- Email: [email protected]
Protein kinase CE translocation in enteric neurons and interstitial cells of cajal in response to muscarinic stimulation
AGAA635
April 2000
3240
3242
IN VIVO RECORDING OF GASTROINTESTINAL MOTILITY IN WSIWS MUTANT RATS.
GLP-l AND GLP-2 ACT IN SYNERGY TO INHIBIT FASTING SMALL BOWEL MOTILITY IN THE RAT.
Takeshi Tomomasa, Atsushi Takahashi, Masahiko Tabata, Hiroaki Kaneko, Toshio Watanabe, Toru Kobayashi, Yasuaki Tsuchida, Akihiro Morikawa, Gunma Univ Sch of Medicine, Maebashi, Japan; Gunma Prefectural Children's Hosp, Hokkitsu, Japan. Background: WslWs rat (Ws) has a mutation at the c-kit gene which has a critical role in the development of interstitial cells of Cajal (ICC). In the gastrointestinal tract of Ws ICCs are either absent or markedly reduced. In vitro studies using intestinal muscle strips showed that regularity of contractions is severely impaired in Ws. Interestingly, however, Ws rats can live and be fertile, while the WlW v mouse with a mutation at the c-kit gene can not survive. In the present study we recorded gastrointestinal mechanical contractions in vivo in Ws and control (wild type: (+1+» rats. Methods: Ws (8weeks old, approximately 250g, n=9) and control rats (Cont, n=8) were operated under nembuthal anesthesia. Miniature strain gauge force transducers developed specifically for in vivo rat motility studieswere sutured onto the serosal side of the gastric antrum and jejunum (5cmto 8cm distal to pylorus). In order to confirm the migration of intestinal phase 3s, two transducers were attached in proximal jejunum 5 em apart in Ws (n=2) and Cont (n=3). After 4 to 7 days recovery period and 24-36 h fasting, lead wires of transducers were connected to transducers and contractions were recorded in computer through an amplifier and an AID converter. Rats were free of any body restrictions. Results: (I) In Ws sporadic or short c1usters(< lOs) of contractions were the dominant gastric contractile pattern, whereas longer (> 1 min) clusters of rhythmic contractions were the dominant pattern in Cont. Migration of those gastric clusters to the duodenum were not clearly observed. (2) In the duodenum and jejunum, MMC patterns were present in 4 out of II Ws and in 6 out of II Cont. The mean duration, interval, and frequency of contractions of phase 3 were 2.3+/-0.6 min, 28.7+1-8.7 min, 29.5+1-2.0 min'! and 4.5+1-0.9 min, 12.3+1-1.6 min, 34.3+1-2.2 min'! in Ws and Cont, respectively. (3) In phase 2 of small intestine, clusters of contractions with various length were the dominant contraction pattern in borh Ws and Cont. (4) Feeding immediately changes motility patterns both in Ws and in Cont. Conclusion: Unlike normal rats, clusters of rhythmic contractions are rare in the stomach of Ws. Although phase 3-like clustered contractions were present in the small intestine in Ws, they are quantitatively different from those in Cont.
Ayhan Bozkurt, Erik Naslund, Per M. Hellstrom, Dept of Physiology, Marmara Univ, Istanbul, Turkey; Div of Surg, Karolinska Inst Danderyd Hosp, Danderyd, Sweden; Dept of Gastroenterology, Karolinska Inst KaroIinska Hosp, Stockholm, Sweden. Background: Glucagon-like peptide-1 (GLP-1) and -2 (GLP-2) are secreted into the circulation in parallel after a meal. GLP-I has been shown to inhibit gastrointestinal motility in both rodents and humans. The aim of the present study was to examine the effect of GLP-2 on fasting motility and if synergy exists between GLP-1 and GLP-2 in inhibiting gut motility. Methods: Electrodes were implanted into the serosa of the small intestine 5 (D), 15 (11), 25 (12) cm distal to the pylorus in male Sprague-Dawley rats. Intravenous infusion of either saline, GLP-1 (1,2, 5, 10, 20 pmol/kgl min), GLP-2 (I, 2, 5, 10,20, 100 pmol/kglmin) or GLP-1 and GLP-2 (I, 2, 5, 10, 20 pmol/kglmin for either peptide) were continued for 60 min. Results: GLP-I infusion resulted in a dose-dependent increase in the migrating motor complex cycle (MMC) length beginning at 5 pmol/kgl min. GLP-2 resulted in an increased cycle length first at 100 pmollkg/min. GLP-1 and GLP-2 resulted in an increased cycle length a I pmol/kglmin (table I). Conclusion: GLP-I and GLP-2 administered simultaneously act in concert to inhibit fasting small bowel motility at concentrations lower than GLP-I alone, increasing MMC cycle length. GLP-2 alone has little effect on fasting small bowel motility. Further studies on the effect of proglucagon products on gut motility have to take into account both GLP-I and GLP-2.
