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.
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+ (Cao2+) 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 Cao2+-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 Cao2+-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 Cao2+-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.
authors.affiliation: Department of Biology, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Caroline
authors.initials: C
authors.lastname: Chan
authors.forename: M Lyn
authors.initials: ML
authors.lastname: Harland
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Webb
authors.forename: Jinglong
authors.initials: J
authors.lastname: Chen
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Miller
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Barritt
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D016503:Drug Delivery Systems; D004721:Endoplasmic Reticulum / Q000502:physiology*; D005029:Ethylenediamines / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D006873:Hydroquinones / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005029:Ethylenediamines; D006873:Hydroquinones; C019359:2,5-di-tert-butylhydroquinone; D000331:Aequorin; D019284:Thapsigargin; D017301:Ca(2+) Mg(2+)-ATPase; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium; D016257:Fura-2