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Measurement of ATP released from a single cell and its application

单细胞释放ATP的测定及其应用

基本信息

批准号：
07556118
负责人：
NAKAZATO Yoshikazu
金额：
$ 2.3万
依托单位：
HOKKAIDO UNlVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556118/
关键词：
ATP secretion catecholamine secretion isolated adrenal chromaffin cell firefly luminescent enzyme electrochemical detector carbon electrode on-line measurement whole-cell voltage clamp 蛍光測定ニュートラルレッド SBFI 光電子増倍管非選択性陽イオンチャネル

项目摘要

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.

1)ATP引起豚鼠肾上腺嗜铬细胞内向电流、细胞内Ca^2+浓度和儿茶酚胺分泌增加。另一方面，它通过G蛋白激活抑制电压依赖性Ca^2+电流。（2）豚鼠肾上腺嗜铬细胞存在N、L、P/Q和R型Ca^2+通道，猪肾上腺嗜铬细胞存在N、L和P/Q型Ca ^2+通道，但不存在R型Ca^2+通道。3)通过ATP光度计连续监测ATP光致发光。将培养在盖玻片上的猪肾上腺嗜铬细胞置于0.5ml的小室中，以2 ml/min的流速灌流，流出液分为两部分，一部分用于ATP光度计测定ATP，另一部分用于电化学检测器测定儿茶酚胺。乙酰胆碱、60 mMKC_1和5 mMBaCl_2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分泌的时程与ATP分泌的时程一致。流出液中出现的儿茶酚胺与ATP的摩尔比为7- 12。4）用HPLC测定的腺嘌呤核苷酸（ATP、ADP、AMP）的含量在流出液中几乎相同，并且流出液中的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)用微碳电极与膜片钳放大器连接，测定嗜铬细胞中儿茶酚胺的量子释放。乙酰胆碱引起的碳电极表面附近的儿茶酚胺的氧化负责的尖峰电流的频率的剂量依赖性增加。6)我们可以检测到从单个肾上腺嗜铬细胞分泌的儿茶酚胺，但没有ATP的分泌。