Intracellular Ca2+ concentration and H2O2 production in mouse peritoneal macrophages are stimulated by platelet activating factor

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Abstract
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+, 10−7M 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 μM) increased H2O2 production in peritoneal macrophages in the presence of extracellular Ca2+, but H2O2 production was not observed in the absence of extracellular Ca2+.