Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its...

Cell Calcium 35 (2004) 317–331

Evaluation, using targeted aequorins, of the roles of the endoplasmicreticulum and its (Ca2+ + Mg2+)ATP-ases in the activation of

store-operated Ca2+ channels in liver cells

Caroline Chana, M. Lyn Harlandb, Sarah E. Webba, Jinglong Chenb,Andrew L. Millera, Greg J. Barrittb,∗

a Department of Biology, The Hong Kong University of Science& Technology, Clear Water Bay, Kowloon, Hong Kong, Chinab Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, SA 5001, Australia

Received 23 December 2002; received in revised form 11 August 2003; accepted 5 September 2003

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 withapo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells werepre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition ofextracellular Ca2+ (Ca2+

o ) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated witha sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Ca2+

o -induced [Ca2+]cyt spike) afterwhich [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 ionomycinand the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQcaused a detectable inhibition or change in shape of the Ca2+

o -induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibitedthe inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanidem-chlorophenyl-hydrazone (CCCP) indicatedthat thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin werecompared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in whichaequorin 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 isnot associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellularCa2+ store), which contains thapsigargin-insensitive (Ca2+ + Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.© 2003 Elsevier Ltd. All rights reserved.

Keywords:Endoplasmic reticulum; Store-operated Ca2+ channels; (Ca2+ + Mg2+)ATP-ases; Targeted aequorins; Liver cells

1. Introduction

Store-operated Ca2+ channels (SOCs) are present in mostnon-excitable animal cells[1–3]. Their physiological role is

Abbreviations: ER, endoplasmic reticulum; SERCA, sarcoplasmicreticulum (Ca2+ +Mg2+)ATP-ase; cytAEQ, aequorin targeted to the cyto-plasmic space; erAEQ, aequorin targeted to the ER; [Ca2+]cyt, the concen-tration of free Ca2+ in the cytoplasmic space; [Ca2+]er, the concentrationof free Ca2+ in the ER; Ca2+

o , extracellular Ca2+; KRB, Krebs–Ringerbicarbonate buffer; DBHQ, 2,5-di-tert-butylhydroquinone; BAPTA,1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; CCCP, car-bonyl cyanidem-chlorophenyl-hydrazone; PBS, phosphate buffered saline;HA1, haemaglutinin; TPEN, tetrakis-(2-pyridymethyl) ethylenediamine

∗ Corresponding author. Tel.:+61-8-8204-4260; fax:+61-8-8374-0139.E-mail address:[email protected] (G.J. Barritt).

thought to be to replenish the endoplasmic reticulum (ER)Ca2+ stores during and after the action of agonists whichinduce the release of Ca2+ from the ER through InsP3and ryanodine receptors[1–3]. The activation of SOCs isinitiated by a decrease in Ca2+ in the ER [1–3]. Underphysiological conditions, this decrease is caused by the ac-tions of InsP3 and Ca2+ in the cytoplasmic space. How-ever, it can be achieved artificially by inhibitors of the ER(Ca2+ + Mg2+)ATP-ase (SERCA) such as thapsigargin and2,5-di-tert-butylhydroquinone (DBHQ)[1,3]. There is evi-dence for several sub-types of SOCs, which can be distin-guished on the basis of their different selectivities for Ca2+compared with Na+ [3]. The SOCs most extensively charac-terised are the Ca2+ release-activated Ca2+ channels (CRACchannels) present in mast cells and lymphocytes[4]. One

0143-4160/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.ceca.2003.09.004

318 C. Chan et al. / Cell Calcium 35 (2004) 317–331

Fig. 1. A schematic representation of the proposed relationship between store-operated Ca2+ channels (SOCs) in the plasma membrane, the endoplasmicreticulum, and the SERCAs. The locations of aequorin targeted to the cytoplasmic space (cytAEQ) and ER (erAEQ) are indicated. Also shown are twoproposed sub-regions of the ER, the “small ER Ca2+ stores” (proposed to be located in close proximity to the SOCs in the plasma membrane and tocontain thapsigargin-insensitive SERCAs) and the “bulk ER Ca2+ stores” (containing thapsigargin-sensitive SERCAs). It is proposed that Ca2+ diffusesslowly between these two types of ER Ca2+ store, and that the activation of SOCs requires a decrease in [Ca2+]er in the “small ER Ca2+ stores”. Thespace between the SOCs and the “small ER Ca2+ stores” is designated the “subplasmalemmal space”, and the majority of the cytoplasmic space “thedeep cytoplasmic space”. The broken lines and arrows indicate that Ca2+ that enters the subplasmalemmal space can diffuse into the deep cytoplasmicspace or be transported into the ER and mitochondria. The pathway will depend on the activity of the SERCAs, the local concentrations of Ca2+ andthe location of the mitochondria. Previous studies by others upon which the scheme is based are summarised in[14] and [51–53].

