- Email: [email protected]
Heat stable toxin of E. coli (STa) stimulates duodenal mucosal bicarbonate secretion in cystic fibrosis knockout mice
the rim of patients with peptic ulcer, whereas all samples taken from healthy tissues were found negative.All samplesanalysedwith in situ hybridization method were negativefor HSV1. Antibodies against the virus were detected by indirect immunofluorescence assay in 77 out of 90 patients with peptic ulcer (86%) and in 44 out of 50 patients of the control group (88%). Statistically significant difference was found between peptic ulcers cases positive for both HSV-1 and positive for HP (67.9%) and those negative for HSV-1 and positive for HP (91.9%) (p value= 0.009). This difference becomeslarger in casesof prepyloric ulcers positive for HSV-1, where the percentageof positive results for HP is limited to 36.4% of the cases. CONCLUSION:The above results suggest that HSV-1 may play a role in the pathogenesis from a subset of peptic ulcer disease.
In contrast, in CF patients, bicarbonate secretory responses in duodenal biopsies mounted in Ussing chambers responded normally to STa (presumablyvia guanylatecyclase C/cGMP). Our aimwas to determine if luminal perfusion of STa stimulates DMBS through a cGMPmediated pathway in CFTR(-/-) mice. Methods:Utilizingthe CF murine model, DMBS was compared in CFTR(-/-) animals and wildtype [CFTR(+/+) & ( + / - ) ] animals; confirmed by PCR. Anesthesia was induced with hypnorm/midazolam (i.p.). The proximal duodenum (5-10mm) was cannulated, perfused with 154 mM NaCI (0.2 ml/min) and [HCO~-]measured by micro-back titration. After measuring basal DMBSfor 45 rain, the duodenal segment was stimulated with either STa (0.1 uM), forskolin (0.1 mM) or 8Br-cGMP (1 mM). Established doses of each agonist were pertused for 30 min. Agonists were tested in separateanimals, and at least 4 animals were studied in each series. Results: Basal HCO3secretionwas about 60% less in CFTR(- / - ) vs. wildtype mice, 4.6 -+ 0.8 vs. 11.5 +_1.3 pmol/cm-h, respectively (P
728 Characterization of Nicotinic Acetylcholine Receptors (oAChRs) In Myenteric Neurons from Guinea Pig Intestine Jim H. Run, Xiaoping Zhou, James J. Galligan, Michigan State Univ, East Lansing, MI Background. Enteric nAChRs play a key role in gastrointestinal reflexes.The a and/3 subunit composition of nAChRsdeterminestheir pharmacologicaland functional properties.Although nAChRs play a critical role in the enteric nervous system, the subunit composition of enteric nAChRs is unknown. Aim. The aim of this study was to characterize nAChRs in guinea pig myentedc neurons using electrophysiologicaland immunohistochemicalmethods.The overall goal was to establish the subunit composition of myenteric nAChRsand to determine if there were developmentalchanges in the properties of myenteric nAChRs. Methods. Whole cell patch clamp recordings were obtainedfrom newborn guinea pig myenteric neurons in primary culture. Conventionalelectrophysiologicalmethods were used to record from neurons in the intact myenteric plexus from newborn and adult guinea pigs. Immunohistochemical methods were used to localize a and/~ subunits cultured neurons in culture and in the intact plexus from newborn and adult guinea pigs. Results. Acetylcholine (ACh) caused an inward current in >90% of neurons in culture. The nAChRagonist rank order potency was: DMPP>ACh>nicotine>cytisine. Dihydro-/3-erythroidine(DH,BE)blocks nAChRscontaining ~ receptorswith an ICsO20/.,,M; the DH/3EICsoversusACh in cultured neuronswas 50 _+4 ~M. e-Bungarotoxin and e-MLA, antagoniststhat block a7 subunit containing nAChRsdid not affect ACh currents. Intracellular recording from neurons in the intact myentedc plexus from adult and newborn guinea pigs showed that ACh, nicotine and cytisine caused equivalent depolarizations in S neurons. ACh, and nicotine but not cytisine depolarizedAH neurons. Depolarizationsevoked by cytisine and nicotine were blocked by mecamylamine.The DH,BEIC~ versus ACh was 13 +_ 4 p,M in adult neurons, tmmunohistochemical studies using an antibody (mAb35) that recognizes a3, ~5 and/34 subunits stained many in neurons in culture, and in the intact plexusfrom newborn and adult guinea pigs. An antibody against p2 subunits and an antibody against ~7 subunits did not revealany neurons in culture or in the intact plexusfrom newborn and adult guinea pigs. Conclusions. Pharmacologicaland immunohistochemicaldata indicate that the subunlt composition of myenteric nAChRs is either a3p4 or ~3a5~. Myenteric neurons do not express many nAChRs containing ~7 or if2 subunits. The properties of myenteric nAChRs in newborn intestine are similar to those in the adult intestine.
