Bombesin Potentiates Taurocholic Acid-Induced Neurotensin Release in Rats C

Bombesin Potentiates Taurocholic Acid-Induced Neurotensin Release in Rats C. HERRMANN*, J. C. CUBER, T. DAKKA, C. BERNARD, AND J. A. CHAYVIALLE Downloaded from https://academic.oup.com/endo/article/128/6/2853/2535156 by guest on 27 September 2021 INSERM Unite 45, Hopital E. Her riot, Pavilion tf)is, 69437 Lyon Cedex 03; and Laboratoire d'Ecologie et de Physiologie du Systeme Digestif, INRA, 78350 Jouy-en-Josas, France

ABSTRACT. The hypothesis of synergistic effects between TC; the integrated response of NT-LI release was 3.2-fold higher luminal stimulants and the intramural neural network in the than the sum of responses to 10'9 M BOM and 10 mM TC alone. control of intestinal neurotensin (NT) release was investigated These cooperative effects were apparent with 10'10 M BOM and with an isolated vascularly perfused rat jejuno-ileum. Luminal TC concentrations over the physiological range 1-10 mM. Nei- administration of low doses of taurocholic acid (TC; 1, 5, and 10 ther tetrodotoxin (106 M) nor atropine (105 M) was able to mM) provoked only a small increase in NT-like immunoreactiv- modify this synergy. Substance-P and methacholine, two other ity (NT-LI) in the portal effluent (50%, 100%, and 130%, re- NT secretagogues, did not potentiate the TC-induced NT respectively, above basal). Increasing the concentration of TC to sponses. In conclusion, BOM potentiated the release of NT 20 mM induced a strong and sustained release of NT-LI (700% induced by TC, thus suggesting that bombesinergic neurons, above basal). The arterial infusion of bombesin (BOM; 10'9 M) among other stimulatory neurons of the enteric nervous system, induced only a transient rise in NT-LI levels (200% above basal), may modulate the sensitivity of N-cells to luminal stimulants. with a rapid return to basal values. In contrast, 10'9 M BOM {Endocrinology 128: 2853-2857,1991) synergistically enhanced NT-LI responses induced by 10 mM

EUROTENSIN (NT), a tridecapeptide widely dis- that SP is also a potent NT secretagogue (15). Addition- N tributed in the gastrointestinal tract of several ally, SP, BOM, and. methacholine (Me) synergistically species (1-3), is mainly located in open-type endocrine stimulated the release of NT (18), thus suggesting a cells, designated N-cells, in the ileal mucosa (4, 5). Cir- strong functional relationship between the intramural culating NT-like immunoreactivity (NT-LI) is thought neural network and the N-cell. to play a role in the control of several gut functions (6). In the present study the hypothesis of cooperative .However, the mechanisms that govern the release of effects between luminal factors and neurotransmitters in intestinal NT are poorly understood. the release of NT was tested with the isolated vascularly On one hand, some luminal nutrients are important perfused rat ileum. stimulants of NT release, since blood levels of NT-LI rise drastically after a carbohydrate- or fat-rich meal (7- Materials and Methods 11). Additionally, the luminal infusion of bile salts in an isolated vascularly perfused rat ileum is capable of in- Materials creasing the secretion of NT-LI (12). BSA (fraction V) was purchased from Biomerieux (Charbon- On the other hand, several neuroactive agents were nieres, France). Azonutril 25, a mixture of all amino acids (3.4% shown to influence the N-cells. The participation of a isoleucine, 9.3% leucine, 8.5% lysine, 6.3% methionine, 8.3% cholinergic pathway in the release of NT was first sus- phenylalanine, 3.4% threonine, 1.7% tryptophan, 8.4% valine, pected, since atropine reduced the food-induced NT re- 2.7% aspartic acid, 3.4% glutamic acid, 6.4% alanine, 16.8% sponses (13). Cholinergic agonists were later shown to arginine, 1% cysteine, 6% glycine, 3.4% histidine, 5.4% proline, modulate the secretion of NT in vitro (4,14,15). Peptides 0.9% serine, 0.2% tyrosine, 2% citrulline, and 1.5% ornithine) of the BOM family are well known stimulants of NT was obtained from Laboratoire Roger Bellon (Neuilly-sur- release (12, 16, 17), and a recent study demonstrated Seine, France). The total amino acid content was 14.8 g/100 ml mixture. Substance-P (SP), bombesin (BOM), NT, acetyl- Received December 11, 1990. j8-methyl-choline-chlorlde [methacholine (Me)], tetrodotoxin Address all correspondence and requests for reprints to: J. C. Cuber, (TTX), atropine sulfate, and taurocholic acid (TC) were pur- bis INSERM Unite 45, Pavilion H , Hopital E. Herriot, 69437 Lyon chased from Sigma (St. Louis, MO). 125Iodine was supplied as Cedex 03, France. * Recipient of Grant He 1725/1-1 from the Deutsche Forschungs- sodium iodide from Amersham (Aylesbury, Buckinghamshire, gemeinschaft. United Kingdom).

