| The human lower esophageal sphincter(LES) is the junction of esophagus and stomach, which is made of sling fibers and clasp fibers and differs from most of mammal. The LES has a higher pressure than the intragastric about 5-10mmHg, the normal resting LES pressure is about 10-30mmHg. LES plays a physiological role of the sphincter.Under normal circumstances, LES is in constantly contractile state for limiting the stomach contents refluxing into the esophagus. However, LES relaxs in a timely manner to allow nutrients into the stomach when eating. Therefore, when LES's sturcture or function changed improperly, it would lead to a variety of esophageal diseases, such as GERD(gastroesophageal reflux disease), loss of cardiac relaxation, et al. LES's regulatory mechanisms were very complex, including the nervous system, humoral and myogenic factors. On physiological conditions, brain-gut peptide as a neurotransmitter or hormone were produced by neurological or endocrine cells and adjust contraction and relaxation of LES. But no matter on which level regulation happens, the brain-gut peptide ultimately binds to receptors on the surface of the LES cells. After the receptors were activated, a series of signaling pathway caused LES contraction and relaxation. Most previous studies focused on central, peripheral and enteric nervous system to regulate gastrointestinal motility. The studies about activities of the intrinsic LES were few. Therefore, it is necessary to further study LES's sling and clasp receptors and postreceptor signal transduction mechanisms at cellcular level in order to elucidate the regulatory mechanism of LES activity. Brain-gut peptide, through inositol lipids and cyclic nucleotide system on the plasma membrane, transmits extracellular signals into the cells to induce LES contraction or relaxation. Brain-gut peptides bind with receptors and activates them to promote guanosine triphosphate(GTP) connecting to different GTP binding protein(G-protein). The activated G protein effects different enzymes to produce a variety of regulatory effects to regulate the contraction or relaxation of LES. G protein plays as a bridge on the progression of cellular information transmission. Clarifying the type of G protein, coupled with brain-gut peptide receptor, is import to understand postreceptor signaling pathway during the contraction or relaxation of LES.Motilin and gastrin are important excitatory brain-gut peptides and play an important role on the regulation of gastrointestinal motility. The study with G protein coupled signaling pathway of motilin and gastrin on sling and clasp fibers was few. Secretin is an inhibitory brain-gut peptide, resulting in gastrointestinal smooth muscle relaxation and delaying gastric emptying. At home and abroad, study about secretin to relax LES was few. Current research about LES's regulation with brain-gut peptide were limited at vitro level. The study of G protein coupled with brain-gut peptide and signal transduction pathway on the sling fibers and clasp fibers was relatively small. In this experiment, the representative excitable brain-gut peptide(motilin and gastrin) and inhibitory peptide(secretin) were applied to single sling fibers and clasp fibers. Research about the role of the receptors, G proteins and postreceptor signal transduction pathway to brain-gut peptide at cellular level will further clarify the signal transduction of excitable brain-gut peptide and inhibitory peptide. That we recognize the regulation mechanism of LES at cellular level is import to understand esophageal motility disorders.In this experiment, excitable brain-gut peptide(motilin and gastrin) and inhibitory peptide(secretin) were used to sutdy isolating sling fibers and clasp fibers of LES. Observing: (1)reaction of sling fibers and clasp fibers to motilin, gastrin and secretin; (2)the type of G protein coupled with these three receptors; (3)relationship between excitable brain-gut peptide and inositol lipid signaling system; relationship between inhibitory brain-gut peptide and cyclic nucleotide signaling system. Through this research to further reveal the role of brain-gut peptide in the LES intracellular signaling pathway at the cellular level. Experiment was divided into three parts:Part one Motilin on human lower esophageal sphincter sling and clasp fibers and role in the regulation of signal transductionObjective: Motilin is a 22-amino acid brain-gut peptide and an important hormone, starting the gastrointestinal interdigestive migrating motor complex (MMO). This article focused on the expression of motilin receptors and signal transduction in the LES's sling fibers and clasp fibers.Methods: Selecting sling fibers and clasp fibers from 5 patients, who underwent surgery due to esophageal cancer in the Fourth Hospital of Hebei Medical University from May 2009 to October 2010. (1)preparation of isolating sling and clasp fibers suspension, solution of motilin at different concentrations(10-6, 10-7, 10-8, 10-9, 10-10, 10-12mol/L) acts on sling and clasp fibers, then using 2.5% glutaraldehyde to stop the reaction, subsequently