731 Bile Salts Are Physiological Stimulants of Trefoil Factor 3 (TFF3) Secretion in the Rat. Rorence Levenez,INRA UEPSD,Jouy en Josas France; StephaneDuroal, Cadne Blancbard, Jean-Claude Cuber, INSERM U45, Lyon France Background: Trefoil peptide 3 (TFF3) is specifically contained in goblet cells of the small intestine and colon.Its plays an important role in the protection and repair of the epithelial barrier.Thefactors that control TFF3secretion are poorly understood.They were investigated in the present study with a model of isolated vascularly perfused rat jejunum.Methods: A loop of the proximal jejunum (10-cm length) was isolated and perfused via the superior mesentericartery with a Krebs-Henseleitbuffer containing 20% washed bovine erythrocytes, 5mM glucose,lOmM amino-acids and 3% BSA at a flow rate of 2.5ml/min. Portal effluent was continuously collected as 2-rain fractions. Isotonic saline was infused into the lumen at a rate of 0.5ml/min. The luminal effluent was continuously collectedthrough the distal catheter as lO-min fractions. The experiments consisted of a 30-rain basal period and a 3g-rain stimulation period with the various luminal factors to be tested. TFF3 immunoreactivity was measured both in the portal and luminal effluents with a specific radioimmunoassay.Results: Luminal infusion of lOmM HCI, 5%glucose or 5%peptone evokeda 2-fold increase in luminal TFF3releasecomparedto isotonic saline alone. In contrast,a40mM oleic acid/2OmMtaurocholic acid (TC) mixture or TC (20mM) alone produced a 4-fold increase in TFF3 secretion.The effect of TC was concentration-dependentover the 5-30ram range.Thefirst significant response was observed upon stimulation with 5mM TC.Amongstthe tested bile acids, TC was was the most potent stimulant followed in decreasing order by cholic acid, deoxycholic acid, glycocholic acid and taurodsoxycholic acid. Tween 20 did not elicit TFF3 secretion, thus eliminating a detergent effect of TC.Intra-arterial infusion of bethanecol,hietamine,serotonin, bombasin or PGE2 also elicited a marked release of TFF3. However, i.a. infusion of TTX, atropine, a bombasin receptor antagonist,mepyramine, indomethacin, methysergide, ketsnserin or SDZ 205,557 in combination with luminal TC did not modify the TFF secretion induced by TC alone. Somatostatin (lOOnM) reduced by 80% the TC-inducedTFF3secretion. Conclusion:Bile salts are physiological stimulants of TFF secretion in the rat jejunum. This effect is a dynamic process which excludes the detergent properties of bile salts and which does not seem to involve the participation of intramural nerves. Somatostatin may restrain TFF3 secretion through a Iocal/paracdneor hormonal pathway.
