Patch-clamp Technique on Human Lower Esophageal Sphincter L-type Calcium Ion Channel Research
|School||Hebei Medical University|
|Keywords||The lower esophageal sphincter Clasp fibers Sling fibers Patch-clamp Calcium channel Acetylcholine|
Objective: 1979 the Liebermann-Meffer scholars through the anatomy of the specimens of 32 cadaver esophagogastric junction (esophagogastric junction, EGJ), which elaborated on the general arrangement of the esophagogastric junction smooth muscle bundles, and thus human esophageal sphincter (lower esophageal sphincter, LES) is a common form of this theory by the hooked the fiber (CLASP fiber) and sling fibers (Sling fiber). Liebermann-Meffert and other scholars believe that the two fibers together to maintain the LES resting high pressure, to form the LES high pressure zone (high-pressure zone, HPZ, 15 ~ 30 mmHg). This discovery laid the anatomical basis for the further LES pathological, pharmacological and physiological characteristics. Subsequent scholars regulatory mechanisms and physiological characteristics of different animals LES function more in-depth study. Has been recognized, smooth muscle intracellular free Ca2 plays an extremely important role of the \Changes in the concentration of intracellular free calcium intake of Ca2 across the cell membrane transport and the calcium intracellular storage pool (sarcoplasmic reticulum, mitochondria, endoplasmic network, etc.), the release of the result of changes in dynamic equilibrium process of calcium. The contractile activity of smooth muscle cells is dependent on the cytoplasmic Ca2 concentration, a low concentration of Ca2 cause smooth muscle relaxation, the high concentration of Ca2 induced smooth muscle contraction. Ca2 caused by smooth muscle contraction comes mainly from the sarcoplasmic reticulum cells and extracellular fluid Ca2. Is generally believed that the transmembrane extracellular calcium into the cell triggers intracellular calcium release, intracellular Ca2 increase was mainly depends on the release of intracellular calcium. Extracellular Ca2 through voltage-dependent calcium channels (voltage dependent calcium channel, VDCC) on the cell membrane into the cell. L-type, T-type and N-type voltage-dependent calcium channels can be divided into subtypes. Mainly in the smooth muscle cells, both L-type and T-type voltage-gated calcium channels. With the L-type calcium channel is different is that the density of T-type calcium channels in cell growth, the T-type calcium channels in normal circumstances the body smooth muscle cells express only a very small amount of. L-type calcium channels have the extracellular calcium concentration-dependent manner, and the L-type calcium channel current increases with increasing extracellular calcium concentration. Calcium channel is prevalent in all the excitement as well as some non-excitable cells and prokaryotic cell membrane. The mechanism of myocardial contraction and relaxation of skeletal muscle, similar to the L-type calcium channel plays a key role in the smooth muscle cell contraction and relaxation mechanism. When the smooth muscle cell membrane depolarization into the intracellular Ca2 through L-type calcium channel is the main triggers contraction of smooth muscle cells. 1976 Neher and Sakmann the patch-clamp technique (patch Clamp Recording Technique), the patch-clamp technique is a record ion flow through ion channels to reflect on the cell membrane a single or a majority of the technology of the molecular activity of ion channels. Since the 1980s began to be used ion channels, not only from the molecular level to understand the dynamics of cell single ion channels open or closed, the membrane select transparent and excitement of providing a direct means, but also from the ion channel level to clarify the mechanism and the mechanism of drug action of certain diseases and provides a direct means of detection. In this study, cell patch clamp technique to analyze the type of human LES smooth muscle cell membrane calcium channels as well as common influencing factors and dive into the sling fibers and the clasp fibers may have different biological characteristics and internal adjustment mechanism. Can be expected, electrophysiological studies will help LES achalasia of the cardia, the lower esophageal sphincter pressure psychosis esophageal motility disorders, and gastroesophageal reflux disease (gastroesophageal reflux disease GERD) and other benign esophageal disease etiology Improved understanding and treatment methods. Methods: 25 cases from November 2008 to December 2009, the Fourth Hospital of Hebei Medical University, due to esophageal cancer esophageal gastrectomy patients. 