Sasaki M, Maeyama K, Watanabe T
Department of Pharmacology I, Tohoku University School of Medicine, Miyagi, Japan.
Lipids. 1991 Dec;26(12):1209-13. doi: 10.1007/BF02536533.
The intracellular calcium concentration ([Ca2+]i) in mouse peritoneal macrophages cultured on a coverglass was measured in the superfusion system using fura-2 as a fluorescent calcium probe and platelet activating factor (PAF) as a stimulant. In the presence of extracellular Ca2+, 10(-7) M PAF sharply increased [Ca2+]i from a basal level of 90 nM to 340 nM. Thereafter, the [Ca2+]i level gradually decreased in two phases, in an initial rapid phase and a subsequent slow phase of decrease. The calcium response was dependent on the PAF concentration. In the absence of extracellular Ca2+, a single sharp peak was observed, suggesting two different modes of Ca2+ movement, one from intracellular stores and the other from the extracellular medium. A simple, sensitive fluorometric assay system was developed for measuring H2O2 in the superfusate of macrophages after stimulation by use of immobilized peroxidase and 3-(p-hydroxyphenyl)propionic acid as a fluorogenic substrate. With this system, as little as 2 pmol of H2O2 could be measured. PAF (1 microM) increased H2O2 production in peritoneal macrophages in the presence of extracellular Ca2+, but H2O2 production was not observed in the absence of extracellular Ca2+.
在灌注系统中，以fura-2作为荧光钙探针，血小板活化因子（PAF）作为刺激剂，测量了培养在盖玻片上的小鼠腹腔巨噬细胞内的钙浓度（[Ca2+]i）。在细胞外Ca2+存在的情况下，10(-7) M的PAF可使[Ca2+]i从90 nM的基础水平急剧增加到340 nM。此后，[Ca2+]i水平分两个阶段逐渐下降，即初始的快速下降阶段和随后的缓慢下降阶段。钙反应取决于PAF的浓度。在无细胞外Ca2+的情况下，观察到一个单一的尖锐峰值，表明Ca2+有两种不同的移动模式，一种来自细胞内储存库，另一种来自细胞外介质。开发了一种简单、灵敏的荧光测定系统，用于在巨噬细胞受到刺激后，利用固定化过氧化物酶和3-（对羟基苯基）丙酸作为荧光底物，测量其灌注液中的H2O2。使用该系统，可检测到低至2 pmol的H2O2。在细胞外Ca2+存在的情况下，1 microM的PAF可增加腹腔巨噬细胞中H2O2的产生，但在无细胞外Ca2+的情况下未观察到H2O2的产生。