729 Galanin and Orexin A Potentiate Intracellular Calcium Responsesto Cholacystokinin and Carbochst in Human and Rat Duodenal Enterocytes. Gunnar Flemstroem, Ramin Shadatmedad,Gunilla Jedstedt, Markus Sjoeblom, Saefsten Bengt, Akerman E. Karl, Uppsala Univ, UppsalaSweden The bicarbonate secretion by the duodenal mucosa protects this epithelium against acid dischargedfrom the stomach and the neurohumoral control of the secretion is a key element in mucosal protection. Duodenal epithelial intracellular and cell-to-cell signaling are final pathways in the secretory control but have not beenclarified. We havethus studied the effects of some secretagogueson duodenal enterocyte (duodenocyte) intracellular calcium ([Ca2+D using interconnectedduodenocytes(isolatedaggregates).Methods. Human duodenalbiopsies and scraped rat duodenal mucosa were cut into 0.5-1.0 mm in diameter pieces followed by mild digestion (coUagenase/dispase)to yield aggregates (10-200 cells) of duodenocytes. Aggregateswere loadedwith fura-2, mounted in a perfusionchamberand [Ca2+]~was measured with fluororescenceimaging. Thosechosenfor study were composed mainly of duodenocytas with enlarged vacuoles characteristic of crypt cells. Results. The use of aggregatesprovide more stable preparationsthan isolatedenterocytes.Superfusionwith cholecystokininoctapeptide (CCK-8; 1-50 nM), caerulin (100 nM), ADP (100/~U) or carbachol (1-100 p,M) induced a transient rise in [Ca2+]~.Responsesto CCK-8, but not those to carbachol, were prevented by CCK~-receptor antagonists. Galanin (1-100 nM) and oraxin A (10-100 nM) causeda small and transient rise in [Ca2*]~only in some duodenocytes but induced a slowly developing, Iong-lastiog and large rise in (Ca2+]~in most aggregatecells. Furthermore, galanin and orexin markedly potentiatedthe rise in [Ca2+],inresponseto CCK-8and carbachol. Eftecta in rat and human aggregateswere very similar. One or two cells in an aggregate were often the first to respond to CCK-8 (or carbachol) with a rise in [Ca2+],and this was followed by spread of the [Ca2+],signal within the aggregate.Conclusions. Galanin and orexin A-induced increases in enterocyte [Ca2÷],may modulate the intestinal responseto secretagogues.Interconnecting enterocytes respond to stimuli increasing [Ca2+]~asa syncytium. These phenomena may be important in the overall function of the intestinal mucosa and in mucosal protection.
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Peripheral CRF Stimulates Colonic Propulsion and Inhibits Gastric Emptying in Mice: Differential Role of CRF Receptor Subtypes 1 (CRF-R1) and 2 (CRF-R2) Vicente Martinez, Cardenal Herrera Univ, Veterinary Sch, Moncada Spain; Lixin Wang, CURE: Digest DiseasesResearch Ctr, Los Angeles, CA; Jean E. Rivier, Salk Institute for Biology Study, La Julia, CA; Yvette Tache, CURE: Digest DiseasesResearch Ctr, Los Angeles, CA BACKGROUND:CRF actions are mediated through CRF-R1 and CRF-R2. In rats, peripheral CRF inhibits gastric emptying (GE) and stimulates colonic motor activity (JPET290:629, 1999). AIMS: To characterizechangesin gastric and colonic motility after intrapefitoneal(i.p.) administration of CRF in mice and the receptor(s) involved by using selective CRF receptor antagonists. METHODS:Changes in gastric emptying (% in 2h, GE) of a solid nutrient meal and colonic propulsion (time of expulsion of a glass bead inserted into the distal colon) were determined simultaneously in conscious mice affer the i.p. injection of CRF (2-60 p.g/kg; n = 4-8), with or without pretreatmentwith specific CRF-Rantagonists:the non selectiveCRFRI/R2 antagonist, astressin (0.12 mg/kg); the selective CRF-R1 antagonists, NBI-27914 and CP-154,526 (5*30 mg/kg); and the selective CRF-R2 antagonist, antisauvagine-30 (0.1 mg/ kg, ASV-30). RESULTS:In i.p. vehicle injected mice GE was 43.7_+7.1% and distal colonic transit 14.6_+2.2 min. Lp. CRF induced a simultaneous dose-related inhibition of GE and stimulation of colonic propulsive activity. Astressin blocked CRF actions on GE (astressin+ CRF: 30.8_+8.6%; Veh +CRF: 2.0+_2.0%; P
73O Heat Stable Toxin of E. coil (STa) Stimulates Duodenal Mucosal Bicarbonate Secretion in Cystic Fibrosis Knockout Mice Debbie Childs, Diane L. Crumble, Vijaya S K Pratha, Zachary Sellers, Daniel L. Hogan, Jon I. Isenberg, Univ of CA, San Diego, San Diego, CA Background:Incystic fibrosis (CF) knockout mice [CFTR(-/-)], duodenal mucosal bicarbonate secretion (DMBS) is impaired in response to all agonists tested including: forskolin, VIP and PGE2(alllikely acting via cAMP), carbachol (acting via Ca2+), and luminal acidification.