3241 PROTEIN KINASE CE TRANSLOCATION IN ENTERIC NEURONS AND INTERSTITIAL CELLS OF CAJAL IN RESPONSE TO MUSCARINIC STIMULATION. Xuan-Yu Wang, William T. Gerthoffer, Kenton M. Sanders, Sean M. Ward, Univ of Nevada, Reno, NV. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (IC-DMP) express receptors for excitatory neurotransmitters and are closely associated with excitatory motor neurons. We tested whether ICDMP are functionally innervated by cholinergic neurons in the murine small intestine. Muscle strips of the murine small intestine were stimulated by intrinsic nerves and acetylcholine (ACh) and processed for immunohistochemistry to determine the effects of stimulation on protein kinase CE (PKCE) activation. Kit and vimentin antibodies were used to determine whether cells with PKCE-like immunoreactivity (PKCE-LI) were ICC. PKCE-LI was observed in myenteric neurons in the murine intestine, but under control conditions PKCE-LI was not detectable in other cell types within the tunica muscularis. Electrical field stimulation (EFS) or application of ACh caused translocation of neural PKCE-LI from the cytosol to a peripheral compartment. After stimulation, PKCE-LI was found in spindleshaped cells in the deep muscular plexus. Kit-LI and vimentin-Ll identified these cells as IC-DMP. PKCE-LI in IC-DMP and translocation of PKCE-LI in neurons was blocked by tetrodotoxin or atropine suggesting these responses were due to neurally released ACh and activation of muscarinic receptors. The responses to nerve stimulation were mimicked by 4-00phorbol ester, but not by substance P or sodium nitroprusside. Western blots confirmed translocation of PKCE-LI from soluble to particulate fractions after EFS or ACh. In conclusion, PKCE is linked to muscarinic receptor activation in IC-DMP and a sub-population of myenteric neurons. PKCE-LI can be used as an indication of muscarinic innervation. These studies demonstrate that IC-DMP in the small intestine are functionally innervated by excitatory motor neurons.
Fig 1:Results ofGLP·1 and GLP-2 on fasting motility inthe rat. Agent Saline GlP·1 Glp·2 GlP·1+GLP·2
0
J1
J2
175(12.7-20.9) (12) 26.6 (14.6-52.0) (8) 730(14.0-116.3) (7) 276(18.1-34.1) (7)
17.9 (13.1-24.1) (12) 259(18.9-49.9) (8) 74.5 (14.0-120.0) (7) 25.6 (17.4-42.2) (7)
17.7 (12.9-24.8) 12) 301 (15.9-47.9)(8) 78.1 (69.0-127.4) (7) 20.8 (16.8-39.4) (7)
Values shown as median (range) (n), GLP-1 dose 5pmollkg/min, GLP-2100 pmol/kg/min, GLP-1+GLP-21+1 pmollkg/min, allp<0.01 vssaline exept J2GLP-1+GLP-2 vs saline p=0.06, Wilcoxon signed rank test.
