Di Virgilio F, Steinberg T H, Swanson J A, Silverstein S C
Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032.
J Immunol. 1988 Feb 1;140(3):915-20.
Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.
以乙酰氧基甲酯形式载入J774.2巨噬细胞的Fura-2，在37℃载入开始后的90分钟内被隔离到细胞内液泡中。该染料也能有效地从细胞中分泌出来。Fura-2的隔离和分泌降低了该细胞系中胞质游离Ca2+浓度测量的准确性。当通过质膜的可逆通透作用将Fura-2以五钾盐形式载入细胞质时，J774.2细胞也会对其进行隔离和分泌。无论Fura-2载入细胞质的机制如何，2.5 mM丙磺舒（一种有机阴离子转运阻滞剂）均可阻止其隔离和分泌。丙磺舒对静息或受刺激的胞质游离Ca2+水平或FcR介导的吞噬作用均无影响。这些发现表明巨噬细胞表达了一种针对Fura-2阴离子形式的转运机制。该转运系统负责清除这种细胞类型细胞质中的Fura-2。此外，我们建议使用丙磺舒来阻断Fura-2的分泌和细胞内隔离，这可能会克服将这种Ca2+指示剂应用于巨噬细胞以及可能应用于其他细胞类型时出现的问题。