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In contrast, in CF patients, bicarbonate secretory responses in duodenal biopsies mounted in Ussing chambers responded normally to STa (presumablyvia guanylatecyclase C/cGMP). Our aimwas to determine if luminal perfusion of STa stimulates DMBS through a cGMPmediated pathway in CFTR(-/-) mice. Methods:Utilizingthe CF murine model, DMBS was compared in CFTR(-/-) animals and wildtype [CFTR(+/+) & ( + / - ) ] animals; confirmed by PCR. Anesthesia was induced with hypnorm/midazolam (i.p.). The proximal duodenum (5-10mm) was cannulated, perfused with 154 mM NaCI (0.2 ml/min) and [HCO~-]measured by micro-back titration. After measuring basal DMBSfor 45 rain, the duodenal segment was stimulated with either STa (0.1 uM), forskolin (0.1 mM) or 8Br-cGMP (1 mM). Established doses of each agonist were pertused for 30 min. Agonists were tested in separateanimals, and at least 4 animals were studied in each series. Results: Basal HCO3secretionwas about 60% less in CFTR(- / - ) vs. wildtype mice, 4.6 -+ 0.8 vs. 11.5 +_1.3 pmol/cm-h, respectively (P
728 Characterization of Nicotinic Acetylcholine Receptors (oAChRs) In Myenteric Neurons from Guinea Pig Intestine Jim H. Run, Xiaoping Zhou, James J. Galligan, Michigan State Univ, East Lansing, MI Background. Enteric nAChRs play a key role in gastrointestinal reflexes.The a and/3 subunit composition of nAChRsdeterminestheir pharmacologicaland functional properties.Although nAChRs play a critical role in the enteric nervous system, the subunit composition of enteric nAChRs is unknown. Aim. The aim of this study was to characterize nAChRs in guinea pig myentedc neurons using electrophysiologicaland immunohistochemicalmethods.The overall goal was to establish the subunit composition of myenteric nAChRsand to determine if there were developmentalchanges in the properties of myenteric nAChRs. Methods. Whole cell patch clamp recordings were obtainedfrom newborn guinea pig myenteric neurons in primary culture. Conventionalelectrophysiologicalmethods were used to record from neurons in the intact myenteric plexus from newborn and adult guinea pigs. Immunohistochemical methods were used to localize a and/~ subunits cultured neurons in culture and in the intact plexus from newborn and adult guinea pigs. Results. Acetylcholine (ACh) caused an inward current in >90% of neurons in culture. The nAChRagonist rank order potency was: DMPP>ACh>nicotine>cytisine. Dihydro-/3-erythroidine(DH,BE)blocks nAChRscontaining ~ receptorswith an ICsO20/.,,M; the DH/3EICsoversusACh in cultured neuronswas 50 _+4 ~M. e-Bungarotoxin and e-MLA, antagoniststhat block a7 subunit containing nAChRsdid not affect ACh currents. Intracellular recording from neurons in the intact myentedc plexus from adult and newborn guinea pigs showed that ACh, nicotine and cytisine caused equivalent depolarizations in S neurons. ACh, and nicotine but not cytisine depolarizedAH neurons. Depolarizationsevoked by cytisine and nicotine were blocked by mecamylamine.The DH,BEIC~ versus ACh was 13 +_ 4 p,M in adult neurons, tmmunohistochemical studies using an antibody (mAb35) that recognizes a3, ~5 and/34 subunits stained many in neurons in culture, and in the intact plexusfrom newborn and adult guinea pigs. An antibody against p2 subunits and an antibody against ~7 subunits did not revealany neurons in culture or in the intact plexusfrom newborn and adult guinea pigs. Conclusions. Pharmacologicaland immunohistochemicaldata indicate that the subunlt composition of myenteric nAChRs is either a3p4 or ~3a5~. Myenteric neurons do not express many nAChRs containing ~7 or if2 subunits. The properties of myenteric nAChRs in newborn intestine are similar to those in the adult intestine.