2853 2854 BOMBESIN POTENTIATES TC-INDUCED NT RELEASE Endo«1991 Voll28«No6 Methods cates in a final incubation volume of 800 fd. After a 24-h incubation with the antiserum at 4 C, labeled NT (2,000 cpm) Surgical preparation. The surgical steps and functional viability was added, and incubation continued over an additional 48-h of the isolated vascularly perfused rat jejuno-ileum were pre- period. Bound and free radioactivities were separated by char- viously reported in detail (12). Male Wistar rats (250-300 g) coal treatment. The sensitivity and IDSO of the assay were 0.6 were anesthetized with pentobarbital-sodium (50 mg/kg, ip). and 4.0 fmol/tube, respectively. The recovery of exogenous NT After opening the abdomen by a midline incision, the right and (2.5-50 fmol/ml perfusate) in the ethanol extraction procedure middle colic arteries were tied and cut off. A Silastic tube was was 75.2 ± 6.8% (n = 24). The antiserum is characterized by inserted into the lumen, about 20 cm proximal of the hindgut, 100% cross-reactivity with intact NT, 75% with NT-(4-13), and a second one was introduced into the terminal ileum. The 7% with NT-(7-13), 12% with NT-(8-13), and less than 0.1% loop was flushed out twice with 10 ml prewarmed isotonic with N-terminal fragments of NT or neuromedin-N. HPLC