3243 GLUCAGON-LIKE PEPTIDE·1 (GLP.l) STRONGLY AFFECTS DUODENO·CECAL TRANSIT TIME, BUT ONLY WEAKLY SMALL INTESTINAL MOTILITY INDEX IN HUMANS. Andreas Franke, Jutta Keller, Gabi Groeger, Ulrich Rosien, Jens 1. Holst, Peter Layer, Israelitisches Krankenhaus, Hamburg, Germany; Univ of, Essen, Germany; Israelitisches Krankenhaus, Hanburg, Germany; Univ of, Copenhagen, Denmark. Introduction: Glucagon-like peptide-1 (GLP-I) is a distal intestinal regulatory peptide which is released in response to a meal or intraileal nutrients. It is not only involved in the regulation of glucose homeostasis, but also decreases endogenously stimulated digestive secretory functions and gastric emptying. The effects of GLP-I on small intestinal motility are unknown. Methods and aims: To determine the effects of GLP-I on human small intestinal motility, 8 healthy subjects were intubated with an orojejunal multilumen-tube for continuous duodenal perfusion of essential amino acids (EAA) and intestinal manometry. In parallel subjects received i.v, infusions of graded doses of GLP-l (0, 40, 80 pmollkgh, each dose for 60 min). In the second part of the study EAA-Perfusion was stopped and 10 g lactitol were given as a bolus intraduodenally during constant i.v. infusion of 40 pmol/kgh GLP- I. Afterwards, subjects were extubated, Hz-exhalation was analysed every 10 min. and duodeno-cecal transit time was calculated (time interval until increase in Hz-exhalation of 20 ppm over basal). 4-7 days later, the second part of the experiment was repeated during i.v. infusion of NaCL Results: Infusion of physiological doses of GLP-I increased duodeno-cecal transit time significantly. There was no signifikant decrease in motility index caused by physiological but only by supraphysiological plasma concentrations of GLP- I. table Conclusions: Physiological postprandial GLP-I plasma concentrations inhibit small intestinal transit. However, this is not reflected by a parallel decrease in overall small intestinal motor activity. Glp·1dose (pmol/kgh)
0 40 80
Plasma GlP·1 (pmoln)
Motility index
Plasma GLp·1 (Pmoln)
Duodeno·cecal transit time(min.)
22.6±3.3 55.1±44' 79.9+6.5"
4.2±0.43 3.6±0.26 3.3±0.39'
138±1.8 42.8±5.9'
97.2±11.7 142.9±17.6'
(integrated mean values±SE, 'p<0.05 vs. 0 pmol/kgh, "p<0.05 vs. 40 pmol/kgh)
April 2000
3240
3242
IN VIVO RECORDING OF GASTROINTESTINAL MOTILITY IN WSIWS MUTANT RATS.
GLP-l AND GLP-2 ACT IN SYNERGY TO INHIBIT FASTING SMALL BOWEL MOTILITY IN THE RAT.
Takeshi Tomomasa, Atsushi Takahashi, Masahiko Tabata, Hiroaki Kaneko, Toshio Watanabe, Toru Kobayashi, Yasuaki Tsuchida, Akihiro Morikawa, Gunma Univ Sch of Medicine, Maebashi, Japan; Gunma Prefectural Children's Hosp, Hokkitsu, Japan. Background: WslWs rat (Ws) has a mutation at the c-kit gene which has a critical role in the development of interstitial cells of Cajal (ICC). In the gastrointestinal tract of Ws ICCs are either absent or markedly reduced. In vitro studies using intestinal muscle strips showed that regularity of contractions is severely impaired in Ws. Interestingly, however, Ws rats can live and be fertile, while the WlW v mouse with a mutation at the c-kit gene can not survive. In the present study we recorded gastrointestinal mechanical contractions in vivo in Ws and control (wild type: (+1+» rats. Methods: Ws (8weeks old, approximately 250g, n=9) and control rats (Cont, n=8) were operated under nembuthal anesthesia. Miniature strain gauge force transducers developed specifically for in vivo rat motility studieswere sutured onto the serosal side of the gastric antrum and jejunum (5cmto 8cm distal to pylorus). In order to confirm the migration of intestinal phase 3s, two transducers were attached in proximal jejunum 5 em apart in Ws (n=2) and Cont (n=3). After 4 to 7 days recovery period and 24-36 h fasting, lead wires of transducers were connected to transducers and contractions were recorded in computer through an amplifier and an AID converter. Rats were free of any body restrictions. Results: (I) In Ws sporadic or short c1usters(< lOs) of contractions were the dominant gastric contractile pattern, whereas longer (> 1 min) clusters of rhythmic contractions were the dominant pattern in Cont. Migration of those gastric clusters to the duodenum were not clearly observed. (2) In the duodenum and jejunum, MMC patterns were present in 4 out of II Ws and in 6 out of II Cont. The mean duration, interval, and frequency of contractions of phase 3 were 2.3+/-0.6 min, 28.7+1-8.7 min, 29.5+1-2.0 min'! and 4.5+1-0.9 min, 12.3+1-1.6 min, 34.3+1-2.2 min'! in Ws and Cont, respectively. (3) In phase 2 of small intestine, clusters of contractions with various length were the dominant contraction pattern in borh Ws and Cont. (4) Feeding immediately changes motility patterns both in Ws and in Cont. Conclusion: Unlike normal rats, clusters of rhythmic contractions are rare in the stomach of Ws. Although phase 3-like clustered contractions were present in the small intestine in Ws, they are quantitatively different from those in Cont.