731 Bile Salts Are Physiological Stimulants of Trefoil Factor 3 (TFF3) Secretion in the Rat. Rorence Levenez,INRA UEPSD,Jouy en Josas France; StephaneDuroal, Cadne Blancbard, Jean-Claude Cuber, INSERM U45, Lyon France Background: Trefoil peptide 3 (TFF3) is specifically contained in goblet cells of the small intestine and colon.Its plays an important role in the protection and repair of the epithelial barrier.Thefactors that control TFF3secretion are poorly understood.They were investigated in the present study with a model of isolated vascularly perfused rat jejunum.Methods: A loop of the proximal jejunum (10-cm length) was isolated and perfused via the superior mesentericartery with a Krebs-Henseleitbuffer containing 20% washed bovine erythrocytes, 5mM glucose,lOmM amino-acids and 3% BSA at a flow rate of 2.5ml/min. Portal effluent was continuously collected as 2-rain fractions. Isotonic saline was infused into the lumen at a rate of 0.5ml/min. The luminal effluent was continuously collectedthrough the distal catheter as lO-min fractions. The experiments consisted of a 30-rain basal period and a 3g-rain stimulation period with the various luminal factors to be tested. TFF3 immunoreactivity was measured both in the portal and luminal effluents with a specific radioimmunoassay.Results: Luminal infusion of lOmM HCI, 5%glucose or 5%peptone evokeda 2-fold increase in luminal TFF3releasecomparedto isotonic saline alone. In contrast,a40mM oleic acid/2OmMtaurocholic acid (TC) mixture or TC (20mM) alone produced a 4-fold increase in TFF3 secretion.The effect of TC was concentration-dependentover the 5-30ram range.Thefirst significant response was observed upon stimulation with 5mM TC.Amongstthe tested bile acids, TC was was the most potent stimulant followed in decreasing order by cholic acid, deoxycholic acid, glycocholic acid and taurodsoxycholic acid. Tween 20 did not elicit TFF3 secretion, thus eliminating a detergent effect of TC.Intra-arterial infusion of bethanecol,hietamine,serotonin, bombasin or PGE2 also elicited a marked release of TFF3. However, i.a. infusion of TTX, atropine, a bombasin receptor antagonist,mepyramine, indomethacin, methysergide, ketsnserin or SDZ 205,557 in combination with luminal TC did not modify the TFF secretion induced by TC alone. Somatostatin (lOOnM) reduced by 80% the TC-inducedTFF3secretion. Conclusion:Bile salts are physiological stimulants of TFF secretion in the rat jejunum. This effect is a dynamic process which excludes the detergent properties of bile salts and which does not seem to involve the participation of intramural nerves. Somatostatin may restrain TFF3 secretion through a Iocal/paracdneor hormonal pathway.
