Conrad G W, Rink T J
J Cell Biol. 1986 Aug;103(2):439-50. doi: 10.1083/jcb.103.2.439.
Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.
将来自经巯基乙酸盐刺激的小鼠的腹膜细胞接种到盖玻片上，使其贴壁2小时，形成一层密集的贴壁细胞单层，其中超过95%为巨噬细胞。在用喹2的四乙酰氧基甲酯孵育后，用无钙生理盐水冲洗盖玻片，将其以45度角放置在含有磁力搅拌棒的方形比色皿中，并在荧光分光光度计中分析喹2荧光的变化。这种荧光被视为细胞内钙离子浓度（[Ca2+]i）的指标，随着外源钙离子浓度（[Ca2+]o）升高至1 mM而增加。当[Ca2+]o约等于10 microM时，[Ca2+]i = 72±14 nM（n = 26）；当[Ca2+]o = 1 mM时，[Ca2+]i = 140 - 220 nM，该水平不会因N,N,N',N'-四（2-吡啶甲基）乙二胺（一种可淬灭喹2的重金属膜通透螯合剂）而升高。添加小鼠α+β成纤维细胞干扰素、脂多糖、凝血酶、胶原蛋白、血管加压素、ADP、化合物48/80或U46619不会改变[Ca2+]i。然而，如果[Ca2+]o = 1 mM，添加血小板活化因子（PAF）（2 - 20 ng/ml）可在1分钟内使[Ca2+]i升高480 nM。在存在5 mM乙二醇双四乙酸（EGTA）的情况下，PAF使[Ca2+]i升高25 nM。这表明PAF可导致外源Ca2+内流，同时也从细胞内储存中释放一些Ca2+。与这些结果一致，当在100 microM Cd2+或Mn2+存在下将PAF添加到1 mM Ca2+中以阻断Ca2+内流时，[Ca2+]i仅适度增加；在这种减弱的峰值反应时，通过用二乙烯三胺五乙酸螯合外源重金属，可在1分钟内将[Ca2+]i升高至正常PAF刺激水平。在吲哚美辛存在下观察到正常的PAF反应。观察到能升高[Ca2+]i的最低PAF剂量为0.1 ng/ml。[Ca2+]i对2 - 20 ng/ml PAF的反应是短暂的，再次应用无效果。在细胞悬液中也观察到了PAF反应。这些结果表明，[Ca2+]i的增加可能是PAF激活巨噬细胞的早期事件。
