Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.
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+].
Hofer
A M
AM
University of Padova, CNR Center for Biomembranes, Viale G.Colombo 3, I-35121 Padova, Italy.
Landolfi
B
B
Debellis
L
L
Pozzan
T
T
Curci
S
S
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006706:Homeostasis; D017136:Ion Transport / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C072323:Mag-Fura-2-acetoxymethyl ester; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2