saline. The remaining upper small intestine was then separated Downloaded from https://academic.oup.com/endo/article/128/6/2853/2535156 by guest on 27 September 2021 analysis of the portal supernatants revealed a single peak of by several ligatures on the supplying vessels and removed. A immunoreactivity coeluting with NT-(1-13) (12). metal cannula was quickly (within 30 sec) inserted into the superior mesenteric artery, and a Silastic one was introduced Calculations and statistics. The data in all figures are presented into the portal vein. The vascular perfusion started immediately as the mean ± SEM and are expressed as femtomoles per time at a rate of 2.5 ml/min. The perfused solution was a Krebs- period. Integrated responses were obtained by subtraction of Hemseleit buffer containing 25% washed bovine erythrocytes, mean basal values of released NT-LI from stimulated values 3% BSA, 8 mM glucose, and 1% Azonutril. This mixture was during the given period. For statistical analysis, a one-way continuously gassed with 95% O2-5% CO2 and warmed at 37 C. analysis of variance was performed, followed by a multiple The vascular perfusion pressure remained stable during the comparison test. experiments at 50-70 mm Hg. The intestinal loop was then transferred into a temperature-stable (37 C) plastic box filled Results with isotonic saline. The luminal perfusion with prewarmed isotonic saline at a flow rate of 0.25 ml/min was then started. Release of NT by TC The venous effluent was collected as 2-min fractions into chilled glass tubes containing 250 ^1 200 mM EDTA. The Luminal administration of TC stimulated the release erythrocytes were eliminated after centrifugation. The super- of NT dose-dependently over the range 1-20 mM. The natants were extracted with 2 vol ethanol, dried, and stored at first significant response was obtained with 5 mM TC -30 C for the subsequent determination of NT by RIA. [integrated response (IR), 96.7 ± 10.8 fmol/30 min]. The output of NT rose to 111.6 ± 25.0 fmol/30 min with 10 Experimental design. The experiments started with a 20-min mM TC and reached a maximal level with 20 mM TC basal period, followed by a 30-min stimulation period and a (IR, 583.3 ± 175.3 fmol/30 min). Upon luminal admin- subsequent 10-min basal period. NT release was induced by the istration of 30 mM TC, the IR of NT release reached luminal perfusion of a solution of TC, adjusted to pH 7.4. The 442.2 ± 101.6 fmol/30 min only. luminal stimulation began with a bolus injection of TC (4 ml over 1 min), followed by perfusion at a rate of 0.2 ml/min. At Infusion of TC in combination with BOM the end of the stimulation, the lumen was flushed out twice with isotonic saline. In basal periods, isotonic saline was infused Whereas vascular infusion of 10"9 M BOM alone pro- in the lumen at a rate of 0.25 ml/min. All drugs that were added duced only a transient increase in NT-LI in the portal to the vascular circuitry were dissolved in a Krebs-Henseleit supernatant (peak, 27.2 ±9.1 fmol/2 min; basal, 5.6 ± buffer supplemented with 3% BSA and delivered at a rate of 0.4 fmol/2 min), luminal administration of 10 mM TC in 0.25 ml/min via a catheter close to the vascular inflow. In some 9 combination with the arterial infusion of 10" M BOM experiments, vascular infusion of TTX (106 M) or atropine (105 M) was started 20 min before the beginning of the stimu- produced a sharp increase in the portal NT-LI level (74.8 lation period and continued throughout the experiment. A ±11.3 fmol/2 min after 2 min, from basal level of 8.0 ± single experiment was performed on each preparation. 2.2 fmol/2 min), followed by a high plateau secretion at about 45 fmol/2 min until the end of the infusion (Fig. RIA. The RIA for NT in the ethanol-extracted portal effluent 1). The integrated response of NT-LI was 599.9 ± 105.1 was performed as previously described with an antiserum (29G) fmol/30 min upon combined infusion of 10'9 M BOM obtained from a New Zealand White rabbit after repeated with 10 mM TC. This is about 3.2-fold higher than the injection of NT conjugated to bovine albumin through ethyl- sum of individual responses produced by the same stim- carbodiimide condensation (12, 19). This antiserum, which ulants given separately (Fig. 2). The effect of 10 mM TC specifically recognizes intact NT, was used in the assay at a was also potentiated by 1010 M BOM (IR, 346.3 ± 78.9 final dilution of 1:200,000. The radioactive ligand was mon- 8 oiodo-[125I-Tyr3]NT, prepared and purified as described by fmol/30 min) and 10 M BOM (IR, 464.6 ± 89.3 fmol/30 Hoist Pedersen et al. (20). On the day of the assay, the ethanol min; Fig. 2). extracts of the perfusate were reconstituted in assay buffer (50 The potentiating effect of 10'9 M BOM was also clear mM phosphate, pH 7.5, containing 2 mM EDTA and 2% porcine upon luminal administration of a concentration of TC serum). Reconstituted samples were assayed as 200-jul dupli- as low as 1 mM (IR, 386.2 ± 42.9 fmol/30 min; Fig. 3). BOMBESIN POTENTIATES TC-INDUCED NT RELEASE 2855