Ayhan Bozkurt, Erik Naslund, Per M. Hellstrom, Dept of Physiology, Marmara Univ, Istanbul, Turkey; Div of Surg, Karolinska Inst Danderyd Hosp, Danderyd, Sweden; Dept of Gastroenterology, Karolinska Inst KaroIinska Hosp, Stockholm, Sweden. Background: Glucagon-like peptide-1 (GLP-1) and -2 (GLP-2) are secreted into the circulation in parallel after a meal. GLP-I has been shown to inhibit gastrointestinal motility in both rodents and humans. The aim of the present study was to examine the effect of GLP-2 on fasting motility and if synergy exists between GLP-1 and GLP-2 in inhibiting gut motility. Methods: Electrodes were implanted into the serosa of the small intestine 5 (D), 15 (11), 25 (12) cm distal to the pylorus in male Sprague-Dawley rats. Intravenous infusion of either saline, GLP-1 (1,2, 5, 10, 20 pmol/kgl min), GLP-2 (I, 2, 5, 10,20, 100 pmol/kglmin) or GLP-1 and GLP-2 (I, 2, 5, 10, 20 pmol/kglmin for either peptide) were continued for 60 min. Results: GLP-I infusion resulted in a dose-dependent increase in the migrating motor complex cycle (MMC) length beginning at 5 pmol/kgl min. GLP-2 resulted in an increased cycle length first at 100 pmollkg/min. GLP-1 and GLP-2 resulted in an increased cycle length a I pmol/kglmin (table I). Conclusion: GLP-I and GLP-2 administered simultaneously act in concert to inhibit fasting small bowel motility at concentrations lower than GLP-I alone, increasing MMC cycle length. GLP-2 alone has little effect on fasting small bowel motility. Further studies on the effect of proglucagon products on gut motility have to take into account both GLP-I and GLP-2.
3241 PROTEIN KINASE CE TRANSLOCATION IN ENTERIC NEURONS AND INTERSTITIAL CELLS OF CAJAL IN RESPONSE TO MUSCARINIC STIMULATION. Xuan-Yu Wang, William T. Gerthoffer, Kenton M. Sanders, Sean M. Ward, Univ of Nevada, Reno, NV. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (IC-DMP) express receptors for excitatory neurotransmitters and are closely associated with excitatory motor neurons. We tested whether ICDMP are functionally innervated by cholinergic neurons in the murine small intestine. Muscle strips of the murine small intestine were stimulated by intrinsic nerves and acetylcholine (ACh) and processed for immunohistochemistry to determine the effects of stimulation on protein kinase CE (PKCE) activation. Kit and vimentin antibodies were used to determine whether cells with PKCE-like immunoreactivity (PKCE-LI) were ICC. PKCE-LI was observed in myenteric neurons in the murine intestine, but under control conditions PKCE-LI was not detectable in other cell types within the tunica muscularis. Electrical field stimulation (EFS) or application of ACh caused translocation of neural PKCE-LI from the cytosol to a peripheral compartment. After stimulation, PKCE-LI was found in spindleshaped cells in the deep muscular plexus. Kit-LI and vimentin-Ll identified these cells as IC-DMP. PKCE-LI in IC-DMP and translocation of PKCE-LI in neurons was blocked by tetrodotoxin or atropine suggesting these responses were due to neurally released ACh and activation of muscarinic receptors. The responses to nerve stimulation were mimicked by 4-00phorbol ester, but not by substance P or sodium nitroprusside. Western blots confirmed translocation of PKCE-LI from soluble to particulate fractions after EFS or ACh. In conclusion, PKCE is linked to muscarinic receptor activation in IC-DMP and a sub-population of myenteric neurons. PKCE-LI can be used as an indication of muscarinic innervation. These studies demonstrate that IC-DMP in the small intestine are functionally innervated by excitatory motor neurons.