feature of CRAC channels is their strong inhibition by in-creased concentrations of Ca2+ at the cytoplasmic mouth ofthe channel[4,5].

Ca2+ inflow through SOCs in hepatocytes and liver celllines has chiefly been characterised using fluorescent Ca2+sensors and patch-clamp recording techniques[6,7]. Recentstudies with H4-IIE cells (a liver cell line derived from therat Reuber hepatoma) have shown that the principal type ofSOC has the same high Ca2+ selectivity and other propertiesas CRAC channels. It was concluded that the properties ofthe principal SOCs in H4-IIE cells are indistinguishable fromthose of the CRAC channels in mast cells and lymphocytes[7].

The mechanism of activation of SOCs has not yet beenelucidated, although numerous experiments have been con-ducted and several hypotheses proposed[1–4,8]. Someexperimental results indicate that pre-requisites for the ac-tivation of SOCs are (i) the location of regions of the ER

close to the plasma membrane, (ii) the maintenance of thenormal integrity of the actin cytoskeleton and ER[8,9], and(iii) the normal function of SERCAs[10–13]. There is alsosome evidence that the activation of SOCs and flow of Ca2+through the channels involves the movement of Ca2+ fromthe extracellular space to the ER through a small region ofthe cytoplasmic space, the subplasmalemmal space, withoutdiffusion to the bulk of the cytoplasmic space (reviewed in[14]) (shown schematically inFig. 1). Other experimentssuggest that the activation of SOCs may require the fusionof vesicles containing SOC proteins with the plasma mem-brane (reviewed in[2,8]). It is presently hypothesised thatthe mechanism of SOC activation involves a small moleculesuch as Ca2+ influx factor, conformation coupling, and/orinsertion of vesicles containing SOCs into the plasma mem-brane[8], or that activation is achieved simply by a decreasein the concentration of Ca2+ in the subplasmalemmal space,which de-inhibits the Ca2+-inhibitable SOCs[14].

C. Chan et al. / Cell Calcium 35 (2004) 317–331 319

The aim of the present experiments was to obtain a betterunderstanding of (i) the relationship between the concentra-tion of Ca2+ in the cytoplasmic space ([Ca2+]cyt) and theconcentration of Ca2+ in the lumen of the ER ([Ca2+]er)during the activation of SOCs and refilling of the ER, and (ii)the role of SERCAs in the activation mechanism. The exper-iments reported have been performed with H4-IIE liver cellsusing aequorin targeted to the cytoplasmic space (cytAEQ)or lumen of the ER (erAEQ) to measure changes in Ca2+ inthese intracellular locations. The results indicate that thereare significant quantitative differences between the changesin [Ca2+]cyt reported by aequorin, on the one hand, and flu-orescent probes on the other. These may be due, in part, todifferences in their Ca2+ buffering, in their affinity, and intheir dynamic range of response to Ca2+. It was also foundthat the changes in [Ca2+]cyt detected by aequorin locatedin the cytoplasmic space (a transient spike and subsequentlow plateau) associated with re-filling of the ER (i.e. whenCa2+

o is added to cells in which the ER has been depleted ofCa2+) are insensitive to inhibition by thapsigargin or DBHQ,whereas these agents inhibit the inflow of Ca2+ to the bulkof the ER. These observations suggest the possibility that asmall sub-region of the ER which possesses thapsigargin-and DBHQ-insensitive SERCAs is responsible for the acti-vation of SOCs in H4-IIE liver cells.

