonist, however, completely abolished the effect of G R P (not shown).

Fig. 2 shows the secretion of somatostatin from the antrum. Vagus stimulation caused a significant increase in somatostatin secretion. After administration of the antagonist the somatostatin response to vagus stimulation was no longer significant, but a tendency towards an increase was still seen. G R P increased somato- statin secretion, weakly at a concentration o f lo-"' mol/l (not shown) and significantly at lo-' mol/l (from 0.24 * 0.02 to an average of 0.37 ? 0.04 pmol/min during the last 5 min of the

pmol/min

ANTRUM VEIN SOMATOSTATIN OUTPUT

1 5 10 15 1 5 10 15 M 25 min

Fig, 2. The secretion of somatostatin from isolated perfused antrum in response to GRP infused to reach a concentration of moi/i or in response to electric stimulation of the vagus nerves at a stimulation frc- quency of 8 Hz (marked 'Va') with or without the con- comitant infusion of a bombesin-receptor antagonist. Mean ? SEM of seven perfusion experiments.

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Neuropeptide GRP 93

W I T H A N T A G O N I S T A N T R A L M O T I L I T Y

8 H2 V M U S 8 HZ VAGUS

t G R P 10-9 t

G R P

I IN 1 M I N

Fig. 3. Contractions of isolated perfused antrum in response to electric stimulations of the vagus nerves or to GRP infused to reach concentrations of lo-"' or 10-'mol/l with or without the concomitant infusion of a bombesin receptor antagonist. The contractions were recorded by means of a force-displacement transducer attached to the serosal surface of the antrum by means of a suture. An upward deflection (arbitrary units) reflects a contraction. The motor events were recorded continuously, but only sections corresponding to a few minutes are shown hcre.

GRP infusion). After antagonist administration the secretory response was lower (from 0.23 5 0.035 to 0.27 2 0.04 pmol/min) but still significant. There was a significant difference between the responses to GRP with and without antagonist.

The results of the motility studies are shown in Figs. 3 and 4. Fig. 3 shows original recordings from a single experiment. In the unstimulated state the antrum contracted at a frequency of 1.5 contractions/min. Vagus stimulation at 8 Hz increased the frequency to 3.9/min. GRP at lo-'" and mol/l both increased the frequency of contractions to 4.O/min and 4.8/min, respect- ively. After antagonist administration there was no effect of either vagus stimulation or GRP administration. Fig. 4 shows a summary of the motility recordings. Both vagus stimulation and

GRP at a concentration of lo-"' mol/l increased the frequency of contraction significantly (from 1.42 * 0.13 to 4.39 * 0.36 and from 1.49 * 0.10 to 3.60 * 0.37 contractions/min, respectively). Administration of the antagonist virtually abol- ished the responses to both vagus stimulation and GRP.

DISCUSSION

The results obtained in this investigation confirm and extend the results from our previous studies of the role of GRP-producing neurons in the gastric antrum in pigs ( 5 , 6). First, it was clear that the new antagonist effectively antagonized the effect on gastrin secretion of GRP at a con- centration of lo-'" mol/l and its effects on motility at this and at a tenfold higher concentration. The

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0-l J 1. ttolst PI al.

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Eig. 4. Summary of data from recordings of motor activity of isolated perfused antrum in response to electric stimulation of the vagus nerves at 8 Hz (marked 'Va') and GRP infused to reach a concentration of 10-"'rnol/l with or without the concomitant infusion of a bombesin receptor antagonist. The mean frequency of contraction was determined for 5-min periods before, during, and after vagus stimulation and for 10-min periods before, during, and after GRP infusion in each of the seven perfusion experiments. and the mean values of these results illustrated as vertical bars ( + 1 SEM)

antagonist also strongly inhibited the effect of G R P on somatostatin secretion (at mol/l), but in this case an increased secretion was observed in spite of infusion of the antagonist to a concentration of 0.5 x 10-6mol/l. We have previously (6) noted that the antral somatostatin- producing cells are less sensitive to G R P than the gastrin cells., which is the reason for using a G R P concentration as high as lo-' mol/l, and we find it most likely that the new antagonist has a similarly lower affinity for the G R P receptor on the D- cells. It has been reported recently that the new antagonist rnay distinguish among receptors for G R P (14). Apparently, some receptors, such as the ti-cell receptors, bind the antagonist with high affinity, whereas others, such as oesophageal

receptors, d o not. In the case of the gastrin cells it is now well established that their affinity for GRP is very high (with a Kd of approximately lo-" mol/l(15)), whereas most other tissues have affinities that are several orders of magnitude lower. A detailed study of eventual G R P receptor subtypes in the porcine antrum wiH, however, require a less complicated experimental model.

