Effect of Bombesin on Lower Esophageal Sphincter Pressure in Humans

Transcription

1 GASTROENTEROLOGY 1982;83:10-4 Effect f Bmbesin n Lwer Esphageal Sphincter Pressure in Humans ENRICO CORAZZIARI, GIANFRANCO DELLE FA VE, CLAUDIO POZZESSERE, ANNA KOHN, LAURA DE MAGISTRIS, FIORELLA ANZINI, and ALDO TORSOL! Cattedra di Gastrenterlgia, Clinica Medica II, Universita di Rma, Rme, Italy Intravenus infusin f bmbesin in humans results in increased serum gastrin levels and increased lwer esphageal sphincter pressure. T differentiate a gastrin- frm a nngastrin-mediated mechanism, the effect f intravenus bmbesin infusin n lwer esphageal sphincter pressure was studied in 5 healthy subjects and in 5 antrectmized patients. Lwer esphageal sphincter pressure was recrded by three cntinuusly perfused catheters with sidepenings lcated 5 mm apart. Bld samples were taken at regular intervals during the study perids t assay gastrin and pancreatic plypeptide. Manmetric tracings and bld samples were cded and evaluated blindly. Lwer esphageal sphincter pressure increased during bmbesin infusin bth in nrmal and antrectmized subjects. The increase had a delayed nset and persisted after discntinuatin f bmbesin. N crrelatin was fund between lwer esphageal sphincter pressure and serum gastrin r pancreatic plypeptide values. These data indicate that the effect f bmbesin n lwer esphageal sphincter pressure is nt gastrin r pancreatic plypeptide mediated. Bmbesin is a tetradecapeptide islated frm the skin f the Eurpean amphibians Bmbina bmbina and Bmbina variegata (1). Like ther bilgically active peptides islated frm frg skin, a bmbesin- Received May 11, Accepted February Address requests fr reprints t: Enric Crazziari, M.D., via Ug De Carlis Rma, Italy. This study was supprted by CNR grant N The authrs thank Prfessr V. Erspamer fr his cnstructive criticism and Mrs. M. Shields fr help with the linguistic style. Bmbesin was kindly supplied by Dr. R. De Castigline (Farmitalia, Italy). This paper was presented at the Third Internatinal Sympsium n Gut Hrmnes, Cambridge. United Kingdm. September 14-18, by the American Gastrenterlgical Assciatin /82/ $02.50 like peptide has been identified in endcrine cells f the human gastrintestinal tract (2). Bmbesin by intravenus (i.v.) infusin prduces an increase in serum gastrin (3), mainly released frm the gastric antrum (4), and in lwer esphageal sphincter (LE5) pressure (5,6). A gastrin-mediated effect f bmbesin n the human LE5 has recently been suggested (6). The aim f the present investigatin was t study the effect f i.v. infusin f bmbesin n LE5 pressure. Nrmal subjects and antrectmized patients were studied t differentiate a gastrin- frm a nngastrin-mediated effect. Materials and Methds Five nrmal male vlunteers (mean age: 37 ± 9 yr, mean ± SD) and 5 patients (4 men and 1 wman; mean age: 52 ± 11 yr) previusly submitted t antrectmy and gastrjejunal (4 patients) r gastrdudenal (1 patient) anastmsis fr dudenal ulcer were studied. At the time f investigatin, nne f the subjects studied had gastrintestinal symptms, r clinical evidence f gastresphageal reflux. Nne f the individuals were taking drugs. This study was apprved by the Clinical Research Cmmittee f the Clinic a Medica II, University f Rme, n Nvember 13, 1979 and infrmed written cnsent was btained frm each subject. Three water filled, plyvinyl catheters, 1 mm ID, were used t transmit intraluminal pressures t Elema-Schnander external transducers. Manmetric variatins were then recrded n a multichannel Hellige plygraph. The catheters had side penings, 1.2 mm in diameter, placed t measure the intraluminal pressures at three distinct pints, 5 mm apart. The pressure recrding tubes were perfused with distilled water at a cnstant rate f mll min using a Braun syringe pump (B. Braun Instruments, San Francisc, Calif.). An additinal radipaque catheter, 3.6 mm ID with multiple rifices ver the distal 5 cm, was intrduced with the manmetric tube and psitined under flurscpic cntrl in the lwest part f the stmach r gastric remnant. Studies were perfrmed upn patients in the supine