± 101.6 fmol/30 min, respectively, compared to the con- 9 T 90 i •o- BOM 10' M trol value of 599.9 ± 105.1 fmol/30 min with TC and -e- TC 10 mM -»- TC lOmM+BOM 10"9M BOM). Infusion of TC in combination with Me or SP The synergistic effect of BOM and TC on the release of NT was specific, since arterial infusion of 10"9 M SP or 10"6 M Me only slightly increased the TC-evoked secretion of NT (Fig. 4). The integrated response of NT 0 J obtained upon the combined infusion of SP with 10 mM -20 20 40 TC was equal to the sum of integrated responses calcu- Downloaded from https://academic.oup.com/endo/article/128/6/2853/2535156 by guest on 27 September 2021 time ( min ) lated from the experiments performed with the two stim- FlG. 1. NT-LI responses (femtomoles per 2 min; mean ± SE) upon ulants given separately (Fig. 5). Similarly, Me did not luminal perfusion of 10 mM TC alone (n = 7), vascular infusion of 9 potentiate the release of NT evoked by 10 mM TC (Fig. 10 M BOM alone (n = 7), or infusion of BOM and TC in combination 5). (n = 8).

750 n Discussion • BOM + TC 10 mM 0 BOM alone The present study demonstrates for the first time that • TCIOmM alone the NT response induced by a luminal stimulant (TC) 500 - can be strongly potentiated by the vascular infusion of BOM, a neurotrartsmitter found in the enteric nervous

250- TC 10 mM 100 -i 9 c -*• +BOM1O" M 9 0 - £ — + SP 10" M -o- + Me 10"6 M -10 -9 -8 CM BOM 10 M BOM 10 M BOM 10 M FIG. 2. Integrated responses of NT-LI release (femtomoles per 30 min; mean ± SE) during luminal administration of 10 mM TC in combination with increasing concentrations of BOM (•). Effects of individual agents are inset in bars. Results represent the mean ± SE of at least six experiments.

0 J 800 -i -20 20 40 c • TC+BOM 10 M 0 BOM 10"9M alone time ( min ) E 600- o 0 TC alone FlG. 4. NT-LI response:? (femtomoles per 2 min; mean ± SE) during K> luminal administration of 10 mM TC in combination with vascular •3 400 H infusion of BOM (n = 7; •), SP (n = 6; •), and Me (n = 6; •).

