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Xenin relaxes precontracted rat ileal smooth muscle in vitro: Structure activity relationship and binding studies
April 1995
Motility and Nerve-Gut Interactions
A585
• XENIN RELAXES P R E C O N T R A C T E D RAT I L E A L S M O O T H MUSCLE IN VITRO: STRUCTURE ACTIVITY RELATIONSHIP AND BINDING STUDIES A, Clemens, S Katsoulis, R. Nustede*, K. Seyfarth*, C. MorysWortmann*, G. E. Feurle~ U. R. F61sch and W. E. Schmidt. Laboratory of Molecular Gastroenterology, I. Dept. of Medicine, Christian-Albrechts-University , Kiel; *Dept. of Surgery, Georg-August-University, G6ttingen; *Dept. of Immunochemistry, Max-Planck Institute for Experimental Medicine, Gtttingen; ~ DRK-Hospital, Neuwied; Germany Recently, the 25-residue peptide Xenin (Xe), a xenopsin-like novel member of the neurotensin (NT) peptide family, was isolated from human gastric mucosa. This study was designed to characterize the effect of Xe compared to NT on rat ileal smooth muscle activity. Methods: Xenin peptides were synthesized by Fmoc chemistry and HPLC-purified Full-thickness longitudinal rat ileal muscle strips were mounted under lg tension in a continuously oxygenated organ bath (pH 7.4, 37°C). Changes in motility were measured isometrically. Displacement of 125INT by Xe and analogues was studied in isolated ileal myoeytes. Results: Xe relaxed in a dose-dependent manner KCl-preeontracted (30 mM) rat ileum segments in vitro (ECso, Xe: 25 ± 2,0 nM, NT: 20 ± 0,8 nM, xenepsin: 15 ± 1,9 nM and neuromedin N: 51 ± 5,0 aM). Kinetensin and NT (1-11) (31aM each) were ineffective. Xe (12-25), Xe (14-25), Xe (19-25), Xe (20-25) and NT (9-131 were equipotent compared to Xe and NT. The order of potency for other fragments was: Xe (21-25) > NT (9-13) > Xe (1-24) >> Xe (1-23). The efficaeies of all peptides were identical. TTX (1 ~M), hexamethonium (1 I~M), atropine (l~tM), tetmethylammonium (1 mM) and 4-aminopyridine (1 mM) did not modulate the effects of Xe (l~tM) and NT (II#M). The bee venom potassium channel blocker apamia (300 nM) abolished NT- or Xe-induced relaxations° respectively. Desensitisation against NT prevented the effect of Xe and vice versa The specific NT receptor antagonist SR 48692 (3 ~M) fully inhibited Xe- or NT-mediated effects. Xe (KD 0,6 aM, B ~ 10,8 pM), Xe (13-25) (KD 1 nM, B~= 7,9 pM) and SR 48692 potently displaced t251-NT binding to ileal myoeyteso Xe (1-23) was inactive. Conclusions: Xe and NT exert direct myogenic relaxant effects on KCIprecontraeted rat ileum strips in vitro. Stmcture-aetivity and binding data suggest that Xe and NT activate the NT receptor which is coupled to an apamm-sensitive KT-chanael. High-affinity binding of Xe requires the C-terminal penmpeptide of NT or Xc. Supported by DFG grants
• INTRAGASTRIC INFUSION OF CHLORHYDRIC ACID (HCI) POTENTIATES GASTRIC SENSITIVITY TO GASTRIC DISTENSION 1N MAN. B. Coffnk R. ChoUet, B. Flourit, M. L~maun, C. Franchisseur, JC. Rambaud, R. Jian. INSERM U290, Htpitaux Saint-Lazare and Saint-Louis, 75010 Paris, France.
• SMALL INTESTINAL MANOMETRY IN PATIENTS WITH THE SHORT BOWEL SYNDROME (SBS) AND INTERPOSITION OF A REVERSED SMALL BOWEL LOOP (RSBL). B. Coffin, B. Flourit, P. Valleur, M. Ltmarm, P. Hautefeuille, JC Rambaud, B. Messing. Services de Gastroenttrologie and Chirurgie, Htpitanx Saint-Lazare and Lariboisitre, 750[0 Paris, France.
