Patch-clamp Technique On Human Lower Esophageal Sphincter L-type Calcium Ion Channel Research

Objective: In 1979, Liebermann-Meffert and colleagues demonstrated that human lower esophageal sphincter was composed of the clasp fibers and sling fibers through autopsy of 32 esophageal and gastric junctions (esophagus-gastric junction, EGJ). Physiological study has revealed that the two fibers formed a LES high-pressure zone (high-pressure zone, HPZ, 15 ~ 30mmHg) which maintained the resting LES pressure. This discovery set up the anatomical basis for the further study of LES pathology, pharmacology, and physiological characteristics. The studies of regulatory mechanisms and physiological characteristics of LES have been conducted worldwide, but most on animals.As well-known, intracellular free Ca2+ in smooth muscle contraction plays an extremely important "second messenger" role. Intracellular free calcium concentration changes depended on Ca2+ transport across the cell membrane and intracellular calcium storage pool (sarcoplasmic reticulum, mitochondria, endoplasmic reticulum, etc.), which formed the calcium homeostasis. Contraction of smooth muscle cells depends on the catabolic Ca2+ concentration; low concentrations of Ca2+ initiated smooth muscle relaxation, high concentrations of Ca2+ cause smooth muscle contraction. Smooth muscle contraction caused by Ca2+ mainly from intracellular sarcoplasmic reticulum and extracellular fluid Ca2+.It is generally believed that influx of extracellular calcium triggers the release of intracellular Ca2+. Influx of extracellular Ca2+ into the cell through voltage-dependent calcium channel (voltage dependent calcium channel, VDCC) which can be divided into L-, T-, N-type and other subtypes. There are mainly two kinds of L-and T-type voltage-gated calcium channels in the smooth muscle cells. Differing from the L-type calcium channel, T-type calcium channel density is related to the cell growth. Smooth muscle cells express a few T-type calcium channels in normal conditions. The current flow of the L type calcium channel currents was elevated with extracellular calcium concentration increase.Calcium channel exists in cell membrane of all excitement and some non-excitable cells, as well as prokaryotes. L-type calcium channel plays a key role in contraction and relaxation of smooth muscle cells, as in skeletal or myocardial muscles. When smooth muscle cell membrane deplored, the intracellular Ca2+ influx through L-type calcium ion channel which is the mainly trigger factor of the contraction of smooth muscle cells.In 1976, Neher and Sakmann established the patch-clamp technique to record the ionic currents passing the ion channel on the membrane which reflected the activity of a single or a majority of the ion channel. Since the 1980s the technique has been provided the measures not only to understand the ion channels on molecular level, but also to expound the pathogenesis and pathological effects on some diseases. In this study, the cell patch-clamp technique was used to analyze the calcium channel types on human LES and their affecting factors, and to further investigate of the biological characteristics and regulating mechanism of LES. It can be predicted that LES electrophysiological studies would be useful for further understanding and treatment of esophageal motor disorders and gastro-esophageal reflux disease (GERD).Methods: Twenty-five patients who underwent esophagectomy for middle esophagus esophageal carcinoma in the Fourth Hospital of Hebei Medical University from November 2008 to December 2009 were selected. There were 17 male and 8 female. And their average age was 54.2 years old. Fresh surgical specimen was taken from the operation room, and then the lower esophageal mucosal layer and submucosal layer were dissection to get sling fibers and clasp fibers. The sling fibers and clasp fibers were cut into pieces in volume 2mm3. The pieces were treated by collagenase II and papaya enzymes to obtain single smooth muscle cells, and then the cell viability was confirmed above 95%. Finally, the single smooth muscle cells were stored in 37℃Hepes buffer reserve for using.Determination of cell survival rate: A drop of 0.2% trypan blue solution was given to five drops of cells suspension. The number of cells which were still alive was counted within 3 minutes after mixing. The dead cells will be stained to red, while these other cells will maintain their colorless state. The cell viability formula: survival (%) = total number of living cells / (live cells + dead cells)×100%.The cells were fixed on the inverted microscope perfusion slot work table. A few drops of smooth muscle cell suspension drops were drew into the perfusion bath. After the cells adhered, the calcium electric current perfusion was used to fill the bath. Dead cells were washed away. The cells with bright and clean surface, neat edge, immobility, and good stereoscopic appearance were observed by the microscope. Electrode was made from hard glasses by glass-level controller (Sutter P-97, USA). After filled with calcium ion electrode liquid, the impedance of the electrode was 2~3M?. Three-dimensional hydraulic micro-manipulator was used to move the electrode to attach the cell. After the success of seal, the negative pressure of the electrode formed a high impedance seal more than 1G? between the electrode and the cell membrane. The capacitive current and the electrode were compensated, which formed the entire cell record pattern. The pulse signal was controlled by the pulse+ pulsefit software (HEKA, version8.53, Pfalz, Germany). The channel signal was amplified through the EPC-9 patch-clamp amplifier (HEKA, Pfalz, Germany), and conducted to the cell by the Ag-AgCl electrode wire and the microelectrode which filled with fluid. The resulting current signal through the EPC-9, was collected by the pulse+ pulsefit software, analysis, and then fitted by Origin7.5. All experiments were at room temperature (25℃) manner. The student t'test was used to analyze the data by SPSS14.0 (SPSS Inc. Chicago, USA). P <0.05 was considered for statistically significant difference.Results: 1 The shape of the lower esophageal sphincter cell.Observed by the inverted microscope, freshly isolated lower esophageal sphincter sling fibers and clasp fibers smooth muscle cells were spindle and varied length. In Hepes buffer solution, some cells were relaxation, and some cells were at different levels of contraction. Both of them have a nucleolus core. The average length of the sling fibers was 99.8±25.4μm (53 ~ 151μm , n=200cells). The clasp cells were in an average length of 100.5±26.9μm (45~143μm, n = 200cells). Comparison of the two was not statistically significant (t = 0.244, P = 0.676).2 I_V curveThe selected cells on holding potential -90mV or -60mV were stimulated by 10mV step depolarized from -80mV to +30 mV, with a band width of 200ms. At this point, an inward current reversed can be recorded at about -50mV. The I-V curves for both were U-shaped, but the peak value was different. When holding potential was at -90mV, the introverted electric current was -7.11±1.01pA/Pf (n=6 cells). And at -60mV, it was -5.75±1.49pA/Pf (n=6 cells). Comparison of the two was statistically significant (t=2.142, P=0.052). Sling fibers and clasp fibers cells recorded calcium currents did not find significantly difference.3 Acetylcholine on the calcium inward currentAcetylcholine can significantly activate the L-type calcium channels, and cause increased peak calcium current. With 1 mmol/L concentration of acetylcholine added, the peak of the introverted electric current on holding potential of -90mV and -60mV were -9.89±1.32pA/Pf (n=6 cells) and -9.21±0.94pA/Pf (n = 6cells). And normal current peaks in the clamp voltage of -90mV and -60mV were -7.11±1.49pA/Pf (n = 6 cells) and -5.75±1.49pA/Pf (n=6). Comparison of the two was (t=8.142, P=0.000), and (t=8.851, P = 0.000).Conclusion:1 Clasp and sling fibers cells, the size and the shape are basically the same, found no significant difference. 2 The inward current is mainly through L-type calcium ion channels into cells of lower esophageal sphincter; in addition, the other subtypes can not afford a major role.3 With acetylcholine added, calcium channel can extend it's opening hours, open probability, and calcium inward current can be markedly enhanced.