2 July 1982 BOMBESIN AND LESP 11 psitin after vernight fasting. The recrding assembly was psitined with all the rifices in the stmach; affer a 20-min rest perid, the manmetric catheter was withdrawn by 0.5-cm statin pull-thrugh and anchred \\Then the three recrding rifices were lcated in the highpressure zne f the LES. Lwer esphageal sphincter pressure was recrded cntinuusly thrughut the 30- min Lv. saline infusin, the 30-min Lv. bmbesin infusin (10 ng. kg- 1. min- 1 ) and the 60-min Lv. saline infusin. In humans, Lv. bmbesin infusin at the dse f 10 ng. kg- 1 min- 1 has been shwn t affect dudenal, jejunal, and gallbladder (7,8) mtr activity and t induce maximal serum gastrin increase (persnal data). After preliminary gastric emptying, cntinuus aspiratin f gastric cntents was perfrmed in all subjects fr 20 min befre, and thrughut the Lv. infusin perids. Lwer esphageal sphincter pressure was recrded as cmhzo with the mean gastric fundic pressure as the zer reference. The highest midinspiratry pressure recrded frm ne f the three manmetric rifices was taken as LES pressure. Lwer esphageal sphincter pressure was measured at 1-min intervals r, if the subject swallwed, 20 s after the end f sphincter inhibitin. Each manmetric tracing was cded, cut int 30-min perids, and evaluated blindly by an external bserver. Bld samples fr serum gastrin and pancreatic plypeptide (PP) radiimmunassays were cllected thrugh an indwelling needle in a frearm vein at 15-min intervals during the basal saline infusin; then 5,10,20, and 30 min after the beginning f bmbesin infusin and every 15 min thereafter until the end f the study. Plasma specimens were cded and evaluated blindly. Gastrin assay was perfrmed using antiserum N (generusly dnated by Dr J. F. Rehfeld) at a final dilutin f 1: 125,000, which measures the sulfated and unsulfated frms f gastrin cmpnents I, II, and III with almst equimlar ptency (9,10). Synthetic human gastrin 17 I (SHG-17 I, Calbichern, USA, batch: L ) used as standard was labeled by a mdified chlramine-t methd. Tw micrgrams f gastrin were disslved in 20 J.Ll f 0.1 M phsphate buffer, ph 7.4. One millicurie (10 J.LI) f P Z5 Ilsdium idide and 20 J.LI (20 J.Lg) f freshly disslved chlramine-t (10 mg in 10 ml phsphate buffer) were then added. The reactin was stpped after 30 s by the additin f 50 J.Lg sdium metabisulfite. The mixture was applied n a diethylaminethyl (DEAE) Sephadex A 50 clumn (0.9 x 20 cm), eluted with a linear gradient f 0.02 M ammnium bicarbnate M NaCI t 0.02 M ammnium bicarbnate M NaCI (Gradient Mixer, Pharmacia, Sweden). The assay was perfrmed in 2 ml final vlume diluted in a 20 nm sdium-barbital buffer, ph % bvine serum albumin (BSA) (Fractin V, Sigma Chemical C., St. Luis, M.). Gastrin values were expressed as pmlll equivalent f SHG Pancreatic plypeptide radiimmunassay (11) was perfrmed using antiserum 248/19 (generusly dnated by Dr. R. Chance) at a final dilutin f 1: 6,000,000. Highly purified human pancreatic plypeptide (HPP) was used as standard and bvine pancreatic plypeptide (BPP) was labeled by a mdified chlramine-t methd. One micrgram f BPP was disslved in 0.1 M sdium-phsphate buffer, ph 7.5, t which 1 mci f [ lz5 Ilsdium idide (10 J.LI) and 10 J.LI f freshly disslved chlramine-t (4 J.Lg) in phsphate buffer were added. The reactin was stpped after 30 s by the additin f 25 J.Lg f sdium metabisulfite. Plasma free f PP (250 J.LI) and ptassium idide (200 J.Lg) were then added. The mixture was applied n a Sephadex G-50 SF clumn (0.9 x 100 cm) eluted with 0.1 M phsphate buffer + 0.1% BSA. The assay was perfrmed in 1.5 ml final vlume diluted by 20 nm sdium barbital buffer, ph % BSA. The values were expressed as pmlil equivalent f HPP. Results f bth peptide assays were evaluated cnsidering the bund/ttal rati, crrected fr damage f the labeled antigens. Manmetric tracings and bld samples were evaluated at the end f the study. Student's t-test fr paired data and crrelatin analysis were used t evaluate data. Results Nrmal Subjects Mean basal LES pressure in nrmal subjects was 25 ± 4 cmhzo (mean ± SEM) (Figure 1). Sphincter pressure prgressively increased during bmbesin infusin and differed significantly frm basal values 15 min after the beginning f peptide administratin (p < 0.05). Lwer esphageal sphincter pressure increased thereafter fr the remaining 15 min during, and fr 30 min after bmbesin infusin. Elevated sphincter pressure values persisted thrughut the study perid. Serum gastrin values rse prmptly during plypeptide administratin and differed significantly frm basal values (p < 0.05) 5 min after the beginning f bmbesin infusin. Peak values were reached after a further 15 min f infusin. Gastrin values then decreased and did nt differ statistically frm basal values 30 min after the end f peptide administratin. Althugh serum PP levels (evaluated in 3 f the 5 subjects) shwed wide individual variability, mean serum PP values rse significantly (p < 0.05) at 20 min during, and reached a peak 10 min after discntinuatin f bmbesin infusin. The PP values then decreased withut reaching basal levels. Antrectmized Patients Mean basal LES pressure in antrectmized patients was 27 ± 3 cmhzo (Figure 2). With the exceptin f the first 10 min f bmbesin infusin during which wide variatins in individual sphincter respnse were recrded, LES pressure differed significantly (p < 0.05) frm basal values 15 min after the beginning f peptide administratin, when peak respnse was reached. Thereafter sphincter pressure values persisted thrughut the study perid. Serum gastrin values did nt vary significantly during the study perids.

3 12 CORAZZIARI ET AL. GASTROENTEROLOGY Vl. 83, N.1, Part 1 pml/i Figure 1. Lwer esphageal sphincter pressure (LESP), gastrin (G), and pancreatic plypeptide (PP) serum levels befre, during, and after bmbesin infusin (black bar) in nrmal vlunteers. Pancreatic plypeptide serum values derived frm 3 patients , min A mderate, but nt significant, increase in serum PP levels was present during bmbesin administratin and in the fllwing saline infusin perid. Crrelatin Between LES Pressure and Serum Gastrin and Pancreatic Plypeptide Levels N crrelatin was fund between LES pressure and serum gastrin r serum PP values in any f the subjects studied in any perid f the investigatin. Discussin Bmbesin has been shwn t have a direct stimulating effect upn smth muscle derived frm varius parts f the human gastrintestinal tract (12). In viv studies in humans, n the ther hand, have shwn an inhibitry effect f bmbesin n the mechanical activity f the small intestine (7,12) and a stimulating effect n the