Measurement of ATP released from a single cell and its application

1)ATP可引起豚鼠肾上腺嗜铬细胞内向电流、细胞内钙离子浓度和儿茶酚胺分泌量的增加。另一方面，它通过激活G蛋白抑制电压依赖性钙电流。2)豚鼠肾上腺嗜铬细胞存在N、L、P/Q和R型钙通道，培养的猪肾上腺嗜铬细胞存在N、L和P/Q通道，但不存在R型钙通道。3)用三磷酸腺苷光度计连续监测三磷酸腺苷的光致发光。三磷酸腺苷的检出限为L-2nM。将培养在盖玻片上的猪肾上腺嗜铬细胞放入0.5ml的小室中，以2ml/min的流速灌流。将出水分成两组，一组用于ATP光度计测三磷酸腺苷，另一组用于电化学检测器连续测定儿茶酚胺。乙酰胆碱、60mMKC1和5mMBaCl2可引起儿茶酚胺和ATP的释放增加，这些刺激引起的儿茶酚胺的分泌与ATP的分泌的时程是一致的。出水中儿茶酚胺与三磷酸腺苷的摩尔比为7~12.4)，用高效液相色谱法测定出水中腺嘌呤核苷酸(ATP、ADP、AMP)的含量基本相同，出水中儿茶酚胺与腺嘌呤核苷酸的摩尔比为4~5。这些值类似于嗜铬颗粒中存在的摩尔比。5)用膜片钳放大器连接的微碳电极测量了嗜铬细胞中儿茶酚胺的定量释放。乙酰胆碱引起碳电极表面附近儿茶酚胺氧化的尖峰电流频率随剂量的增加而增加。6)单个肾上腺嗜铬细胞可检测到儿茶酚胺的分泌，但未检测到ATP的分泌。

1) ATP caused increases in inward currents, intracellular Ca^<2+> concentration and catecholamine secretion in adrenal chromaffin cells of the guinea-pig. On the other hand, it inhibited voltage-dependent Ca^<2+> currents through G protein activation. 2)N,L,P/Q and R types of Ca^<2+> channels were present in the adrenal chromaffin cell of the guinea-pig and N,L and P/Q but not R types of Ca^<2+> channels in the cultured chromaffin cell of the pig. 3) ATP photoluminescence was continuously monitored by an ATP photometer. The detection limit for ATP was l-2nM.The pig adrenal chromaffin cells cultured on coverslips were put in a chamber (0.5ml) and superfused at a flow rate of 2ml/min. The effluent was divided into two, the one was applied to the ATP photometer to measure ATP and the other to an electrochemical detector to measure catecholamine continuously. Acetylcholine, 60mMKC1 and 5mMBaCl_2 caused increases in the release of catecholamine and ATP.The time courses of catecholamine secretion induced by these stimuli were consistent with those of ATP secretion. The molar ratio of catecholamine to ATP appearing in the effluent was 7-12.4) The content of adenine nucleotides (ATP,ADP,AMP) measured with HPLC was almost the same in the effluent, and the molar ratio of catecholamine to adenine nucleotides in the effluent was 4-5. These values resembled the molar ratio present in the chromaffin granules. 5) The quantal release of catecholamine from a chromaffin cell was measured with a micro-carbon electrode in connection with a patch clamp amplifier. Acetylcholine elicited a dose-dependent increase in the frequency of spike currents responsible for the oxidation of catecholamine near the surface of the carbon electrode. 6) We could detect catecholamine secretion from a single adrenal chromaffin cell but not ATP secretion so far.