Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process

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Abstract
Free [Ca2+] in agonist‐sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK‐21 fibroblasts were loaded with the low‐affinity fluorescent calcium indicator mag‐fura‐2‐AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist‐ and thapsigargin‐sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA‐AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre‐loading the cells with BAPTA‐AM permitted efficient refilling in Ca2+‐free, EGTA‐containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA‐AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].