The Effects Of Acetylcholine On Isolated Lower Esophageal Sphincter Smooth Muscle Cells And Intracellular Calcium Concentration

Objective: The lower esophageal sphincter (LES) is a thicken -ed region of the circular muscle layer of the distal esophagus, extending over an axial distance of 2³cm in human. Lieber- mann-Meffer proposed that the muscular equivalent of the LES consists of a thickened inner circular muscle layer configured as semicircular or clasp fibers adjacent to the lesser curvature and sling like muscle bundles on the greater curvature side formed by long oblique gastric fibers. These separate muscle groups create a zone of high pressure (15³0mmHg) and maintain sphincter closure. In vivo studies using manometric catheter with three paired holes in the left and right orientations within LES have shown a significant radial asymmetry in the distribution of the high-pressure zone. The highest pressures were recorded in the left lateral direction of the LES region which was sling fibers. Blockade of muscarinic cholinergic transmission with atropine caused a significant reduction of LES in the left side of gastroesophageal junction. In vitro the sling is more sensitive to cholinergic stimulation, whereas the clasp muscle develops greater spontaneous tension. It is well known that the appropriate function of the LES is controlled by excitatory and inhibitory neuronal mechanisms that cause relaxation during swallowing and normally prevent reflux but permit vomiting and venting of gas. This study was conducted to investigate the different response of sling and clasp fiber cells to Acetylcholine and the reason of this difference, with the purpose of further understanding of the physiological and pharmacological characteristics of the LES, and providing theoretical bases for the clinical treatment of esophageal motility disorders.Methods: Twenty patients undergoing subtotal esophage -ctomy for middle thoracic esophageal carcinoma in our hospital between August 2005 and December 2006 were selected in this study, including 10 male and 10 female patients,ages ranging from 52 to 64 yr (mean 57.7yr). Fresh specimens of the GEJ were obtained in the operating room. After the surrounding fascia and mucosa were removed by sharp dissection, muscle strips were prepared from various regions of the GEJ and adjacent structures. The sling and clasp fibers could be readily identified as a thickened band of circularly oriented smooth muscle adjacent to the greater and lesser curvature of the stomach, respectively. The isolated smooth muscle cells of the sling and clasp fibers were obtained by enzymatic digestion, conforming that the activity of cells were above 95%. 1 A drop of the cell-containing medium was placed on a glass slide and covered with a cover slip. The length of 50 consecutive intact cells, encountered at random, in each slide was measured with a phase-contrast microscope. The average length of 50 cells, measured in the absence of agonists, was taken as"control"length. In addition, average cell length was measured after addition of test agents. For each experiment, contraction was expressed as percent shortening of the average of 50 consecutive cells compared with the average of 30 untreated (control) cells. (1) Progressive concentrations of Ach were added to the mixture cells, and the differential contraction of the two kinds of cells to Ach and the maximal effective dose of Ach were observed. (2) When muscarinic receptor antagonists were used, cells were incubated in appropriate concentration of the antagonist for 1 min before the addition of Ach. We observed the effects of the M1, M2, M3 and M4 antagonists, pirenzepine, methoctramine, and 4-DAMP tropicamide respectively on the contraction induced by a maximally effective dose of Ach in LES smooth muscle cells, so that the antagonist resulting in the largest inhibition and difference between sling and clasp cells were defined. 2 [Ca²⁺]_i was measured by laser scanning confocal microscopy in single smooth muscle cells which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM.The changes in [Ca²⁺]_i were represented by fluorescent intensity (Fi) or relative fluorescent intensity (F_i/F₀%). (1) In HEPES-Ringer solution (CaCl₂ 1.5 mmol/ L), progressive concentration of Ach were added to the mixture cells. The difference between the two kinds of cells in calcium concentration was observed. (2) Appropriate concentration of Ach was added to the absence of extracellular Ca2+ solution, observing the effect of EGTA on the elevation of [Ca2+]i in the sling and clasp cells.