The Research Of Calcium Channel Application Of The Patch-clamp Technique To The Human Lower Esophageal Sphincter

Objective: The existence and structural equivalent of the lower esophageal sphincter (LES) in human beings have been a matter of speculation for many years. After studying the anatomy of 32 cadavers, 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 and clasp fibers. LES clasp fibers is a semi-circular in the right side, while sling fibers is U-shaped band hoisted in the gastroesophageal junction. Both muscle muscles maintain the closure status of the LES, and form a high-pressure zone (HPZ, 15³0mmHg). This theory laid the foundation for further studies on the physiology, pathology and pharmacology of the LES. From then on, some reseachers have inveinvestigated the physiology and regulation mechanism of LES from different animals, and found that LES had differnt property different from other smooth muscle structure. And more importantly, there are also great differences in the sling fibers and clasp fibers. Therefore, the abnormal of LES structure and function may be the pathological basis of a variety of esophageal diseases. It has been demonstrated that the liberation of intracellular Ca²⁺ is important in the smooth muscle contraction process, just like"the second messenger". The contaction of smooth muscle cells depends on intracellular concentration of Ca²⁺. High concentration of Ca²⁺ causes the smooth muscle contraction, low concentration of Ca²⁺ causes sdiastole. Ca²⁺ generating smooth muscle contraction is from extracellular fluid Ca²⁺ influx and the release of the intracellular Ca²⁺ (mainly from sarcoplasmic reticulum, SR). The extracellular fluid Ca²⁺ influx through the protoplasmic membrane calcium ion channel, and the release of the Ca²⁺ from intracellular stores also passes the calcium ion channel. The calcium ion channel exists in all the excitatory cells as well as some non-excitatory cells and even the nucleus biology. Similar to the skeletal muscle and the cardiac muscle, the calcium ion channel is playing the pivotal role in contraction and diastole of the smooth muscle cells. And L-calcium channel in the mebrane of the smooth muscle cells has been shown to affect excited-contraction pair. When depolarization of smooth muscle cell membrane, Ca²⁺ flows through the L-calcium channel is a triggering factor generating smooth muscle cell contraction.This research using the patch clamp technique to investigate the types of the calcium ion channel in the human LES smooth muscle cells and their affecting factors. It can be imagined that the study on LES will be helpful in understanding the etiology, pathology and the choice of the treatment for esophageal motility disorders, and gastroesophageal reflux disease.Methods: Smooth muscles of the sling and clasp fibers were obtained from 22 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between January 2007 and December 2007, including 13 males and 9 females with a mean age of 57.3 years. Fresh specimens of EGJ was collected from operating room. Sharp dissection of mucosal and submucosal layers were performed. The muscle strips of sling and clasp fibers were seperated and then cut to small pieces of 2 mm3. Single cells was isolated using cell separation solution containing collagenase enzymeâ…¡and papain. After more than 95 percent viability of the cells was confirmed, the cells were resuspended Hepes buffer at 37℃for subsequent use.Cell viability determination: Five drops of cell suspention and 1 drop of 0.2% the trypan blue was mixed for 3 min. Dead cells were dyed red, and living cells colorless. The percentage of cell viability was calculated with the following formula: survival percentage (%)= number of viable cells /total number of cells (viable cell + dead cell)×100%.The cell launder was fixed on the mounting rack of inverted microscope, and then launder was filled with acutely separated smooth muscle cell suspention. After the cells adhered, the calcium electric current perfusate was used to fill the class. At last, the cells with bright and clean surface, neat edge, immobility, and good stereoscopic appearance was observed with the microscope. The recording electrode was made from rough glass by glass-level controller (Sutter P-97, USA). After filled sufficient calcium ion electrode fluid, impedance of the electrode was 2³M?. The cells were approached by the electrode using three dimensional hydraulic pressure micro-manipulator. The negative pressure of the electrode brought about sealing-in impedance more than 1G?, broke the cell membrane, compensated the capacitive current and the electrode series resistance, which formed the entire cell record pattern. The impulse signal was controlled by pulse + pulsefit software (HEKA, version 8.53, Pfalz, Germany). The channel signal was amplified through the EPC-9 patch clamp amplifier (HEKA, Pfalz, Germany), and conducted to the cell through the Ag-AgCl electrode and the micro electrode packing with fluid. The electric current signal was transferred to pulse signal by EPC-9, collected and analysed by pulse + pulsefit software using the Origin 7.5 to fit the data. All experiments were carried out at room temperature (25℃). The obtained data were expressed as mean±standard deviation, and student t test was usd to analyse the data with SPSS 13.0 software package. The difference was considered as significance if P value was less than 0.05.Results: 1 The shape of singal LES cellWe found that fresh LES cells were spindle and varied length with the inverted microscope. In Hepes buffer solution, some cells were in condition of contraction and others in relaxation, both have a central core (Fig.2³). The average length of sling muscle cells was 101.1±27.2μm (51¹48μm,n=200), and the clasp was 99.5±23.6μm(48～154μm,n=200). There was no significan ddifferences in cell length between the two kinds of muscle cells P>0.05(t=0.247, P=0.676).2 I-V curvesCells on holding potential -60 mV or -90 mV, use 10mV step stimulation depolarized from -80⁺10mV with a band width of 200ms. When 5¹3ms after the stimulation start was imposed, introverted electric current could be recorded in I-V curves. It reached the peak value at 0mV and turned over at approximately -50mV. The I-V curves for both were U-shaped, but the peak value is different. When holding potential was - 60mV, the introverted electric current was -5.58±1.53pA/pF (n=6 cells), compared to - 7.02±0.93pA/pF (n=6 cells), P<0.05(t=1.970, P=0.043) at the holding potential on -90mV. There were no significant differences in recorded electic current between sling and clasp muscle cells.3 The effects of Nifedipine to inward electric currentNifedipine could obviously suppress the introverted electric current on both holding potential of -60mV and -90mV, and 10μm Nifedipine nearly cancel the introverted electric current(-0.44±0.73pA/pF&-1.47±1.23 pA/pF,n=6 cells), P<0.05(t=7.427, P=0.000&t=8.816, P=0.000) compared to normal current. The same trend was found at 0.1, 1, 5μm.Conclusion:1. The shap and length of sling and clasp muscle cells have the similar shape and length.2. The introverted electric current of human LES cells was predominantly through the L calcium channel, and no other calcium channel is found in the formation of the introverted electric current of human.3. Nifedipine can obviously suppress the introverted electric current, and hypersensitivity to the dihydropyridines medicine such as Nifedipine is one of characteristics of L calcium channel.