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Measurement of Free [Ca2+] Changes in Agonist-Sensitive Internal Stores Using Compartmentalized Fluorescent Indicators

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The fact that acetoxymethyl (AM)-ester derivatives of fluorescent Ca2+ indicators accumulate not only in the cytoplasm but also in organelles was recognized long ago as a potential source of artifacts during measurements of cytoplasmic [Ca2+ ] (1 ,2 ). Later it was observed that high-affinity dyes such as fluo-3 and fura-2, normally saturated in the high-[Ca2+ ] environment of the agonist-sensitive Ca2+ store, could register [Ca2+ ] changes in this compartment under special conditions (e.g., when pools were already partially empty) (3 6 ). The propensity of indicators to become compartmentalized was fully exploited, however, when investigators began to use lower affinity probes such as magfura-2 (7 ), to monitor [Ca2+ ] changes in the inositol(1,4,5)trisphosphate [Ins(1,4,5)P3 ]-sensitive store (a compartment largely accepted to be the endo-plasmic reticulum [ER]). Thus, the release and reloading of this organelle with Ca2+ , as reported by ER-trapped dye, could be directly visualized in single permeabilized cells with high spatiotemporal resolution (8 ,9 ). This basic approach has been adopted by a number of laboratories to investigate phenomena ranging from Ca2+ oscillations (10 12 ) to quantal release (13 15 ), and the subcellular localization of Ca2+ storage sites (16 18 ).
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