(3) Effect of nisoldipine on [Ca2+]i elevation of sling and clasp cells induced by Ach: the preparation was pretreated with the calcium channel blocker nisoldipine for 1 min, and Fi was measured after adding Ach. All data were expressed as mean±SD, and analyzed by one-way ANOVA followed by the paired t test for comparison within group and the unpaired t test for group comparison. P values less than 0.05 were considered statistically significant.Results: 1 Freshly isolated LES SM cells were spindle shaped and appeared phase bright contained the nucleus under phase-contrast microscopy. In HEPES-Ringer solution, some cells were in condition of contraction and others in relaxation. The average length of sling muscle cells was 120.1±14.7μm (51²28μm) in normal buffer and the clasp was 119.5±11.2μm(48²24μm); 2 Ach (10-12-10-7mol/L) elicited a rapid and marked contraction in isolated LES smooth muscle cells in HEPES-Ringer solution. The contraction was dose-dependent contraction and reached the peak level at 30s. The degree of contraction was greater in sling SM cells than in clasp (P<0.01) with maximal contraction occurring at 10-8mol/L;3 Pretreatment the sling SM cells and clasp SM cells with M1-M4 receptor antagonist Pirenzepine, Methoctramine, 4-DAMP, Tropicamide(10^-6mol/L), then added Ach 10^-8mol/ L. M1^M4 receptor antagonists inhibited 31.12±1.24%,12.35±0.91%,72.13± 1.33%,20.25±1.51% of contraction effect of Ach in sling SM cells and 29.21±0.73%,14.23±1.14%,71.02±0.81%,21.42±1.02% in clasp SM cells. There was significant difference in terms of effect inhibition between any different 2 antagonists in the same muscle cells (F=52.73, P=0.00). The rank order of inhibition for antagonists was 4-DAMP, Pirenzepine, Tropicamide and Methoctrmine. There was no significant difference of inhibition between any 2 SM cells in the same antagonist (P>0.05); 4 The cells loading with fluo-3 were detected with rod shaped and visible striations by confocal laser scanning system. [Ca²⁺]_i changes were represented with fluorescent intensity (Fi) or relative fluorescent intensity (Fi/F0 %). Fi/F0 % is the ratio of change in Fi to the fluorescence under quiescent condition (F0). Therefore, the distribution of [Ca²⁺]_i was recorded both in the quiescent condition and after application of Ach. Ach evoked [Ca²⁺]_i significant elevation in sling and clasp SM cells in the presence of extracellular Ca2+ (1.5mmol/L), and Ach-induced [Ca²⁺]_i elevation were of greater in LES sling cells than in LES clasp cells (1.6 times) (t=15.17,P=0.01); 5 In Ca2+-free HEPES-solution, Ach (10^-8 mol/L) induced Ca2+ elevation were inhibited in a time- and challenge-dependent manner in LES clasp cells (81.62±1.25%)(P<0.01), but calcium-free medium did not reduce Ach- induced [Ca²⁺]_i elevation in sling cells (P> 0.05). There was a significant difference between these two cells. (P<0.01); 6 Addition of Nifedipine (1μM) to isolated clasp cell solution caused significant inhibition of Ach-induced [Ca²⁺]_i elevation in the LES clasp smooth muscle cells (33.53±11.24% of controls) (P<0.01). However, Nifedipine (1μM) did not inhibit Ach-induced Ca2+ elevation in sling smooth muscle cells (P>0.05). Even when sling smooth muscle cells were exposed to higher concentrations of Nifedipine (10μM) and for longer durations, no significant inhibition of Ach induced [Ca²⁺]_i elevation was observed. There was a significant difference of inhibition between any 2 muscle cells in this inhibitor (P<0.01).Conclusion: 1 Ach elicited a rapid and marked contraction in isolated LES smooth muscle cells. The extant of contraction was greater in sling SM cells than in clasp; 2 The cell contraction was demonstrated to be mediated by the M3 receptor subtype. There are no differences in function of mAChRs between sling and clasp cells. M1 and M4 receptors mediate the contraction of muscle strips partially, and the M2 receptor plays a minor role in mediating the smooth muscle cells contraction; 3 Ach evoked [Ca²⁺]_i significant elevation in sling and clasp SM cells in the presence extracellular Ca2+, Ach-induced [Ca²⁺]_i elevation were of greater in sling cells than in clasp cells; 4 Ach induced [Ca²⁺]_i elevation in sling smooth muscle cells is due to continuous release of Ca2+ from SR stores. Ach induced [Ca²⁺]_i elevation in LES Clasp smooth muscle cells is due to continuous influx of extracellular Ca2+ primarily via LCa.