[Problems on the determination of intracellular free calcium concentration when measured by fura-2/AM in mast cells].
When rat peritoneal mast cells which had been loaded with fluorescent Ca2+ indicator fura-2/AM were exposed to compound 48/80, fura-2 fluorescence was increased in the presence of 1mM extracellular Ca2+, but remained unchanged in the Ca2(+)-eliminated medium. Only in the presence of 1mM extracellular Ca2+, both fura-2 fluorescence and histamine release were increased in response to compound 48/80, depending on the concentration of the stimulant. Both of these responses were attenuated in the same degree by such release inhibitors as disodium cromoglycate and HSR-6071. Fluorescent microscopic observation of fura-2/AM-loaded mast cells revealed that fura-2 fluorescence was densely localized in the granules and the nucleus, but less in the cytoplasm. All these results strongly suggest that increase in the fura-2 fluorescence might not result from the increase in cytoplasmic free Ca2+ concentration, but from binding of fura-2, which had been released together with chemical mediators from granules following stimulation, to extracellular Ca2+. Therefore, it seems likely that the fura-2/AM loading method is inadequate to investigate whether or not the increase in cytoplasmic free Ca2+ concentration triggers release of chemical mediators from mast cells.
Ohashi
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Azuma
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D000818:Animals; D002118:Calcium / Q000032:analysis*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D008407:Mast Cells / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D051381:Rats
C049925:fura-2-am; D002118:Calcium; D016257:Fura-2