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Title: Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2+ + Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells
Authors: Chan, Caroline
Harland, M. Lyn
Webb, Sarah E.
Chen, Jinglong
Miller, Andrew L.
Barritt, Greg J.
Keywords: Endoplasmic reticulum
Store-operated Ca2+ channels
(Ca2+ + Mg2+)ATP-ases
Targeted aequorins
Liver cells
Issue Date: Apr-2004
Citation: Cell calcium, vol. 35, iss. 4, April, 2004, p. 317-331
Abstract: The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2+ + Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Ca2+o) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Ca2+o-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Ca2+o-induced [Ca2+]cyt spike was: inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Ca2+o-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2+ + Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.
Rights: Cell calcium © copyright (2004) Elsevier. The Journal's web site is located at
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