729 Galanin and Orexin A Potentiate Intracellular Calcium Responsesto Cholacystokinin and Carbochst in Human and Rat Duodenal Enterocytes. Gunnar Flemstroem, Ramin Shadatmedad,Gunilla Jedstedt, Markus Sjoeblom, Saefsten Bengt, Akerman E. Karl, Uppsala Univ, UppsalaSweden The bicarbonate secretion by the duodenal mucosa protects this epithelium against acid dischargedfrom the stomach and the neurohumoral control of the secretion is a key element in mucosal protection. Duodenal epithelial intracellular and cell-to-cell signaling are final pathways in the secretory control but have not beenclarified. We havethus studied the effects of some secretagogueson duodenal enterocyte (duodenocyte) intracellular calcium ([Ca2+D using interconnectedduodenocytes(isolatedaggregates).Methods. Human duodenalbiopsies and scraped rat duodenal mucosa were cut into 0.5-1.0 mm in diameter pieces followed by mild digestion (coUagenase/dispase)to yield aggregates (10-200 cells) of duodenocytes. Aggregateswere loadedwith fura-2, mounted in a perfusionchamberand [Ca2+]~was measured with fluororescenceimaging. Thosechosenfor study were composed mainly of duodenocytas with enlarged vacuoles characteristic of crypt cells. Results. The use of aggregatesprovide more stable preparationsthan isolatedenterocytes.Superfusionwith cholecystokininoctapeptide (CCK-8; 1-50 nM), caerulin (100 nM), ADP (100/~U) or carbachol (1-100 p,M) induced a transient rise in [Ca2+]~.Responsesto CCK-8, but not those to carbachol, were prevented by CCK~-receptor antagonists. Galanin (1-100 nM) and oraxin A (10-100 nM) causeda small and transient rise in [Ca2*]~only in some duodenocytes but induced a slowly developing, Iong-lastiog and large rise in (Ca2+]~in most aggregatecells. Furthermore, galanin and orexin markedly potentiatedthe rise in [Ca2+],inresponseto CCK-8and carbachol. Eftecta in rat and human aggregateswere very similar. One or two cells in an aggregate were often the first to respond to CCK-8 (or carbachol) with a rise in [Ca2+],and this was followed by spread of the [Ca2+],signal within the aggregate.Conclusions. Galanin and orexin A-induced increases in enterocyte [Ca2÷],may modulate the intestinal responseto secretagogues.Interconnecting enterocytes respond to stimuli increasing [Ca2+]~asa syncytium. These phenomena may be important in the overall function of the intestinal mucosa and in mucosal protection.
732
Peripheral CRF Stimulates Colonic Propulsion and Inhibits Gastric Emptying in Mice: Differential Role of CRF Receptor Subtypes 1 (CRF-R1) and 2 (CRF-R2) Vicente Martinez, Cardenal Herrera Univ, Veterinary Sch, Moncada Spain; Lixin Wang, CURE: Digest DiseasesResearch Ctr, Los Angeles, CA; Jean E. Rivier, Salk Institute for Biology Study, La Julia, CA; Yvette Tache, CURE: Digest DiseasesResearch Ctr, Los Angeles, CA BACKGROUND:CRF actions are mediated through CRF-R1 and CRF-R2. In rats, peripheral CRF inhibits gastric emptying (GE) and stimulates colonic motor activity (JPET290:629, 1999). AIMS: To characterizechangesin gastric and colonic motility after intrapefitoneal(i.p.) administration of CRF in mice and the receptor(s) involved by using selective CRF receptor antagonists. METHODS:Changes in gastric emptying (% in 2h, GE) of a solid nutrient meal and colonic propulsion (time of expulsion of a glass bead inserted into the distal colon) were determined simultaneously in conscious mice affer the i.p. injection of CRF (2-60 p.g/kg; n = 4-8), with or without pretreatmentwith specific CRF-Rantagonists:the non selectiveCRFRI/R2 antagonist, astressin (0.12 mg/kg); the selective CRF-R1 antagonists, NBI-27914 and CP-154,526 (5*30 mg/kg); and the selective CRF-R2 antagonist, antisauvagine-30 (0.1 mg/ kg, ASV-30). RESULTS:In i.p. vehicle injected mice GE was 43.7_+7.1% and distal colonic transit 14.6_+2.2 min. Lp. CRF induced a simultaneous dose-related inhibition of GE and stimulation of colonic propulsive activity. Astressin blocked CRF actions on GE (astressin+ CRF: 30.8_+8.6%; Veh +CRF: 2.0+_2.0%; P
73O Heat Stable Toxin of E. coil (STa) Stimulates Duodenal Mucosal Bicarbonate Secretion in Cystic Fibrosis Knockout Mice Debbie Childs, Diane L. Crumble, Vijaya S K Pratha, Zachary Sellers, Daniel L. Hogan, Jon I. Isenberg, Univ of CA, San Diego, San Diego, CA Background:Incystic fibrosis (CF) knockout mice [CFTR(-/-)], duodenal mucosal bicarbonate secretion (DMBS) is impaired in response to all agonists tested including: forskolin, VIP and PGE2(alllikely acting via cAMP), carbachol (acting via Ca2+), and luminal acidification.
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