FREE [CA2-SENSITIVE STORES OF SINGLE LIVING INTACT-CELLS - A NEW LOOKAT THE REFILLING PROCESS(] DYNAMICS MEASURED IN AGONIST)

Free [Ca2+] in agonist-sensitive internal stores of single intact cell s was measured in situ in order to examine the role of [Ca2+] in modul ating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such tha t >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal stor e. The rates of store reloading following stimulation by 100 nM bradyk inin were essentially unchanged when cytosolic [Ca2+] was clamped to r esting values with BAPTA-AM. In control cells, recharging of stores to tally depended on the presence of external Ca2+, but pre-loading the c ells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-co ntaining external medium. Our results show: (i) Ca2+ stores normally a re recharged by Ca2+ which must first transit the cytoplasm; (ii) an e levation in cytoplasmic [Ca2+] is not required to replenish Ca2+ store s; (iii) the activation of the plasma membrane Ca2+ pump during the Ca 2+ spike ordinarily results in complete extrusion of released Ca2+; an d (iv) the buffering capacity of the cytoplasm is an essential compone nt of the store refilling process, An interesting finding was that acu te 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+].