2. Materials and methods

2.1. Materials

Reagents were obtained from the following sources:Dulbecco’s modified Eagle’s medium (DMEM), penicillin,streptomycin, foetal calf serum (FCS), Lipofectamine 2000and G418 from Life Technologies Inc. (GIBCO-BRL)Rockville, MD, USA; monoclonal antibody 12CA5 fromRoche Diagnostics Australia Pty. Ltd., Castle Hill, NSW,Australia; Cy3-conjugated donkey anti-mouse IgG sec-ondary antibody from Jackson ImmunoResearch LabInc., Baltimore, USA; rabbit anti-calnexin antibody fromStressgen Biotechnologies Corp., San Diego, CA, USA;FITC-conjugated anti-rabbit IgG from Zymed LaboratoriesInc.; San Francisco, USA; DBHQ from Sapphire Bio-science Pty. Ltd., Crows Nest, NSW, Australia; thapsigargin,caffeine, ATP, ionomycin, 2-aminoethoxydiphenylborate(2-APB), GdCl3, collagen and dimethylsulphoxide fromSigma; coelenterazine and coelenterazine derivatives(f andn), tetrakis-(2-pyridymethyl) ethylenediamine (TPEN) andthe acetoxymethylesters of fura-2, 5,5′-dibromo BAPTA,5,5′-dimethyl BAPTA, EGTA, erAEQ/pcDNA1 and cy-tAEQ/pcDNA1 from Molecular Probes.

2.2. Transfection of H4-IIE liver cells and selection ofstable clones expressing cytAEQ and erAEQ

H4-IIE rat hepatoma cells (ATCC CRL 1548) weremaintained in DMEM, supplemented with penicillin

(100 units/ml), streptomycin (100�g/ml), 10 mM HEPES(pH 7.4) and 10% (v/v) foetal calf serum (completeDMEM), as described previously[6,15]. H4-IIE cells weresubcultured for a maximum of 20 passages.

cDNA encoding a cytoplasmic space-targeted apo-aequorin (cytAEQ/pcDNA1) was comprised of cDNAencoding the HA1 (haemaglutinin) epitope-tagged apo-aequorin (cytAEQ) in the pcDNA1 vector. cDNA en-coding an ER-targeted apo-aequorin (erAEQ/pcDNA1)was comprised of cDNA encoding a peptide leader se-quence fused to HA1 epitope-tagged low Ca2+ affinitymutated apo-aequorin (erAEQ) in the pcDNA1 expres-sion vector [16]. cytAEQ/pcDNA1 and erAEQ/pcDNA1were sub-cloned into the pcDNA3 vector containing theneomycin geneneo. Transfection of H4-IIE cells with er-AEQ/pcDNA3 and cytAEQ/pcDNA3 was carried out usingLipofectamine 2000. Cells (1× 105) were plated onto a35-mm tissue culture dish 24 h before transfection. DNA(6�g diluted in 200�l DMEM) was mixed with Lipofec-tamine 2000 (12�l diluted in 200�l DMEM) incubatedfor 20 min, then added to the cells and incubated for a fur-ther 24 h. Two days after transfection, selection was startedwith 0.8 mg/ml G418. Single colonies were transferred toa 24-well plate, expanded and tested for the presence ofDNA encoding aequorin using PCR. Positive clones har-bouring cDNA encoding apo-erAEQ or apo-cytAEQ wereidentified, and the levels of aequorin luminescence in eachdetermined. Clones cyt241 (cytAEQ) and er334 (erAEQ)which exhibited the highest luminescence were used in thepresent experiments. The clones were maintained in thepresence of G418 (0.4 mg/ml).

2.3. Immunolocalization of expressed erAEQ and cytAEQ

To determine the intracellular localisation of cytAEQ anderAEQ, H4-IIE cells stably expressing cytAEQ or erAEQwere fixed for 30 min with 4% (v/v) paraformaldehyde inphosphate buffered saline (PBS) (2.9 mM KCl, 137 mMNaCl, 1.5 mM KH2PO4, 9.5 mM Na2HPO4). They werethen washed twice with PBS and permeabilised with 0.1%(v/v) Triton-X 100 in PBS for 4 min on ice followed bya 45 min incubation in 15% (w/v) FCS in PBS. The cellswere then incubated for 30 min with the monoclonal anti-body 12CA5 which recognizes the HA1 epitope tag, at adilution of 1:100. Staining was carried out by incubationfor 30 min with the Cy3-conjugated donkey anti-mouse IgGsecondary antibody at a dilution of 1:100. After each anti-body incubation, cells were washed 4–5 times with PBS.For double immunostaining of both erAEQ and calnexinin cells expressing erAEQ, cells were incubated for 30 minwith a mixture of 12CA5 (at a dilution of 1:100) and rab-bit anti-calnexin antibody (at a dilution of 1:200) followedby incubation with a mixture of Cy3-conjugated donkeyanti-mouse IgG (at a dilution of 1:100) and FITC-conjugatedanti-rabbit IgG (at a dilution of 1:100). The cells were thenmounted under 80% glycerol in PBS, pH 8.6 and examined


