Intracellular Signal Transduction Mechanism Of Acetylcholine, Bombesin And Substance P On The Human Lower Esophageal Sphincter

The lower esophageal sphincter (LES) in human beings lies between the esophagus and the stomach. It not only guarantees that food bolus in the esophagus pass into the stomach, but also prevent the gastric contents reflux into the esophagus. Thus, the structural or functional abnormality in the human LES may result in a number of disorders. Although the lower esophageal sphincter has great significance regarding the diagnosis and the treatment of esophageal benign disorders, the understandings of the lower esophageal sphincter in human beings have been a matter of speculation for many years. In 1979, Liebermann-Meffert et al described the arrangement of the smooth muscles at the esophagogastric junction (EGJ) in detail. They demonstrated that the musculature of the human LES consisted of sling fibers at the greater curvature and clasp fibers at the lesser curvature. This theory laid the foundation for further studies on the physiology, pathology and pharmacology of the LES. Over the ensuing years, many studies on the physiological feature and functional regulation of the LES in several kinds of animals were extensively conducted. These studies have suggested that the sling fibers and clasp fibers forming the LES varied significantly in many aspects.Sohn et al. previously found that, Ach -induced contraction of cat esophageal circular muscle is mediated by a muscarinic M2 receptor linked to a PTX -sensitive Gi3 protein and depends on extracellular Ca2+-induced activation of PLD, production of DAG and activation of PKC. In contrast, in LES circular muscle, ACh acts through M3 muscarinic receptors linked to a PTX insensitive Gq-G11 protein, which activates PLC causing production of IP3, release of intracellular Ca2+ and activation of a calmodulin-dependent pathway.Biancani et al. have found that two distinct contractile signal-transduction pathways are present in LES muscle cells. A PI-PLC, IP3, calmodulin-dependent pathway is activated by stimulation with a maximally effective dose of ACh. In this pathway, M3 muscarinic receptors link to Gq/11-type G proteins stimulate PLC, resulting in the formation of IP3 and DAG. IP3 causes the release of Ca2+ from intracellular stores, producing a calciumcalmodulin complex, myosin light chain phosphorylation, and contraction. This pathway is PKC independent, because maximal activation of calmodulin inhibits PKC activity. A distinct PKC-dependent pathway is activated by submaximal doses of ACh or during maintenance of LES tone. In this pathway, contraction is mediated by low levels of PLC activity, resulting in low levels of IP3, which cause the release of low levels of Ca2+ from intracellular stores. These low Ca2+ levels are insufficient to activate a calmodulindependent contraction, which requires micromolar Ca2+ concentrations. In addition, concurrent activity of a phosphatidylcholine -specific (PC-PLC) in the LES contributes to the production of DAG. Low levels of IP3 act synergistically with DAG to activate a PKC -dependent pathway. Thus the amount of Ca2+ available for contraction determines which pathway will be followed, with low Ca2+ levels activating a PKC-dependent pathway and high levels activating a calmodulin-dependent pathway.As one of the most important neural transmitters, it is still not clear that which pathway or both in the LES tension regulation acetylcholine acts through. It is also not clear that the intracellular pathways activated by ACh may depend on the difference in receptors. Or alternatively, it is possible that distinct pathway may be a characteristic of the muscle types and that multiple receptors may activate only one pathway in one type of muscle cells and a different one in another.In the present investigation, we examine bombesin and substance P along with acetylcholine, which are present in the enteric nervous system and utilize different receptors to cause contraction of gastrointestinal muscle. We examined the intracellular signal transduction pathways activated in human lower esophageal sphincter, both the sling fibers and the clasp fibers, as well as the circular layer muscle of the esophagus and gastric fundus. The differences of mechanisms in cellular level between the sling fibers and the clasp fibers in human lower esophageal sphincter tension regulation are also analyzed.Part 1 Regulation of acetylcholine, bombesin and substance P on the sling fibers and clasp fibers of the human LES1 Regulation of acetylcholine on the sling fibers and clasp fibers of the human LESObjective: Acetylcholine is one of the most important neural transmitters regulating human lower esophageal sphincter tone. The research was conducted to investigate it's regulation of the contraction of human sling fibers and clasp fibers forming LES, therefore to lay the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of acetylcholine (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 13.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-9mol/L for acetylcholine. The shortening percentages in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 21.8±0.9%,23.1±1.9%,22.2±2.0% and 21.9±1.2%. There were no significant differences in terms of the maximal response to acetylcholine between muscle types (P>0.05).Conclusion: Acetylcholine causes contraction in a concentration- dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to acetylcholine occurs at a 10-9mol/L concentration.2 Regulation of bombesin and substance P on the sling fibers and clasp fibers of the human LESObjective: Bombesin and substance P which are present in the enteric nervous system differ from acetylcholine which binds to different receptor to cause contraction of gastrointestinal muscle. The research was conducted to investigate the effects of these agonists on the contraction of human sling fibers and clasp fibers forming LES, therefore to set up the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of bombesin and substance P (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 16.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-7mol/L for both bombesin and substance P. The shortening percentages caused by 10-7mol/L bombesin in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 23.2±1.1%,23.9±2.1%,23.9±1.2% and 24.8±1.9% ; The shortening percentages caused by 10-7mol/L substance P in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 24.1±1.2%,25.8±1.1%,25.1±1.1% and 24.3±2.1%. There were no significant differences in terms of the maximal response to both agonists and acetylcholine in any muscle type (P>0.05). And there were no significant differences between muscle types (P>0.05).Conclusion: Bombesin and substance P cause contraction in a concentration-dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to bombesin and substance P occur at a 10-7mol/L concentration. The responses to bombesin and substance P are not different form the maximal response to acetylcholine.Part 2 The expression and function of guanine nucleotide-binding protein on the human LES'sling fibers and clasp fibers1 The expression of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LESObjective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore the expression levels of four subtypes of guanine nucleotide-binding protein in the sling fibers and clasp fibers.Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. The expressions of Gi3,Gq-G11,Go, Gs subtypes G proteins in these muscles were detected by Western blot. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant (by ANOVA).Results: Gq/11 and Gi3 were detected as thick bands in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Go appeared as a thin band. Gs was barely visible.Gel-analysis program showed the expression of Gq/11-subtype G protein in the sling fibers (0.486±0.082), in the clasp fibers (0.492±0.109), in the ESO (0.417±0.161) and in the gastric fundus (0.494±0.101); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Go-subtype G protein in the sling fibers (0.011±0.008), in the clasp fibers (0.016±0.012), in the ESO (0.010±0.007) and in the gastric fundus (0.009±0.008). Gs-subtype G protein was trace content in all four groups. The differences between groups were not statistically significant (P>0.05, by ANOVA).Conclusion:These data suggest that Gq/11 and Gi3 are present in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Differences in each of four subtypes of G proteins in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus tissue are not apparent.2 The function of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LES Objective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore that which of four subtypes of guanine nucleotide-binding protein play an important role in the sling fibers and clasp fibers.Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the four subtypes G protein antibodies before agonists were used. The original length and length changes of all kinds of muscle cells to different treatments were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. The maximal dose of acetylcholine, bombesin and substance P were used to induce contraction. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.Results: In the cells of the circular layers of the esophagus and the clasp fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gi3 antibodies (P < 0.001 by ANOVA), but not by Go, Gq/11 antibodies, which indicated that, in the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. While in the cells of the circular layers of the gastric fundus and the sling fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gq/11 antibodies (P < 0.001 by ANOVA) but not by Go, Gi3 antibodies, which indicated that, in the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11.Conclusion:In the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. In the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11. Guanine nucleotide-binding proteins linked with acetylcholine, bombesin or substance P receptors are unique and muscle-type dependent.Part 3 The role of phospholipases on the contraction of the human LES'sling fibers and clasp fibersObjective:Activation of the phospholipases is responsible for hydrolysis of lipid pool and production of second messengers after guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P. Which kind of phospholipases is activated in human lower esophageal sphincter's sling fibers and clasp fibers, and the circular layers of the esophagus and gastric fundus when stimulated by a maximally effective dose of Ach, bombesin or substance P is still not certain. This study was conducted to explore that the role of PC-PLC, PI-PLC, and PC-PLD on the human LES'sling fibers and clasp fibers.Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the three phospholipases before agonists were used. When inhibitors (10-6M U-73122, 10-4M propranolol, 10-4M D609 and combination of propranolol and D609) were used, the cells were incubated in the antagonists for 5 min before addition of agonist, either acetylcholine, bombesin or substance P.Results: (1) PI-PLC antagonist U-73122 (10-6 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the gastric fundus and the sling fibers, but not of the cells of the circular layers of the esophagus and the clasp fibers. The differences among four groups are significant (P<0.05). Which indicated that, PI-PLC played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the gastric fundus and the sling fibers. (2) Conversely, the PC- PLC inhibitor D609 (10-4 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). Which indicated that, PC-PLC plays an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers. (3) PC-PLD inhibitor propranolol (10-4M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). which indicated that, PC-PLD played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers.(4) In acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, the inhibitory effects of D609 and propranolol were additive. (P=0.014,P=0.027)by paired t test against D609 or propranolol alone.Conclusion:PC-PLC, PI-PLC, and PC-PLD play different roles in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and gastric fundus, and the sling fibers and clasp fibers. The contraction of four muscle type cells in response to acetylcholine, bombesin or substance P is differentially inhibited by different phospholipase specific inhibitors. PI-PLC antagonist inhibits the contraction of the cells of the circular layers of the gastric fundus and the sling fibers; PC-PLC or PLD antagonists inhibits the contraction of the cells of the circular layers of the esophagus and the clasp fibers. And the inhibitory effects result in almost complete abolition of contraction.PART 4 The role of calmodulin-dependent and PKC-dependent pathways on the contraction of the human LES'sling fibers and clasp fibersObjective:To investigate the roles of two distinct intracellular signal transduction pathways: Protein Kinase C (PKC)-dependent and calmodulin- dependent, in the contractile mechanism of the clasp and sling fibers, the two parts of human lower esophageal sphincter, in comparison with circular muscle layers of gastric fundus and esophagus. Meanwhile the roles of extracellular and intracellular Ca2+ in this process were studied.Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and October 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. When Protein Kinase C(PKC)-dependent pathway inhibitors H7, calmodulin-dependent pathway inhibitor CGS9343B, Ca2+-free medium or substitution of 4mmol/L Sr2+ for Ca2+ were used, the cells were incubated in appropriate solution for 5 min before addition of agonists. The length of cells was measured by video-based motion edge-detection system.Results: Contraction of the cells of the clasp muscles and the esophagus was blocked by H7 and by incubation in Ca2+-free medium, but not by the CGS9343B and 4mmol/L Sr2+. By contrast, contraction of the cells from sling and gastric fundus was blocked only by CGS9343B and 4mmol/L Sr2+.Conclusion: Contraction of the sling fiber and the circular muscle of the gastric fundus depends on release of intracellular calcium and activation of a calmodulin-dependent pathway. While contraction of the clasp fiber and the circular muscle of the esophagus depends on influx of extracellular calcium and activation of a PKC-dependent signal transduction pathway.