In agreement with previous studies (5), administration of the G R P antagonist abolished the gastrin response to vagus stimulation. This observation corroborates the conclusion put for- ward previously by us and others (4-6) that GRP is likely to be the responsible transmitter of the effect of vagal activation on gastrin secretion. However, not only did the antagonist abolish

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Neuropeptide GRP 95

the stimulatory effect of vagus activation; the response was reversed to a significant inhibition. Thus, a vagal, inhibitory mechanism must also exist. Indirect evidence of inhibitory effects trans- mitted to the G-cells by vagal fibers has been derived from studies of postgastrectomy gastrin secretion and selective vagotomies (16), but the mechanism has never been elucidated.

In agreement with previous studies, the vagus effect on somatostatin secretion, which is gen- erally stimulatory in the antrum (6), was also strongly inhibited by the GRP antagonist, sug- gesting that this effect of vagus stimulation is also transmitted by GRP released from GRP- containing nerves, often observed in close associ- ation with the antral D-cells (6). Results from studies of canine D-cells and results obtained from preparations of rat somach have indicated that the effect of GRP on antral somatostatin secretion may be indirect, being sensitive to the effects of the ganglionic blocker hexamethonium and to the neurotoxin tetrodotoxin (17, 18). This question was not addressed in the present inves- tigation. However, because of the pronounced effects of the antagonist, it appears that GRP must play an important role in the somatostatin response to vagus stimulation, indirectly or not. The incompleteness of antagonist-induced inhi- bition of the somatostatin response to vagus stimulation and the concomitant inhibition of gastrin could indicate that somatostatin, acting in a paracrine manner, was responsible for the gastrin inhibition. Unfortunately, antagonists of somatostatin action sufficiently potent to enable an experimental study of this hypothesis are not available at present.

The most conspicuous result of this inves- tigation, however, is the marked effect of the antagonist not only on GRP-induced motility but also on the motor effects of vagus stimulation. GRP and bombesin have for some time been known to affect gastrointestinal motility (for a review see Ref. 19). Bombesin has been reported to induce a tetrodotoxin-resistant contraction of canine, gastric circular smooth muscle strips or isolated cells (20,21). However, the gastric motor effects resulting from close arterial injections were abolished by tetrodotoxin (22). Similarly,

atropine has been reported to antagonize bombe- sin’s motor effects (23), suggesting an effect mediated by activation of cholinergic mechanisms (21,23). In the present preparation GRP potently influenced the frequency of contractions of the antrum, whereas an effect on the strength of the contractions was not apparent. GRP/bombesin is known to cause the release of many other factors capable of influencing gastric motility/emptying, such as gastrin (24), motilin (25), neurotensin (26), and cholecystokinin (27). Obviously, the motor effects elicited by GRP in this investigation were independent of its effect on these peptides. Because of the single-pass perfusion technique, any motor effects of gastrin released by GRP would imply a paracrine mode of action, which would seem unlikely.

In agreement with studies in other species (28) we found GRP containing nerve fibers in the muscular layers of the antrum and in the mucosa. In fact, when present, nerve cell bodies were only found in the myenteric plexuses (5,6). The presence in the muscular layers and the motor effects of GRP suggest that the peptide may be involved in the motor control of the gastric antrum. Our results with the new antagonist strongly support this. There were no effects of the antagonist when given alone. It completely abolished motor effects of GRP and it strongly and significantly interfered with the motor responses to vagus stimulation. The stimulatory effect on the frequency of antral contraction is in agreement with the reported inhibitory effect of GRP/bombesin on gastric emptying in man and animals (29,30), because an increased antral motor activity generally delays gastric emptying

Thus, the application of the bombesin antag- onist to the porcine gastric antrum strongly sup- ports the concept that GRP-producing nerves participate in the vagally induced control of motor activity and gastrin and somatostatin secretion.

(31).

REFERENCES

1. McLennan H. Synaptic transmission. W . B. Saun- ders, Philadelphia, 1963, 17-24

2. Holst JJ. Identification of peptidergic nervous con- trol of gastrointestinal functions. Verh Deutsch Ges Inn Med 1987, 93, 132-136

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Oh J . J . H d s t er a[.

3. Hokfelt T, Fuxe K, Pernow B, eds. Coexistence of neuronal messengers: a new principle in chemical transmission. Prog Brain Res 1986, 68, 1-405

4. Schubert MI<, Saffouri B, Walsh JH, Makhlouf G. Inhibition of neurally mediated gastrin secretion by bombesin antiserum. Am J Physiol 1985, 248, G456G462

5. Holst JJ. Knuhtsen S, Brskov C, et al. GRP nerves in pig antrum: role of GRP in vagal control of gastrin secretion. Am J Physiol 1987, 253. G643- G649

6. Holst JJ, Knuhtsen S, Brskov C, Skak-Nielsen T, Poulsen SS. Vagn Nielsen 0 . GRP-producing nerves control antral somatostatin and gastrin secretion in pigs. Am J Physiol 1987, 253, G767- G77-1