800 -i _ 200 - c I 0 TC 10mM alone 0 transmitter alone J I 1 0 • TC 10 mM + transmitter 10 o TC concentration ( mM) FlG. 3. Integrated responses of NT-LI (femtomoles per 30 min; mean ± SE) during stimulation with 10'9 M BOM in combination with increasing concentrations of TC (1, 5, 10, and 20 mM; •). Effects of individual agents are inset in bars. Results represent the mean ± SE of 1 six experiments. 0 J '///A 9 However, the effect of 10" M BOM was not synergisti- 6 9 9 Me IO~ M SP10" M B0M1(T M cally increased by luminal administration of 20 mM TC 9 FIG. 5. Integrated responses of NT-LI (femtomoles per 30 min; mean (Fig. 3). The potentiating effect of 10 M BOM on TC- ± SE) during combined infusion of 10 mM TC with 10 mM Me {left), induced NT secretion was not modified by vascular in- SP (middle), and BOM (right). The values (D) are compared to the fusion of TTX or atropine (IR, 696.5 ± 182.8 and 594.0 sum of responses to TC and neurotransmitter given separately (H). 2856 BOMBESIN POTENTIATES TC-INDUCED NT RELEASE Endo• 1991 Vol 128 • No 6 system, while two other neurotransmitters, namely SP Ca2+-dependent mechanisms, such as BOM, but does not and Me, were not potentiating factors of NT release. interact synergistically with TC on NT release, it is likely Luminal nutrients were shown to be strong stimulants that the potentiating effect of BOM on TC-induced NT of intestinal NT in several species, suggesting that the secretion is a mechanism specifically related to the acti- secretion of NT-LI is mainly dependent on factors acting vation of BOM receptors. However, the validation of this at the apical pole of the N-cell. Glucose and fatty acids hypothesis requires extensive investigations with other increased plasma NT-LI levels (7, 8, 11, 12, 21). Addi- regulatory peptides found in enteric neurons and endo- tionally, the presence of pancreatic juice and bile salts crine cells, since peptides of the BOM family are capable in luminal chyme was presumed to be an important of stimulating the release of several gut peptides, which, component of the stimulatory effect of the nutrients (22). in turn, may affect the release of NT. Indeed, TC a naturally occurring bile salt in rats, was In conclusion, the present study demonstrates that the Downloaded from https://academic.oup.com/endo/article/128/6/2853/2535156 by guest on 27 September 2021 capable of stimulating the release of large amounts of release of NT provoked by a luminal stimulant may be NT in this species (12). Confirming these results, our greatly enhanced by a neurotransmitter of the intramural data showed a dose-dependent rise in portal NT-LI levels network, thus underlying a strong functional relationship after the luminal perfusion of TC over the range 1-20 between factors operating at the apical side of the N-cell mM. These TC concentrations correspond to those meas- and the intramural network in the release of intestinal ured in the intestinal chyme of the rat (23). NT. Whether this mechanism operates under physiolog- BOM-induced NT secretion was unaffected by the ical conditions remains to be elucidated. arterial infusion of TTX (our unpublished finding). This observation and the fact that BOM stimulates the secre- Acknowledgment tion of NT from isolated canine N-cells (24) consistently We thank P. Kitabgi for critical reading of an earlier draft suggest the presence of BOM receptors on ileal NT- of this paper. producing cells. However, the NT response to BOM was transient. In contrast, the combined infusion of TC with References BOM produced a sustained secretion of NT, which was 1. Carraway R, Leeman SE 1973 The isolation of a new peptide, about 3- to 4-fold higher than the sum of individual neurotensin, from bovine hypothalami. J Biol Chem 248:6854-6861 responses. Since this response was unmodified by arterial 2. Hammer R, Leeman SE, Carraway R, Williams R 1980 Isolation infusion of TTX, interactions of TC with BOM at the of human intestinal neurotensin. J Biol Chem 255:2476-2480 3. Kitabgi P, Carraway R, Leeman SE 1976 Isolation of a tridecapep- level of the N-cell may be speculated. Additional work tide from bovine intestinal tissue and its partial characterization with isolated N-cells purified to homogeneity is required as neurotensin. J Biol Chem 251:7053-7058 to elucidate the intracellular events underlying the po- 4. Helmstaedter V, Taugner C, Feurle GE, Forssmann WG 1977 Localization of neurotensin-immunoreactive cells in the small in- tentiating effect of BOM on TC-induced secretion. The testine of man and various mammals. Histochemistry 53:35-41 synergy between BOM and TC was not revealed when 5. Polak J, Sullivan S, Bloom S, Buchan ANJ, Facer P, Brown MR, TC (20 mM) was administered, suggesting that the max- Pearse AGE 1977 Specific localisation of neurotensin to the N cell' in human intestine by radioimmunoassay and immunocytochem- imal secretory rate of NT was reached. This is consistent istry. Nature (Lond) 270:183-184 with our observation that infusion of 30 mM TC did not 6. Ferris CF 1989 Neurotensin. In: Schultz SG, Makhlouf GM (eds) produce additional release of NT compared to that ob- Handbook of Physiology, vol 2, sect 6. American Physiological Society, Bethesda, pp 559-586 tained with 20 mM TC. 7. Mashford ML, Nilsson G, Rokaeus A, Rosell S 1977 The effect of Cholinergic agonists were also shown to be potent food ingestion on circulating neurotensin-like immunoreactivity stimulants of NT release in rats (14). The hypothesis of (NTLI) in the human. Acta Physiol Scand 104:244-246 8. Rosell S, Rokaeus A 1979 The effect of ingestion of amino acids, a potentiating effect of Me on TC-induced secretion was, glucose and fat on circulating neurotensin-like immunoreactivity thus, tested. Me interacted with TC in an additive man- (NTLI) in man. Acta Physiol Scand 107:263-267 ner. Additionally, the possibility that acetylcholine re- 9. Flaten O, Hanssen LE 1982 Concentration of neurotensin in hu- lease from nerve terminals is involved in the potentiating man plasma after glucose, meals and lipids. Acta Physiol Scand 114:311-313 mechanism of BOM may be excluded, since atropine did 10. Hammer RA, Carraway RE, Leeman SE 1982 Elevation of plasma not modify the secretion of NT induced by TC and BOM neurotensinlike immunoreactivity after a meal. J Clin Invest 70:74- in combination. 81 11. Go VLW, Demol P 1981 Role of nutrients in the gastrointestinal SP was shown to be a potent stimulant of NT release release of immunoreactive neurotensin. Peptides [Suppl 2] 2:267- (15). A direct effect of SP on N-cells was speculated, 269 12. Cuber JC, Herrmann C, Kitabgi P, Bosshard A, Bernard C, De since TTX or atropine did not significantly reduce SP- Nadai F, Chayvialle JA 1990 Neuromedin-N is not released with induced NT secretion (25). However, activation of SP neurotensin from rat ileum. Endocrinology 126:1584-1592 receptors along with luminal administration of TC re- 13. Fletcher D, Shulkes A, Bladin P, Booth D, Hardy K 1983 Cholin- ergic inhibition of meal stimulated plasma neurotensin like im- sulted in NT release that did not exceed the sum of munoreactivity in man. Life Sci 33:863-869 individual responses. Since SP is assumed to act via 14. Gill SS, Lee YC, Ghatei MA, Ghiglione M, Uttenthal LO, Bloom BOMBESIN POTENTIATES TC-INDUCED NT RELEASE 2857