• ABDOMINAL SURGERY-INDUCED INHIBITION OF GASTRIC EMPTYING IN THE RAT: R E S P E C T I V E R O L E OF C G R P AND IL-115. C. Coimbra J. McDuff, M. Boivin, V. Plourde. Laboratory of Gastrointestinal Neurobiology and Motility, Andre-Viallet Research Center, University of Montreal, Montreal, QU., Canada.
In patients with SBS we have prevfously shown that the interposition of RSBL increases the intestinal absorption of nutrients (Gastroenterology 1994, 106 A601). In this work. we studied the intestinal motility in 5 patients (21-70 yrs) with SBS (length 50 "12 era) (M*SEM) who had undergone interposition of RSBL (length 10-15 can) belwe~mthejejnaum and colon and we Compared them with 5 matched control subjects (29-72yrs) with SBS (length 56*25 cm) but without interposition of RSBL. Methods : Duodeno-jejunal motility was recorded in the fasting state for 6 h using 4 perfused catheters loeated 10, 20, 30 and 45 cm from the pylorus. Recordings were visually analyzed for phases HI of the migrating motor complex, discrete clustered contractions (DCCs) and prolonged propagated contractions (PPCs). Sp~ifie motility patterns such as prolonged bursts of nonpropagated contractions or retropropagated (emily) contractions were also analyzed. Results : Phase HI DCCs PPCs Number duration(rain) Nb/h duration(s) Nb/h SBS+RSBL 2.0±0.8 7.0±0.6 7.0+2.4 54*7 4.0±2.3 SBS 4.6±1.2 6.2±1.2 1.7±0.8 32~13 1.0-~-0.4 In addition,, in the 5 patiants with RSBL prolonged and nonpropagated bursts of phasic contractions occurring at a frequency of 10-12 contractious/min were recorded at the most distal perfused catheter : their number was 3.6-~1.1 per subject with a mean total duration of 26.7±8.5 min/6h. In one subject, orally propagated DCCs and PPCs were noted. Conelusion : In patients with SBS, interposition of RSBL produces a specific motor pattern charaete*iz~ by prolonged and nonpropagated bursts of phasic contractions located above the reversed segment. This additional alteration in the motility of the short gut could slow down intestinal transit end explain the improved intestinal absorption noted in patients with SBS and interposition of RSBL.
We have previously demonstrated that Calcitonin-Gene-related-Peptide (CGRP) (Peptides, 14:1225, 1993) and Interleukine 115 (IL-115) (Gastroenterology, 106:A481, 1994) mediate part of the inhibition of gastric emptying observed after abdominal surgery (AS) in the rat. However, the mechanism of action through which these peptides exert their inhibitory action remains elusive. AIMS: To examine the possibility of an interaction between CGRP and IL-115 in the final expression of inhibiting gastric emptying after AS in the rat. METHODS: Male Sprague-Dawley rats, weighing 250-300 g were used. They were fasting for 18 hrs with free access to water. Some animals were treated with capsaicin (125 mg/kg, SC) 10-15 days before initiation of experiments. Ketorolac (0.075-0.3 mg) was given IP 10 to 60 min prior to AS. CGRP antagonist, CGRP s-37(30 #g), IL-115 receptor antagonist, (IL-115ra) (200 #g) or both CGRP and IL-115 antagonists were administered IV 5 min prior to AS. AS consisted of laparotomy followed by 1 min cecal exteriorization and manipulation under enflurane anesthesia. In animals not submitted to AS, IL-115 (1.0 izg) or CGRP 8-37were also given IV under enflurane anesthesia. After a 5 min recovery period from anesthesia, a solution of methylcellulose (1.5%)phenol red was given intragastrically in conscious rats and gastric emptying was assessed over a 20 min period of time. RESULTS: Prior administration of ketorolac (0.3 rag, 60 rain before AS) completely prevented the inhibition of gastric emptying induced by AS. IL-1Bra reversed the AS-induced inhibition of gastric emptying by 34.8 %. Simultaneous use of IL-1Bra and CGRP 8-37did not further increase gastric emptying after AS. In capsaicinized animals not submitted to AS, IL-115 was significantly less potent in inhibiting gastric emptying as compared to normal rats, although capsaicin had no effect on basal gastric emptying. Finally, CGRP 8-37 (0-60 /zg) completely reversed the IL-115-induced inhibition of gastric emptying. CONCLUSIONS: Taken together, these data suggest that after AS, IL-1B decreases gastric emptying through the release of CGRP from visceral afferent nerves, via a prostaglandindependent pathway.