17 male, 8 females, mean age 54.2 years. Fresh surgical specimens taken from the operating room, after sharp dissection cardia and mucous layer and submucosa of the lower esophagus, clipping sling fibers and clasp fibers muscle strips and muscle strips cut into volume of 2mm3 small, containing collagenase II and papain cell separation medium single smooth muscle cells. Verify that the cell survival rate is above 95%. And then stored at 37 ° C HEPES buffer set aside. Determination of cell viability: pipette 5 drops of cell suspension, add 1 drop of 0.2% trypan blue solution, and the count in less than 3 minutes after mixing, the dead cells will be stained red, while still living cells able to maintain a colorless state. Cell viability formula: survival rate (%) = the total number of live cells / (number of viable cells of dead cells) × 100%. The cell perfusion slot in the inverted microscope stage, to learn a few drops of smooth muscle cells in suspension drops in perfusion tank stand for 5 minutes, until the adherent cells after perfusion and perfusion corresponding to the calcium current, flush out the death suspended cells, select the mural, the surface smooth, clean, neat edge, stationary, three-dimensional sense of strong cells for the study. Electrode from hard glass blank by the level puller (Sutter P-97, USA) in four steps drawn from, filled with calcium in the pipette solution impedance of 2 to 3MΩ. Three-dimensional hydraulic micromanipulator movable electrode and close to the selected cell sealing. After sealing, the negative pressure caused by suction attracted so that the tip of the electrode within the cell membrane rupture pipette electrode and intracellular liquid pass between the electrode and membrane is formed 1G? The above high impedance sealing, adjusting fast compensation to offset the capacitance of the electrode holder and the electrode wall suction break the cell membrane, and then applied to the electrodes, see capacitor discharge increases suddenly and accompanied by noise increases the formation of the whole-cell recording mode, adjust the slow capacitance compensation and series compensation in order to reduce the instantaneous charge and discharge current, and a clamping error. Pulse signal by the pulse pulsefit software (HEKA, version8.53, Pfalz, Germany) controlled channel signal is amplified with the EPC-9 patch clamp amplifier (HEKA, Pfalz, Germany), Ag-AgCl electrode wire and The filled pipette electrode microelectrode introduced into a cell, the current signal generated by the EPC-9 conversion, PULSE pulsefit software collection, analysis, and collected data using Origin7.5 fitting. All experiments were performed at room temperature (25 ° C). The resulting data were expressed as mean ± standard deviation, the two groups were compared using t-test was used for statistical analysis SPSS14.0 software (SPSS Inc., Chicago, USA) is completed. To P LT; 0.05 for the difference statistically significant. Results: 1 lower esophageal sphincter cell shape was observed under an inverted microscope, freshly isolated lower esophageal sphincter the lasso and clasp fibers smooth muscle cells showed a spindle-shaped, large and small, of different lengths. In Hepes buffer, placed in a high-powered microscope, the diastolic part of the cell, some of the cells at different levels of shrinkage condition. The lasso and clasp fibers smooth muscle cells only one central nucleolus. The average length of the lasso fibroblasts as 99.8 ± 25.4μm (53 to 151μm, n = 200); the clasp fibers smooth muscle cells the average length of 100.5 ± 26.9μm (45 to 143μm, n = 200). The comparison of the two was not statistically significant (t = 0.244, P = 0.676). 2 IV curve of the selected cells were clamped at-90mV and-60mV from-80mV to 30mV 10mV step, the schedule 200ms stimulate cell depolarization. At this point can be recorded to an inward current at approximately-50mV reverse. Two different clamping voltage IV curve showed a U-shaped, but the peak in the inward current -7.11 ± 1.01pA/Pf holding potential of-90mV (n = 6). Holding potential of-60mV the -5.75 ± 1.49pA/Pf (n = 6) were significantly lower than the clamping voltage of-90mV within the current -7.11 ± 1.01pA/Pf (n = 6). The comparison of the two was statistically significant (t = 2.142, P = 0.041). And sling fibers of smooth muscle cells and clasp fibers smooth muscle cells recorded calcium current is not significantly different. 3 of acetylcholine on calcium inward currents affect acetylcholine can significantly activate L-type calcium channels, causing calcium current peak increases. Add 1μmmol / L acetylcholine clamping voltage of-60mV and-90mV peak were -9.21 ± 0.94pA/Pf (n = 6) and the -9.89 ± 1.32pA/Pf (n = 6). Normal peak current at a holding potential of-60mV and-90mV were -5.75 ± 1.49pA/Pf (n = 6) and -7.11 ± 1.01pA/Pf (n = 6). The comparison of the two, respectively (t = 8.142, P = 0.000) and (t = 8.851, P = 0.000). For 5μmmol / L and 10μmmol / L in the concentration of acetylcholine has the same trend. Conclusion: The hook-like fiber cells with lasso fiber cells size, shape substantially the same, we have not found that there are significant differences. 2 lower esophageal sphincter inward current through L-type calcium channels into the cell, L subtypes other subtypes not play a major role. 3 acetylcholine can extend the opening hours of the calcium channel open probability increases significantly enhanced calcium inward current.