under a Leica DMRBE fluorescence microscope (LeicaMicrosystems, Heidelberg, Germany) using a Leica PLFLUOTAR 100×/1.30 NA objective, and excitation filters575 nm (Cy3) and 490 nm (FITC). Fluorescent images werecaptured with the LEICA DC 100 software. Cell nucleiwere identified using Hoechst 33342[17].

2.4. Measurement of aequorin luminescence

Glass tubes (12 mm× 75 mm) (Falcon) were coated withcollagen (500�g/ml rat tail collagen type 1) for 20 min, thenUV sterilized. H4-IIE cells, suspended in complete DMEM,were transferred into collagen-coated tubes at 1× 105 cellsper tube and incubated for 24 h (erAEQ transfected cells)or 2 h (cytAEQ transfected cells) at 37◦C in a humidifiedincubator (5%, v/v, CO2 in air) in order to establish a cellmonolayer on the bottom of the tube. Except where in-dicated otherwise, one of two protocols was employed toreconstitute holo-aequorin. In the first, the [Ca2+]er wasreduced by incubating the monolayer of erAEQ or cytAEQcells for 5 min with the SERCA inhibitor, DBHQ (25�M)in Ca2+-free Krebs–Ringer modified buffer (KRB; 125 mMNaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM MgSO4, 5.5 mMglucose and 20 mM HEPES, pH 7.4), supplemented with3 mM EGTA. The cells were then washed with KRB (in theabsence of added Ca2+). Holo-aequorin reconstitution wasthen carried out by incubating the cells at 22◦C for 1 h witheither coelenterazinen (erAEQ) or coelenterazine (cytAEQ)at 5�M in KRB containing 1 mM EGTA. In the secondprotocol, the monolayer of cytAEQ cells was washed inKRB then holo-aequorin reconstitution was carried out byincubating the cells at 22◦C with coelenterazine (5�M) inKRB containing 1 mM CaCl2.

After holo-aequorin reconstitution, cells were washedonce with KRB. Specific subsequent treatments are de-scribed in the figure legends. Aequorin luminescence wasmeasured at 22◦C using an FB15 luminometer (Zylux Cor-poration, Maryville, TN, USA) fitted with reagent injectorsand linked to an IBM-compatible PC and Zylux Corpora-tion FB12 software. Reagents were added manually by aGilson pipette. At the end of the experiment, the cells werelysed by addition of CaCl2 (10 mM final concentration) anddigitonin (100�M), both added via the reagent injectors.

Stock solutions of thapsigargin, DBHQ, TPEN, 2-APBand the acetoxymethylesters of 5,5′-dibromo BAPTA,5,5′-dimethyl BAPTA and EGTA were prepared in dimethyl-sulphoxide (DMSO) whilst stock solutions of caffeine, ATPand GdCl3 were made up in ultra-pure Milli Q water. Thefinal concentration of DMSO was≤1% (v/v). For exper-iments involving Gd3+, Na2HPO4 was omitted from theKRB to prevent Gd3+ precipitation.

Values of luminescence were converted to [Ca2+]cyt or[Ca2+]er using the calibration curves published by Brini et al.[18] (cytAEQ) and Barrero et al.[19] (erAEQ). These em-ployed the same cytAEQ and erAEQ proteins and the sametemperature (22◦C) as in the present experiments. Values