SR 1984 The use of a rat isolated preparation to investigate the individual food components and effects on exocrine pancreas se- release of neurotensin. Clin Exp Pharmacol Physiol 11:457-466 cretion. Pancreas 5:306-313 20. Hoist Pedersen J, Stadil F, Fahrenkrug J 1983 Preparation of 125I- 15. Herrmann C, Cuber JC, Abello J, Dakka T, Bernard C, Chayvialle 125 JA 1991 Release of ileal neurotensin in the rat by neurotransmitters (Tyr 3)- and I-(Tyr ll)-neurotensin for radioimmunoassay. and neuropeptides. Regul Peptides 32:181-192 Scand J Clin Invest 43:483-491 16. Barber DL, Buchan AMJ, Walsh JH, Soil AH 1986 Isolated canine 21. Ferris CF, Hammer RA, Leeman SE 1981 Elevation of plasma ileal mucosal cells in short-term culture: a model for study of neurotensin during lipid perfusion of rat small intestine. Peptides neurotensin release. Am J Physiol 250:G374-G384 [Suppl 2] 2:263-266 17. Rokaeus A, Yanaihara N, McDonald TJ 1982 Increased concentra- 22. Rokaeus A, Al-Saffar A 1983 The importance of bile and pancreatic tion of neurotensin-like immunoreactivity (NTLI) in rat plasma juice for fat-induced release of neurotensin-like immunoreactivity (NTLI) from the small intestine of the rat. Acta Physiol Scand after administration of bombesin and bombesin-related peptides 119:33-37 (porcine and chicken gastrin-releasing peptides). Acta Physiol 23. Dietschy J 1968 Mechanisms for the intestinal absorption of bile Scand 114:605-610

acids. J Lipid Res 9:297-309 Downloaded from https://academic.oup.com/endo/article/128/6/2853/2535156 by guest on 27 September 2021 18. Herrmann C, Cuber JC, Bernard C, Dakka T, Chayvialle JA 1990 24. Barber DL, Buchan AMJ, Walsh JH, Soil AH 1986 Regulation of Cooperative effects of bombesin, substance P and methacholine in neurotensin release from canine enteric primary cell cultures. Am the release of intestinal neurotensin in the rat. Gastroenterology J Physiol 250:G385-G390 98:A498 (Abstract) 25. Herrmann C, Cuber JC, Bernard C, Chayvialle JA 1990 Substance 19. Cuber JC, Philippe C, Abello J, Corring T, Levenez F, Chayvialle P releases intestinal neurotensin in rats. Digestion [Suppl 1] 46:43 JA 1990 Plasma neurotensin in the conscious pig: release by (Abstract)