Hypersensitivity to gastric distension has been evidenced in patients with lion ulcer dyspepsia. Interactions between gastric distension and other sensitive gastric stimulations have not been studied. The aims of this work were tO determine :the effect intragastricinfusion of HCl at a fl0w rate similar to the maximal gastric acid output on gastric sensitivity to distension in healthy subjects during fasting and after eating. Methods : 9 healthy subjects (age 23-34 yrs) were included in a randomized double-blind cross-over study. Gastric distensions, isobaric and then isovolumic, were performed with an electronic barostat on 2 occasions, separated by 7 days during the intragastric infusion ofeither NaCi or HCI in the fasting state and after the ingestion of a liquid meal (376 ml, 500 cat). Gastric sensitivity was measured by a perception questionaire (0-6), distensions were interrupted when the threshold of discomfort was reached. Results : thresholds of discomfort (mean ± SEM) (Wilcoxon's test, *P<0.05 vs NaCI, § P<0.05 vs fasting). Distensions Fasting Post-prandially NaCI HCI NaCI HCt Isobaric (mmHg) 9.6±(I.8 7.1±0.8' 6.0±1.3§ 5.3±0.9 Isovolumic ~ml) 811±75 789±74 611±90§ 711±79 In the fasting State; HCI infusion decreased the threshold of discomfort during isobaric gastric distension. The meal also decreased thresholds of discomfort, but the sensitizing effeCtof HC1 was abolished after eating. HCI did not modify gastric compliance as Compared tO that observed during NaCI infusion. Conclusion : Gastric acidity is able to potentiate the gastric sensitivity to distension without modifying compliance. HCI could act by decreasing the stimulation threshold of stress-receptors or polymodal receptors. These results could explain why anti-H2 have been reported in some stuides to be effective in improving patients with functionnai dyspepsia.
Motility and Nerve-Gut Interactions
A585
• XENIN RELAXES P R E C O N T R A C T E D RAT I L E A L S M O O T H MUSCLE IN VITRO: STRUCTURE ACTIVITY RELATIONSHIP AND BINDING STUDIES A, Clemens, S Katsoulis, R. Nustede*, K. Seyfarth*, C. MorysWortmann*, G. E. Feurle~ U. R. F61sch and W. E. Schmidt. Laboratory of Molecular Gastroenterology, I. Dept. of Medicine, Christian-Albrechts-University , Kiel; *Dept. of Surgery, Georg-August-University, G6ttingen; *Dept. of Immunochemistry, Max-Planck Institute for Experimental Medicine, Gtttingen; ~ DRK-Hospital, Neuwied; Germany Recently, the 25-residue peptide Xenin (Xe), a xenopsin-like novel member of the neurotensin (NT) peptide family, was isolated from human gastric mucosa. This study was designed to characterize the effect of Xe compared to NT on rat ileal smooth muscle activity. Methods: Xenin peptides were synthesized by Fmoc chemistry and HPLC-purified Full-thickness longitudinal rat ileal muscle strips were mounted under lg tension in a continuously oxygenated organ bath (pH 7.4, 37°C). Changes in motility were measured isometrically. Displacement of 125INT by Xe and analogues was studied in isolated ileal myoeytes. Results: Xe relaxed in a dose-dependent manner KCl-preeontracted (30 mM) rat ileum segments in vitro (ECso, Xe: 25 ± 2,0 nM, NT: 20 ± 0,8 nM, xenepsin: 15 ± 1,9 nM and neuromedin N: 51 ± 5,0 aM). Kinetensin and NT (1-11) (31aM each) were ineffective. Xe (12-25), Xe (14-25), Xe (19-25), Xe (20-25) and NT (9-131 were equipotent compared to Xe and NT. The order of potency for other fragments was: Xe (21-25) > NT (9-13) > Xe (1-24) >> Xe (1-23). The efficaeies of all peptides were identical. TTX (1 ~M), hexamethonium (1 I~M), atropine (l~tM), tetmethylammonium (1 mM) and 4-aminopyridine (1 mM) did not modulate the effects of Xe (l~tM) and NT (II#M). The bee venom potassium channel blocker apamia (300 nM) abolished NT- or Xe-induced relaxations° respectively. Desensitisation against NT prevented the effect of Xe and vice versa The specific NT receptor antagonist SR 48692 (3 ~M) fully inhibited Xe- or NT-mediated effects. Xe (KD 0,6 aM, B ~ 10,8 pM), Xe (13-25) (KD 1 nM, B~= 7,9 pM) and SR 48692 potently displaced t251-NT binding to ileal myoeyteso Xe (1-23) was inactive. Conclusions: Xe and NT exert direct myogenic relaxant effects on KCIprecontraeted rat ileum strips in vitro. Stmcture-aetivity and binding data suggest that Xe and NT activate the NT receptor which is coupled to an apamm-sensitive KT-chanael. High-affinity binding of Xe requires the C-terminal penmpeptide of NT or Xc. Supported by DFG grants
• INTRAGASTRIC INFUSION OF CHLORHYDRIC ACID (HCI) POTENTIATES GASTRIC SENSITIVITY TO GASTRIC DISTENSION 1N MAN. B. Coffnk R. ChoUet, B. Flourit, M. L~maun, C. Franchisseur, JC. Rambaud, R. Jian. INSERM U290, Htpitaux Saint-Lazare and Saint-Louis, 75010 Paris, France.
• SMALL INTESTINAL MANOMETRY IN PATIENTS WITH THE SHORT BOWEL SYNDROME (SBS) AND INTERPOSITION OF A REVERSED SMALL BOWEL LOOP (RSBL). B. Coffin, B. Flourit, P. Valleur, M. Ltmarm, P. Hautefeuille, JC Rambaud, B. Messing. Services de Gastroenttrologie and Chirurgie, Htpitanx Saint-Lazare and Lariboisitre, 750[0 Paris, France.
• ABDOMINAL SURGERY-INDUCED INHIBITION OF GASTRIC EMPTYING IN THE RAT: R E S P E C T I V E R O L E OF C G R P AND IL-115. C. Coimbra J. McDuff, M. Boivin, V. Plourde. Laboratory of Gastrointestinal Neurobiology and Motility, Andre-Viallet Research Center, University of Montreal, Montreal, QU., Canada.
In patients with SBS we have prevfously shown that the interposition of RSBL increases the intestinal absorption of nutrients (Gastroenterology 1994, 106 A601). In this work. we studied the intestinal motility in 5 patients (21-70 yrs) with SBS (length 50 "12 era) (M*SEM) who had undergone interposition of RSBL (length 10-15 can) belwe~mthejejnaum and colon and we Compared them with 5 matched control subjects (29-72yrs) with SBS (length 56*25 cm) but without interposition of RSBL. Methods : Duodeno-jejunal motility was recorded in the fasting state for 6 h using 4 perfused catheters loeated 10, 20, 30 and 45 cm from the pylorus. Recordings were visually analyzed for phases HI of the migrating motor complex, discrete clustered contractions (DCCs) and prolonged propagated contractions (PPCs). Sp~ifie motility patterns such as prolonged bursts of nonpropagated contractions or retropropagated (emily) contractions were also analyzed. Results : Phase HI DCCs PPCs Number duration(rain) Nb/h duration(s) Nb/h SBS+RSBL 2.0±0.8 7.0±0.6 7.0+2.4 54*7 4.0±2.3 SBS 4.6±1.2 6.2±1.2 1.7±0.8 32~13 1.0-~-0.4 In addition,, in the 5 patiants with RSBL prolonged and nonpropagated bursts of phasic contractions occurring at a frequency of 10-12 contractious/min were recorded at the most distal perfused catheter : their number was 3.6-~1.1 per subject with a mean total duration of 26.7±8.5 min/6h. In one subject, orally propagated DCCs and PPCs were noted. Conelusion : In patients with SBS, interposition of RSBL produces a specific motor pattern charaete*iz~ by prolonged and nonpropagated bursts of phasic contractions located above the reversed segment. This additional alteration in the motility of the short gut could slow down intestinal transit end explain the improved intestinal absorption noted in patients with SBS and interposition of RSBL.
