平滑肌细胞钙钳制技术

Calcium Clamping of Smooth Muscle Cell

用金黄色葡萄球菌α毒素（α－ｔｏｘｉｎ）或β溶血皂素（β－ｅｓｃｉｎ）处理平滑肌小肌条，可将细胞膜穿通许多小孔，使膜对某些离子及物质的屏障作用消失，但不影响受体－Ｇ蛋白－第二信使系统的耦联。根据ｃａ￣（２＋）ＥＧＴＡ的平衡结合常数Ｋ进行计算，将高浓度（１０ｍｍｏｌ／Ｌ）ＥＧＴＡ和具一定Ｃａ￣（２＋）浓度的溶液按一定比例混合，可获得所需Ｃａ￣（２＋）浓度的缓冲溶液。后者通过膜小孔进入细胞内可将胞浆游离Ｃａ￣（２＋）浓度“钳制”（ｃｌａｍｐ）在某一要求的水平上。在该模型上可方便地观察一些不依赖胞浆游离Ｇａ￣（２＋）浓度变化的细胞活动过程。该法最适宜于研究激动剂对平滑肌的钙增敏作用及其机制。如适当良，亦可用于心肌、骨骼肌及其它细胞，对研究细胞内信号传导、药物作用机理等方面，有独到的优点。

he permeabilized smooth muscle can be obtained by using staphylococcal α-toxin or β-escinwithout disrupting the agonist-receptor-messenger coupling. The buffer solution which have a de-sired Ca￣(2+)concentration can be made up by mixing 10mmol/L EGTA with a certain Ca￣(2+)-con-taining solution according to the Ca￣(2+)-EGTA stability constant K.The cytoplasmic Ca￣(2+)concen-tration can be clamped to a desired level by the influx of the buffer solution into the cytoplasm.Itis helpful by using such a technique to identify any agonist-initiated mechanism that may modu-late contraction without a change in cytoplasmic Ca￣(2+).This method can also be used on non-smooth muscle preparations only through a little modification.It is a simple and valuable tech-nique for studying cellular signal transduction and the mechanism of drug effect,for it provides aeasy way to conduct drugs into the cytoplasm.