of aequorin luminescence,L (relative luminescence units/s),from the FB15 luminometer were transferred to MicrosoftExcel 2000 (Microsoft Corp, Redmond, WA, USA). Totalluminescence,Lmax (relative luminescence units/s), was es-timated from the sum of the luminescence observed dur-ing each component of the experiment and the lumines-cence induced when cells were lysed in the presence of10 mM CaCl2. The value ofL at a given time was con-verted toL/Lmax using Microsoft Excel 2000. The calcu-lated logL/Lmax ratio was then imported into the GraphPadPRISM 3 software and analysed by non-linear regressioncurve fitting using a polynomial second order equation in or-der to convert values of logL/Lmax to [Ca2+]cyt or [Ca2+]er.Values of [Ca2+]cyt or [Ca2+]er were then exported to Mi-crosoft Excel 2000 and values of [Ca2+]cyt or [Ca2+]er plot-ted as a function of time. Means±S.E.M. of replicate exper-iments were calculated and the S.E.M. bars are shown on thegraphs at 15-s or -min intervals. Degrees of significance weredetermined using the Student’st-test for unpaired samples.

2.5. Imaging of endoplasmic reticulum-targetedaequorin luminescence

For photon imaging, erAEQ cells were plated onto plas-tic coverslips (22 mm× 22 mm; Scienceware, Bel-Art Inc.,Pequannock, USA) at approximately 1×105 cells per cover-slip. The coverslips were then mounted underneath a hole cutin a 35-mm petri-dish lid using high vacuum silicone grease(Dow Corning Corp., Midland, MI, USA) and the cells in-cubated for 24 h at 37◦C in a humidified incubator with 5%(v/v) CO2 in air in order to create a monolayer of cells on thecoverslip. Holo-aequorin was reconstituted with 5�M coe-lenterazinef [20] after incubation of the cells with 25�MDBHQ as described above. (The relative light intensity emit-ted from holo-aequorin reconstituted from coelenterazinef isabout 130 times greater than that emitted from holo-aequorinreconstituted from coelenterazinen [20].) Photons were im-aged as described previously[21].

2.6. Measurement of the cytoplasmic Ca2+concentrationusing fura-2

H4-IIE cells were loaded with fura-2 using the ace-toxymethyl ester, and the fluorescence ratio (proportional to[Ca2+]cyt) was measured using 340 and 380 nm excitationfilters (Chroma Technology Corp., Vt., USA) as describedpreviously[22]. The proportion of fura-2 in the cytoplasmicspace was estimated to be about 70%.

3. Results

3.1. Intracellular location of cytoplasmic and ER-targetedaequorins

Stable H4-IIE cell lines expressing haemaglutinin(HA1)-tagged aequorin targeted to the cytoplasmic space


Fig. 2. Intracellular locations of cytAEQ (A, B) and erAEQ (C–F) inH4-IIE cells stably-transfected with cDNA encoding cytAEQ and erAEQ,respectively. (A) cytAEQ cells labelled by immunocytochemistry withthe monoclonal anti-HA1 antibody. (B) cytAEQ cells treated as in (A)but with the omission of the anti-HA1 antibody. (C–E) The same erAEQcell double-stained with monoclonal anti-HA1 antibody (C) and withpolyclonal anti-calnexin antibody (D). (E) The merged images of (C)and (D). (F) An erAEQ cell treated as in (C) but with omission of theanti-HA1 and anti-calnexin antibodies and viewed using the FITC filter.The scale bar represents 5�m. The results are representative of thoseobtained from five cells which each gave results similar to those shown.

(cytAEQ cells) and ER (erAEQ cells) were prepared as de-scribed inSection 2. The intracellular locations of erAEQand cytAEQ were determined via immunocytochemistrywith an anti-HA1 antibody and a Cy3-conjugated secondaryantibody. The intracellular distribution of immunofluores-cence attributed to cytAEQ is shown inFig. 2A (cf. Fig. 2B,the conjugate control in which the anti-HA1 antibody wasomitted). The results obtained for the cell shown and forother cells examined indicated that cytAEQ is chiefly lo-cated in the cytoplasmic space. The fluorescence image forthe location of ER-targeted aequorin is shown inFig. 2C(cf.Fig. 2F, the conjugate control in which the secondary anti-body was omitted) and the image of calnexin, an ER-protein[23], in Fig. 2D. As indicated by the merged images shownin Fig. 2E, there is considerable co-localisation of erAEQwith calnexin. The conclusion that erAEQ is located in theER is consistent with this result. Thus, it is concluded thatcytAEQ and erAEQ are located in the cytoplasmic spaceand ER, respectively.