recording the length changes of the smooth muscle cells, analysing the data with software GraphPad prism5.0, using nonlinear regression analysis, getting the concentration-response curve, obtaining motilin concentration corresponding to the maximum contraction amplitude. (2)by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting (Western blot), We detected expression in the sling and clasp fibers of motilin receptors. (3)applying saponins to prepare permeable single sling and clasp suspension, observing the effect of phospholipase C inhibitor U-73122(10-6mol/L), motilin antiserum(1:100) and IP3 receptor antagonist(heparin 10μg/mL) on these cells which were pretreated by motilin. (4)by [35S]GTPγS binding experiments, observing the G protein changes. (5)observing specific G protein antibodies(Gαi3,Gαs,Gαq/11) on the effects of motilin, analysing the type of G protein which coupled with motilin receptor.Results: (1) Motilin, in the range of 10-6-10-12mol/L, had a dose-response relationship with sling fibers and clasp fibers, and the maximum response concentration was 10-6 mol/L. Maximal contraction of sling fibers and clasp fibers, induced by motilin, were (24.1±1.2)% and (21.1±1.0)%, contraction of sling fibers higher than clasp fibers(t=3.18, P=0.004); (2)RT-PCR and Western blot showed that sling fibers and clasp fibers both expressed motilin receptors, the former the higher(t=5.16, P=0.002). (3)motilin antiserum limited contractile response on sling fibers and clasp fibers, cell contraction decreased to (5.1±0.5)%(t=5.9, P=0.001) for sling fibers and (7.6±0.4)%(t=8.4, P=0.001) for clasp fibers. The sling fibers were limited largely(t=3.02, P=0.040); heparin limited contractile response on sling fibers and clasp fibers to (13.8±1.2)% (t=6.91, P=0.002) and (12.9±1.4)% (t=7.12, P=0.002). There were no significant difference between them(t=0.37, P=0.730); U-73122 limited contractile response on sling fibers and clasp fibers to (13.3±0.8)% (t=9.68, P=0.001) and (12.1±1.5)% (t=6.31, P=0.003). There was no significant difference between them(t=1.29, P=0.268). (4)[35S]GTPγS banding rate significantly increased (66.5±28.4)%(t=2.86, P=0.046) and (60.1±17.2)%(t=3.20, P=0.033) after stimulating by motilin. There was no significant difference between them(t=0.32, P=0.764). (5) Gαi3 antibody decreased contractile response on sling fibers and clasp fibers to (11.1±1.4)%(t=3.22, P=0.032) and (7.9±3.3)%(t=3.52, P=0.025). No significant difference between them(t=1.10, P=0.333); Gαs and Gαq/11 antibody had no effect on the fibers. The data showed that the Gi protein coupled with motilin.Conclusion: Motilin receptor expressed on sling fibers and clasp fibers, the former was higher than the later. The contractile response of sling fibers and clasp fibers induced by motilin were obviously different. The former contracted stronger than the later. This experiment showed the signal transduction pathway of LES: Motilin binded with motilin specific receptor, then activating Gi protein which coupling with phospholipase C, producing inositol triphosphate which stimulated inositol trisphosphate-sensitive intracellular calcium store to release Ca2+, causing sling and clasp fibers contracting.Part two Gastrin on human lower esophageal sphincter sling and clasp fibers and role in the regulation of signal transductionObjective: A brain-gut peptide-gastrin is an important hormone to promote gastrointestinal motility. Studies had shown that LES's relaxation was related to gastrin. This article focused on contraction rule and signal transduction mechanism of sling and clasp fibers.Methods: Collecting sling fibers and clasp fibers from 5 patients, who underwent surgery due to esophageal cancer in the Fourth Hospital of Hebei Medical University from May 2009 to October 2010. (1)preparing isolating sling and clasp fibers suspension, gastrin solution at different concentrations act on sling and clasp fibers, then using 2.5% glutaraldehyde to stop reaction, subsequently recording the length of the smooth muscle cells, analysing the data with software GraphPad Prism5.0, using nonlinear regression analysis, getting the dose-response curve, finally having gastrin concentration with maximum contraction amplitude. (2)using saponins to prepare permeable single sling and clasp suspension, observing the effect of phospholipase C inhibitor U-73122(10-6mol/L), motilin antiserum(1:100) and inositol trisphosphate receptor antagonist(heparin 10μg/mL) on these cells which were pretreated by gastrin. (3)by [35S]GTPγS binding experiments, observing the changes in G protein levels. (4)using specific G protein antibodies(Gαi3,Gαs,Gαq/11) to inhibit gastrin effect, analysing the type of G protein, coupled with gastrin receptor.Results: (1)Gastrin, in the range of 10-6-10-12mol/L, had a dose-response relationship with sling fibers and clasp fibers, and the maximum response concentration was 10-6mol/L. Maximal contraction of sling fibers and clasp fibers were (27.3±0.7)% and (23.7±0.6)%, the former the stronger(t=3.88, P=0.018). (2)gastrin antiserum decreased contractile response on