gallbladder (8). Bmbesin stimulates release f antral gastrin (3,4), pancreatic plypeptide (13-15), glucagn (16), gastric inhibitry plypeptide (GIP) (17), angitensin (18), and prstaglandins (19), but a pssible inhibitry effect n vasactive intestinal plypeptide (VIP) release has als been reprted (20). Bmbesin may als stimulate chlecystkinin (4,21,22) release. It is therefre difficult t assess in viv the relative effects f the released r inhibited substances and the direct effects n gut smth muscle during bmbesin infusin. Data emerging frm the present study cnfirm that bmbesin infusin prduces an increase in LES basal tne bth in nrmal and antrectmized subjects. Hwever peak LES pressure values, measured pml/i 60cmHp 40 Figure 2. Lwer esphageal sphincter pressure (LESP), gastrin (G), and pancreatic plypeptide (PP) serum levels befre, during, and after bmbesin infusin (black bar) in antrectmized patients b min

4 July 1982 BOMBESIN AND LESP 13 during the infusin perid, were reached in a shrter time in antrectmized patients cmpared with that f nrmal subjects. It is nt pssible n the basis f data available t ffer an explanatin fr this difference. The lack f any significant increase in gastrin levels in the antrectmized patients indicates that the bmbesin effect n LES is nt gastrin mediated. These data are in agreement with thse btained by Mukhpadhyay and Kunnemann (5) in the pssum, but d nt cnfirm results reprted by Marletta et al. (6). Bvine PP has been shwn t stimulate LES pressure in the pssum (23). The present study cnfirms (14,15) that bmbesin mderately stimulates PP release in humans and shws that human LES pressure was nt affected by a PP-mediated mechanism. It wuld appear that bmbesin differs in its ability t release PP in the tw grups studied. Hwever cautin shuld be used in interpreting these data wing t the fact that a wide distributin f individual values was btained in 3 and 5 subjects f each grup, respectively. Due t the lack f sensitive circulating bmbesinlike immunreactivity assays, crrelatin between serum bmbesin values and LES pressure culd nt be made. Thus, n definitive cnclusins n the actin f bmbesin, either direct r indirect, can be drawn frm these results. Of the ther substances released r pssibly released by bmbesin infusin, chlecystkinin (24, 25), GIP (26), and glucagn (27-29) have inhibitry effects n LES pressure in viv. These are, therefre, nt respnsible fr the increment in LES pressure during bmbesin infusin. Bmbesin might affect LES pressure by inhibiting VIP release (20) r by stimulating prstaglandins (PG) (19) and angitensin (18) release. In this study the pssible effects f bmbesin n LES, thrugh its actin n VIP, PG, and angitensin, have nt been investigated. The actin f bmbesin n LES pressure in viv may thus represent the final respnse t different effects, which partly act n smth muscle and are partly mediated by the release f excitatry and inhibitry substances. Findings f this study cannt discriminate a direct effect f bmbesin n the sphincter muscle frm an indirect, but they exclude that it is mediated by either gastrin r pancreatic plypeptide. References 1. Anastasi A, Erspamer V, Bucci M. Islatin and structure f Bmbesin and Alytesin, tw analgus peptides frm the skin f the Eurpean amphibians Bmbin and Alytes. Experientia 1971 ;27: Plak JM, Blm SR, Hbbs S. et al. Distributin f a bmbesin-like peptide in human gastrintestinal tract. Lancet 1976;1: Delle Fave GF. Khn A. De Magistris L. et al. Effects f bmbesin stimulated gastrin n gastric acid secretin in man. Life Sci 1980;27: Bass N. Lezche