| Objective: The human lower esophageal sphincter (LES) is a special thickening cluster of the muscle in the esophagogastric junction. In the 1970s, Liebermann-Meffert et al proposed that LES was composed of the sling fibers and clasp fibers, which keep LES closed and form the high-pressure zon at the esophagogastric junction. This high-pressure zone could prevent reflux from the stomach into the esophagus and allow passage of food from the esophagus to stomach when swallowing. The maintenance of the LES function depends on the regulation of neurohumoral and muscular factors. Calcium ions play a crucial role in the contraction of lower esophageal sphincter. According to our previous study, we showed that the sling fibers were more sensitivity than clasp fibers at the same concentration of acetylcholine (10-8 mol/L), and the intracellular calcium concentration ([Ca2 +] i) of clasp fibers elevated by Ach mainly caused by the L-Ca channels induced Ca2 + influx, but the effects in the sling fibers was mainly due to the intracellular Ca2 + releasing. As an important second messenger, inositol 1,4,5-triphosphate (1,4,5-IP3) could bind with its specific receptor in the signal transduction, inositol 1,4,5-triphosphate receptor (IP3R), and trigger intracellular calcium store released to elevate the intracellular calcium concentration. There are three kinds of IP3R subtypes had been established, IP3R 1, IP3R 2 and IP3R 3. Several cell types of animal and human studies showed that subtypes of IP3R had different functions because of different tissues and cell types of different distributions even in the same body. At present, there are less researches on the mechanisms of signal transduction of sling and clasp fibers contraction in the lower esophageal sphincter, especially for the Chinese and foreign literature about the function of different subtypes of inositol 1,4,5- triphosphate receptors in the lower esophageal sphincter. In this study, we explored the expression of the three kinds of IP3R subtypes in the human lower esophageal sphincter by laser scanning confocal microscope imaging and Western blot, and compared the expression pattern of IP3R subtypes in sling fibers (S) and clasp fibers (C) with esophageal circular muscle (E) and gastric circular muscle (G). The aim of the study is to lay the foundation for further studies of the role of IP3Rs in the contractile regulation in the human lower esophageal sphincter.Methods:1 Thirty-one patients undergoing subtotal esophagectomy for high and middle thoracic esophageal carcinoma in the Fourth Hospital of Hebei Medical University during March,2009 to February,2010 were selected in this study. Fresh specimens were obtained from the gastroesophageal junction in the operating room. After sharpe stripped mucosa and submucosa of cardia and lower esophagus, the preparation of muscle strips of sling fibers (S), clasp fibers (C), esophageal circular muscle (E ) and gastric circular muscle (G) was easy to be done.2 Cell suspension could be prepared by digestion of the sling fiber (S) and clasp fiber (C) strips. After hatched into Fluo-3/AM, each kinds of cell suspension should be divided into two parts with equal content and be held on the central slide of Confocal dishes. Cells of the experimental groups was hatched by adding a certain amount of saponin solution and heparin solution compared with the same dose of calcium-free buffer and heparin solution for the control groups. After scanning the fluorescence intensity of intracellular calcium by confocal microscope, a certain amount of Ach solution was added into the dishes. Five seconds later, fluorescence intensity of intracellular calcium in the same cell was scanned and recorded.3 Protein could be obtained by membrane and cytoplasmic protein extraction kit, and then it was quantitated by BCA and adjusted into the identical concentration. After electrophoresis, protein could be isolated by different molecular weight, then detection of the protein could be operated by IP3R antibodies in the darkroom via light-emitting and light-detecting after trarsmembrane. IOD should be recorded and analyzed for the expression of IP3R subtypes.Results:1 Cell fluorescence intensity increase was not obvious after the addition of Ach in experimental group by laser scanning confocal microscope imaging(S 8.29±5.03,C 12.98±6.24), but it was significantly increased in the control group after addition of Ach(S 37.50±15.03,C 28.49±6.57). There was a significant difference and statistically significance between the two groups of the sling and clasp fiber cells (t = 11.944, P = 0.00).2 There were at least two kinds of IP3R subtypes expressed in cells: one was IP3R 1, it imaged in the membrane and cytoplasmic protein, and the IOD in the four parts were C 9118.11±4733.73, S 8665.15±3197.56, E 8597.43±3624.52, G 9013.00±5058.29; the other was highly probable for IP3R 3, it mainly imaged in the cytoplasmic protein and the IOD in the four parts were C 20686.58±9478.28, S 20016.14±7764.18, E 11200.29±10381.24, G 11817.99±10498.55; expression levels of these two proteins were statistically different in the clasp fibers (C) and sling fibers (S) (FC = 16.543, FS = 29.628, p = 0.00), but there was no significant difference in esophageal smooth muscle (E) and gastric smooth muscle (G) (FE = 1.139, p = 0.309; FG = 1.105, p = 0.336).Conclusions:1 The specific antagonist of IP3R, heparin, could reduce the ascended intracellular calcium concentration which Ach-induced in the sling and clasp fibers of LES, and on the other hand it proved the presence of IP3R in the LES and its function is to affect the concentration of intracellular calcium.2 At least two kinds of IP3R subtype express in sling and clasp fibers: IP3R 1 exists in the membrane and cytoplasm but the concentration is relatively lower. By contrast, IP3R 3, exists in cytoplasm with a higher concentratrion. The concentration of the two subtyps of IP3Rs is higher in clasp fibers than in sling fibers.
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