sling fibers and clasp fibers to (10.9±1.1)% and (14.2±0.5)%. The sling fibers was decreased largely(t=3.2, P=0.033); heparin limited contractile response on sling fibers and clasp fibers to (13.5±0.7)%(t=9.1, P=0.001) and (12.2±2.1)%(t=3.08, P=0.037). No significant difference between them(t=0.57, P=0.602); U-73122 limited contractile response on sling fibers and clasp fibers decreased to (13.1±2.1)%(t=6.15, P=0.004) and (11.4±2.0)%(t=4.71, P=0.009). No significant difference between them(t=0.54, P=0.620). (3) [35S]GTPγS banding rate of sling fibers and clasp fibers significantly increased to (72.1±14.5)%(t=3.11, P=0.036) and (36.5±14.2)%(t=3.01, P=0.039) after stimulating by gastrin. No statistic significance between them(t=1.61, P=0.182). (4) Gαi3 antibody inhibited contractile response on sling fibers and clasp fibers to (10.6±1.2)%(t=7.41, P=0.002) and (8.8±0.6)%(t=4.56, P=0.010), no statistic significance between them(t=1.03, P=0.362); Gαs and Gαq/11 antibody had no effect on smooth muscle cells, treated with gastrin. The data showed that the Gi protein coupled with gastrin.Conclusion: Gastrin receptor expressed both on sling fibers and clasp fibers. The contractile response of sling fibers and clasp fibers, induced by gastrin, were obviously different, the former was stronger than the later. This experiment partly clarified the signal transduction pathway of LES, induced by gastrin: gastrin binded with gastrin specific receptor, then activating Gi protein and coupling with phospholipase C, producing inositol triphosphate which stimulated inositol trisphosphate-sensitive intracellular calcium store to release Ca2+, causing sling and clasp fibers shortening.Part three Secretin on human lower esophageal sphincter sling andclasp fibers and role in the regulation of signal transduction Objective: Secretin is an important gastrointestinal regulating hormone. Studies showed that secretin with physiological doses inhibited gastric motility and made the stomach relaxation. This article focused on relaxation rule and signal transduction mechanism of sling and clasp fibers, induced by secretin.Methods: Collecting sling fibers and clasp fibers from 5 patients, who underwent surgery due to esophageal cancer in the Fourth Hospital of Hebei Medical University from May 2009 to October 2010. (1)preparing sling and clasp fibers isolating suspension, secretin solution with different concentrations(10-6, 10-7, 10-8, 10-9, 10-10, 10-12mol/L) acts on sling and clasp fibers, then using 2.5% glutaraldehyde to terminate the reaction, subsequently recording the length of smooth muscle cells, analysing the data with software GraphPad prism5.0, using nonlinear regression analysis. Then we got the concentration-response curve and secretin concentration corresponding with the maximum relaxation amplitude. (2)using saponins to prepare permeable single sling and clasp suspension, observing the effect of secretin antiserum, AC agonist(Forskolin 10-6mol/L) and cAMP inhibitor on these cells which were pretreated by gastrin. (3)by [35S]GTPγS binding experiments, observing the G protein changes. (4)using specific G protein antibodies(Gαi3,Gαs,Gαq/11) to deal with secretin, analysing the type of G protein which coupled with secretin receptor.Results: (1)Secretin, in the range of 10-6-10-12mol/L, had a dose-response relationship with sling fibers and clasp fibers, and the maximum response concentration was 10-6mol/L. Maximal relaxation of sling fibers and clasp fibers, induced by motilin, were (29.2±0.6)% and (23.9±0.5)%. Relaxation of sling fibers was higher than clasp fibers(t=6.05, P=0.026). (2)Gastrin antiserum decreased relaxation on sling fibers and clasp fibers to (7.5±0.9)% (t=40.2, P=0.001) and (11.2±1.0)%(t=21.6, P=0.002). There was statistic significance between them(t=6.39, P=0.024); AC agonist(Forskolin) promoted relaxation response on sling fibers and clasp fibers, cell relaxation increased to (40.0±2.3)%(t=4.8, P=0.041) and (30.7±1.8)%(t=4.70, P=0.040); cAMP inhibitor limited relaxation on sling fibers and clasp fibers to (17.2±1.8)%(t=5.14, P=0.036) and (14.7±4.3)%(t=4.76, P=0.041). (3) [35S]GTPγS banding rate of sling and clasp fibers were significantly increased to (52.8±19.5)%(t=3.33, P=0.029) and (44.9±20.9)%(t=3.23, P=0.032) stimulated by secretin. (4)Gαs antibody decreased relaxation on sling fibers and clasp fibers to (9.9±2.33)% (t=7.78, P=0.016) and (8.7±1.9)%(t=4.38, P=0.048); Gαi3 and Gαq/11 antibody had no significant effect on cell relaxation. The data showed that Gαs protein was coupled with secretin.Conclusion: Secretin receptor expressed both on sling fibers and clasp fibers. The relaxation response of sling fibers and clasp fibers to gastrin was obviously different. The former was stronger than the later. This experiment partly showed the signal transduction pathway of LES, induced by secretin: secretin binding with secretin specific receptor, then activating Gs protein and coupling with AC which changed ATP into cAMP, the later made the sling and clasp fibers relaxation indirectly.
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