E. Materia A. et al. Effect f bmbesin n extragastric gastrin in man. Am J Dig Dis 1975;20: Mukhpadhyay AK, Kunnemann M. Mechanism f lwer esphageal sphincter stimulatin by bmbesin in the pssum. Gastrenterlgy 1979;76: Marietta F, Mnell S, Catalan F. et al. Relatinship between lwer esphageal sphincter pressure and serum gastrin levels during bmbesin infusin. Ital J Gastrenterl 1979; 11: Crazziari E, Trsli A, Delle Fave GF, et al. Effects f bmbesin n the mechanical activity f the human dudenum and jejunum. Rend GastrenterI1974;6: Crazziari E. Trsli A. Melchirri p. et al. Effect f bmbesin n human gallbladder emptying. Rend Gastrenterl 1974; 6: Rehfeld JF. Stadil F, Rubin B. Prductin and evaluatin f antibdies fr radiimmunassay f gastrin. Scand J Clin Lab Invest 1972;30: De Magistris L. Rehfeld JF. A simple enzymatic prcedure fr radiimmunchemical quantitatin f the large mlecular frms f gastrin and chlecystkinin. Anal Bichem 1980;102: Schwartz TW, Hlst JJ. Fahrenkrug J. et al. Vagal chlinergic regulatin f pancreatic plypeptide secretin. J Clin Invest 1978;61: Bertaccini G. Impicciatre M. Mlina E. et al. Actin f bmbesin n human gastrointestinal mtility. Rend GastrenterI1974;6: Taylr IL. Walsh JH, Carter D, et al. Effects f atrpine and bethanecl n bmbesin-stimulated release f pancreatic plypeptide and gastrin in dg. Gastrenterlgy 1979; 77: De Magistris L. Delle Fave GF. Khn A. et al. Stimulatin f pancreatic plypeptide and gastric secretin by bmbesin in man. Ital J GastrenterI1979;11: De Magistris L, Delle Fave GF. Khn A, et al. Differential stimulatin f pancreatic plypeptide and gastrin secretin by bmbesin in man. Life Sci 1981;28: Delle Fave GF, Tamburran G. De Magistris L. et al. Glucagnlike immunreactivity induced by bmbesin in man. Regul Pept 1980;Suppll: Becker HD. Brger HW. Schafmayer A, Werner M. Bmbesin releases GIP in dgs. Scand J Gastrenterl 1978;13(Suppl 49): Erspamer V. Melchirri P. Spranzi N. The actin f bmbesin n the kidney f the anaesthetized dg. Br J Pharmacl 1973;48: Impicciatre M, Iavarini M. Razzetti R, Omini C. Relatinship between spasmgenic activity f bmbesin and prstaglandins n the rat pylrus. Pharmacl Res Cmm 1981;13: Melchirri p. Imprta G, Spranzi N. Inibizine della secrezine di VIP da parte della bmbesina nel cane. nel gau e nell'um. Rend Gastrenterl 1975;7(Suppll): Erspamer V. Imprta G. Melchirri P, et al. Evidence f chlecystkinin release by bmbesin in the dg. Br J Pharmacl 1974;52: Knturek SJ, Krl R. Tasler J. Effect f bmbesin and related peptides n the release and actin f intestinal hrmnes n pancreatic secretin. J Physil 1976;257:

5 14 CORAZZIARI ET AL. GASTROENTEROLOGY Vl. 83, N.1, Part Rattan S, Gyal RK. Effect f bvine pancreatic plypeptide n the pssum lwer esphageal sphincter. Gastrenterlgy 1979;77: Fisher R, Di Marin AJ, Chen S. Mechanism f chlecystkinin-induced inhibitin f lwer esphageal sphincter pressure. Am J Physil1975;228: Resin H, Stern DH, Sturdevant RAL, et al. Effect f the C terminal ctapeptide f chlecystkinin n lwer esphageal sphincter pressure in man. Gastrenterlgy 1973;65: Sinar D, D'Orisi TM, Thmas FB, et al. Effect f gastric inhibitry peptide n lwer esphageal sphincter pressure. Clin Res 1976;24: Jennewein HM, Waldeck F, Siewert R, etal. The interactin f glucagn and pentagastrin n the lwer esphageal sphincter in man and dg. Gut 1973;14: Hgan WF, Ddds WJ, Hke SE, et al. Effect f glucagn n esphageal mtr functin. Gastrenterlgy 1975;69: Jaffer SS, Makhluf GM, Schrr BA, et al. Nature and kinetics f inhibitin f lwer esphageal sphincter by glucagn. Gastrenterlgy 1974;67:42-6.