Fig 1:Results ofGLP·1 and GLP-2 on fasting motility inthe rat. Agent Saline GlP·1 Glp·2 GlP·1+GLP·2
0
J1
J2
175(12.7-20.9) (12) 26.6 (14.6-52.0) (8) 730(14.0-116.3) (7) 276(18.1-34.1) (7)
17.9 (13.1-24.1) (12) 259(18.9-49.9) (8) 74.5 (14.0-120.0) (7) 25.6 (17.4-42.2) (7)
17.7 (12.9-24.8) 12) 301 (15.9-47.9)(8) 78.1 (69.0-127.4) (7) 20.8 (16.8-39.4) (7)
Values shown as median (range) (n), GLP-1 dose 5pmollkg/min, GLP-2100 pmol/kg/min, GLP-1+GLP-21+1 pmollkg/min, allp<0.01 vssaline exept J2GLP-1+GLP-2 vs saline p=0.06, Wilcoxon signed rank test.
3243 GLUCAGON-LIKE PEPTIDE·1 (GLP.l) STRONGLY AFFECTS DUODENO·CECAL TRANSIT TIME, BUT ONLY WEAKLY SMALL INTESTINAL MOTILITY INDEX IN HUMANS. Andreas Franke, Jutta Keller, Gabi Groeger, Ulrich Rosien, Jens 1. Holst, Peter Layer, Israelitisches Krankenhaus, Hamburg, Germany; Univ of, Essen, Germany; Israelitisches Krankenhaus, Hanburg, Germany; Univ of, Copenhagen, Denmark. Introduction: Glucagon-like peptide-1 (GLP-I) is a distal intestinal regulatory peptide which is released in response to a meal or intraileal nutrients. It is not only involved in the regulation of glucose homeostasis, but also decreases endogenously stimulated digestive secretory functions and gastric emptying. The effects of GLP-I on small intestinal motility are unknown. Methods and aims: To determine the effects of GLP-I on human small intestinal motility, 8 healthy subjects were intubated with an orojejunal multilumen-tube for continuous duodenal perfusion of essential amino acids (EAA) and intestinal manometry. In parallel subjects received i.v, infusions of graded doses of GLP-l (0, 40, 80 pmollkgh, each dose for 60 min). In the second part of the study EAA-Perfusion was stopped and 10 g lactitol were given as a bolus intraduodenally during constant i.v. infusion of 40 pmol/kgh GLP- I. Afterwards, subjects were extubated, Hz-exhalation was analysed every 10 min. and duodeno-cecal transit time was calculated (time interval until increase in Hz-exhalation of 20 ppm over basal). 4-7 days later, the second part of the experiment was repeated during i.v. infusion of NaCL Results: Infusion of physiological doses of GLP-I increased duodeno-cecal transit time significantly. There was no signifikant decrease in motility index caused by physiological but only by supraphysiological plasma concentrations of GLP- I. table Conclusions: Physiological postprandial GLP-I plasma concentrations inhibit small intestinal transit. However, this is not reflected by a parallel decrease in overall small intestinal motor activity. Glp·1dose (pmol/kgh)
0 40 80
Plasma GlP·1 (pmoln)
Motility index
Plasma GLp·1 (Pmoln)
Duodeno·cecal transit time(min.)
22.6±3.3 55.1±44' 79.9+6.5"
4.2±0.43 3.6±0.26 3.3±0.39'
138±1.8 42.8±5.9'
97.2±11.7 142.9±17.6'
(integrated mean values±SE, 'p<0.05 vs. 0 pmol/kgh, "p<0.05 vs. 40 pmol/kgh)