We have previously demonstrated that Calcitonin-Gene-related-Peptide (CGRP) (Peptides, 14:1225, 1993) and Interleukine 115 (IL-115) (Gastroenterology, 106:A481, 1994) mediate part of the inhibition of gastric emptying observed after abdominal surgery (AS) in the rat. However, the mechanism of action through which these peptides exert their inhibitory action remains elusive. AIMS: To examine the possibility of an interaction between CGRP and IL-115 in the final expression of inhibiting gastric emptying after AS in the rat. METHODS: Male Sprague-Dawley rats, weighing 250-300 g were used. They were fasting for 18 hrs with free access to water. Some animals were treated with capsaicin (125 mg/kg, SC) 10-15 days before initiation of experiments. Ketorolac (0.075-0.3 mg) was given IP 10 to 60 min prior to AS. CGRP antagonist, CGRP s-37(30 #g), IL-115 receptor antagonist, (IL-115ra) (200 #g) or both CGRP and IL-115 antagonists were administered IV 5 min prior to AS. AS consisted of laparotomy followed by 1 min cecal exteriorization and manipulation under enflurane anesthesia. In animals not submitted to AS, IL-115 (1.0 izg) or CGRP 8-37were also given IV under enflurane anesthesia. After a 5 min recovery period from anesthesia, a solution of methylcellulose (1.5%)phenol red was given intragastrically in conscious rats and gastric emptying was assessed over a 20 min period of time. RESULTS: Prior administration of ketorolac (0.3 rag, 60 rain before AS) completely prevented the inhibition of gastric emptying induced by AS. IL-1Bra reversed the AS-induced inhibition of gastric emptying by 34.8 %. Simultaneous use of IL-1Bra and CGRP 8-37did not further increase gastric emptying after AS. In capsaicinized animals not submitted to AS, IL-115 was significantly less potent in inhibiting gastric emptying as compared to normal rats, although capsaicin had no effect on basal gastric emptying. Finally, CGRP 8-37 (0-60 /zg) completely reversed the IL-115-induced inhibition of gastric emptying. CONCLUSIONS: Taken together, these data suggest that after AS, IL-1B decreases gastric emptying through the release of CGRP from visceral afferent nerves, via a prostaglandindependent pathway.
Hypersensitivity to gastric distension has been evidenced in patients with lion ulcer dyspepsia. Interactions between gastric distension and other sensitive gastric stimulations have not been studied. The aims of this work were tO determine :the effect intragastricinfusion of HCl at a fl0w rate similar to the maximal gastric acid output on gastric sensitivity to distension in healthy subjects during fasting and after eating. Methods : 9 healthy subjects (age 23-34 yrs) were included in a randomized double-blind cross-over study. Gastric distensions, isobaric and then isovolumic, were performed with an electronic barostat on 2 occasions, separated by 7 days during the intragastric infusion ofeither NaCi or HCI in the fasting state and after the ingestion of a liquid meal (376 ml, 500 cat). Gastric sensitivity was measured by a perception questionaire (0-6), distensions were interrupted when the threshold of discomfort was reached. Results : thresholds of discomfort (mean ± SEM) (Wilcoxon's test, *P<0.05 vs NaCI, § P<0.05 vs fasting). Distensions Fasting Post-prandially NaCI HCI NaCI HCt Isobaric (mmHg) 9.6±(I.8 7.1±0.8' 6.0±1.3§ 5.3±0.9 Isovolumic ~ml) 811±75 789±74 611±90§ 711±79 In the fasting State; HCI infusion decreased the threshold of discomfort during isobaric gastric distension. The meal also decreased thresholds of discomfort, but the sensitizing effeCtof HC1 was abolished after eating. HCI did not modify gastric compliance as Compared tO that observed during NaCI infusion. Conclusion : Gastric acidity is able to potentiate the gastric sensitivity to distension without modifying compliance. HCI could act by decreasing the stimulation threshold of stress-receptors or polymodal receptors. These results could explain why anti-H2 have been reported in some stuides to be effective in improving patients with functionnai dyspepsia.