7. Jenscn Rl, Jones SW. Folkers K. Gardner JD. A synthetic peptide that is a bombesin receptor antagonist. Nature 1984, 309, 61-63

S. C‘oy DH. Heinz-Erian P, Jiang N-Y. et al. Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity. J Biol Chem 1988, 263, SOSh-50h(l

9. Holst JI , Jensen SL, Knuhtsen S. Nielsen OV, Rchleld JF. Effect of vagus, gastric inhibitory poly- peptide, dnd HCI on gastrin and somatostatin rcleiise from perfused pig antrum. Am J Physiol 1983. 244. G51S-GS22

10. Stadil F. Rehfeld JF. Determination of gastrin in serum. Scand J Gastroenterol 1973, 8, 101-112

I I . IJilsted I-. Holst JJ. On the accuracy of’ radioimrniinological determination of somatostatin in plasma. Regul Pept 19x2, 4, 13-31

17. Rehfeld JF, Stadil F. Vikelscle J . Iminunoreactive gastrin componcnts in serum. Gut 1974. 15, 101- I12

13. Baldissera FGA, Holst JJ, Jensen SL, Krarup 7’. 1)istribution and molecular forms of peptides con- taining somatostatin immunodeterminants in cxtracts from the entire gastrointestinal tract of pig and man. Biochim Biophys Acta 1985, 838, 132- 133

14. von Schrenk ‘T. Coy DH. Gardner JD, Jensen RT. Differcnt classes of bombesin receptor antagonists distinguish bornbesin (BN) receptor subtypes. Biomed Res 1988 (suppl I ) , 77

15. Vigna SR. Giraud A, Sol1 AH, Walsh JH, Mantyh PW. Characterization of bombesin receptors on canine antral gastrin cells. Regul Pept 1987, 19, 144

10. Debas HT. Hollinshead J , Seal A, Soon-Shiong P, Walsh JH. Vagal control of gastrin release in the dog: Pathways for stimulation and inhibition. Sur- gery 1984. 95. 3+37

17. Martindale R, Kauffman GL, Levin S, Walsh JH, Yamada T. Differential regulation of gastrin and somatostatin secretion from isolated perfused rat stomachs. Gastroenterology 1982, 83. 240-244

18. McDonald TJ, Fox JET. Effects of porcine gastrin releasing peptide (GRP) on canine antral motility and gastrin release in vivo. Life Sci 1984, 35, 14-15

19. McDonald TJ. The gastrin-releasing polypeptide (GRP). Adv Metab Disord 1988, 11, 199-250

20. Mayer EA, Elashoff J , Walsh JH. Characterization of bornbesin effects on canine gastric muscle. Am J Physiol 1982, 243, G141-Gl47

21. Fox JET, McDonald TJ. Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine. Life Sci 1984,35. 1667- 1673

22. Fox JET, McDonald TJ, Collins SM. Comparison of motility and secretagogue actions of bombesin and gastrin releasing peptide (GRP). Dig Dis Sci 1984, 29(suppl), 27s

23. Bartho L, Holzer P, Donneer J, Lembeck F. Effects of substance P, cholecystokinin oetapeptide, bom- besin, and neurotensin on the peristaltic reflex o f the guinea pig ileum in the abscence and in the presence of atropine. Naunyn Schmidebergs Arch Pharmacol 1982, 321, 321-328

24. Mulholland MW, Debas HT. Physiology and patho- physiology of gastrin: a review. Surgery 1988. 103,

25. Poitras P, Tasse D, Laprise P. Stimulation of motilin release by bombesin in dogs. Am J Physiol 1983.

26. Holst Pedersen J , Knuhtsen S, Bernarbei M, Brskov C, Holst JJ. Secretion of neurotensin from the isolated perfused porcine ileum. Regul Pept

27. Cantor P, Holst JJ, Knuhtsen S, Rehfeld JF. Effect of neuroactive agents on cholecystokinin release from the isolated perfused porcine duodenum. Act;i Physiol Scand 1987, 130. 627-632

28. Moghimzadeh E , Ekman R, Hikanson R, Yanai- hara N , Sundler F. Neuronal gastrin-releasing pep- tide in the mammalian gut and pancreas. Ncuro- science 1983, 10, 553-563

29. Scarpignato C, Micali, Vitulo F, Zimbaro G, Bert- acini G . The effect of bombesin on gastric emptying of solids in man. Peptides 1981, 2, 199-203

30. Vagne M, Collinet M, Cuber JC, et al. Effect of porcine gastrin releasing peptide on gastric secretion and motility and the release of hormonal peptides in conscious cats. Peptides 1987, 8 , 423-430

31. Minami H, McCallum RW. The physiology and pathophysiology of gastric emptying in humans. Gastroenterology 1984, 86, 1592-1610

135-147

245, G249-G2S6

1988, 21, 13-19

Recei\ed 17 March 1989 ALccpted 30 Junt 1989

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