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

Cell Calcium. 2004 Apr;35(4):317-31. doi: 10.1016/j.ceca.2003.09.004.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aequorin / metabolism*
  • Animals
  • Ca(2+) Mg(2+)-ATPase / physiology*
  • Calcium / metabolism*
  • Calcium Channel Agonists / pharmacology
  • Calcium Channel Blockers / pharmacology
  • Calcium Channels / metabolism*
  • Carcinoma, Hepatocellular / metabolism*
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism
  • Drug Delivery Systems
  • Endoplasmic Reticulum / physiology*
  • Ethylenediamines / pharmacology
  • Fura-2 / metabolism
  • Hydroquinones / pharmacology
  • Liver / cytology
  • Liver / metabolism*
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Rats
  • Thapsigargin / pharmacology


  • Calcium Channel Agonists
  • Calcium Channel Blockers
  • Calcium Channels
  • Ethylenediamines
  • Hydroquinones
  • 2,5-di-tert-butylhydroquinone
  • Aequorin
  • Thapsigargin
  • Ca(2+) Mg(2+)-ATPase
  • N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
  • Calcium
  • Fura-2