3.2. Characterisation of the ability of cytAEQ to sensechanges in the cytoplasmic-free Ca2+ concentration

To characterise the ability of cytAEQ to sense changesin [Ca2+]cyt, DBHQ was added to cytAEQ cells incubatedin the presence of extracellular Ca2+ (Ca2+

o ) to releaseCa2+ from the ER and induce Ca2+ inflow. A peak of in-creased [Ca2+]cyt with a tail of about 2 min duration was

Fig. 3. Transient increases in [Ca2+]cyt induced by the addition of DBHQto cytAEQ cells. Holo-aequorin was reconstituted by incubation of cy-tAEQ cells with coelenterazine in the presence of 1 mM Ca2+

o , as de-scribed inSection 2. After washing, the cells were incubated in KRBeither in the absence of added Ca2+

o or in the presence of 2 mM Ca2+o ,

as indicated. For experiments involving Gd3+, Na2PO4 was omitted fromthe KRB for incubations in both the presence and absence of Gd3+. (A)DBHQ (20�M) addition in the presence (broken line) or absence (solidline) of 2 mM Ca2+

o . (B) DBHQ (20�M) addition in the presence of2 mM Ca2+

o and in the presence (broken line) or absence (solid line)of 10�M Gd3+. (C) DBHQ (20�M) addition in the absence of addedCa2+

o and in the presence (broken line) or absence (solid line) of 10�MGd3+. The traces shown are the means± S.E.M. of three to eight exper-iments. For the results shown in (A) and (B), comparison of the valuesof [Ca2+]cyt at each of 105, 165 and 225 s after DBHQ addition withthe value of [Ca2+]cyt just before DBHQ addition (Student’st-test forunpaired samples) showed no significant difference.


Fig. 4. Changes in [Ca2+]cyt and [Ca2+]er induced by the addition of Ca2+o to cytAEQ and erAEQ cells previously incubated in the absence of added

Ca2+o . cytAEQ or erAEQ cells were treated with DBHQ (except in (C)), and holo-AEQ was reconstituted by incubation with coelenterazine (cytAEQ)

or coelenterazinen (erAEQ) in the presence of 1 mM EGTA, as described inSection 2. Cells were initially incubated in KRB which contained 100�MEGTA and no added Ca2+. For experiments involving Gd3+, EGTA and Na2PO4 were both omitted from the KRB for the experiments conducted inthe presence and absence of Gd3+. (A) The addition of 2 mM Ca2+

o to erAEQ cells and the effect of Gd3+ (10�M; present at the beginning of theincubation) on the increase in [Ca2+]er induced by addition of 2 mM Ca2+

o . (B) The addition of 2 mM Ca2+o to cytAEQ cells. (C) The sequential addition

of 1 and 10 mM Ca2+o to cytAEQ cells (not pre-treated with DBHQ). (D) The effect of Gd3+ (10�M; present at the beginning of the incubation) on the

transient increase in [Ca2+]cyt induced by addition of 2 mM Ca2+o to cytAEQ cells. The traces shown are the means± S.E.M. of 5–11 experiments. The

degrees of significance for comparison (Student’st-test for unpaired samples) of the values of [Ca2+]cyt (with and without Gd3+) at the time shown (∗)in (A) and at the peak (∗) in (D) are P ≤ 0.0001 andP ≤ 0.01, respectively. In (B), comparison of the values of [Ca2+]cyt at 15 s after Ca2+

o additionwith the value of [Ca2+]cyt immediately before Ca2+

o addition gaveP ≤ 0.001. In (C), comparison of values of [Ca2+]cyt after Ca2+ addition with thevalue of [Ca2+]cyt immediately before the first Ca2+

o addition gaveP ≤ 0.001 andP ≤ 0.05 at 15 and 30 s, respectively, after the first Ca2+o addition,

and P ≤ 0.001 at 30 s after the second Ca2+ addition. In (D), for either Gd3+ present or Gd3+ absent, there was no significant difference between thevalue of [Ca2+]cyt at 15, 30, 45, 60 or 75 s after Ca2+

o addition and the value of [Ca2+]cyt immediately before Ca2+o addition.

observed (Fig. 3A, KRB, Ca2+). When the experiment wasperformed in the absence of added Ca2+

o , the resulting plotwas not significantly different from that obtained in thepresence of 2 mM Ca2+

o (Fig. 3A, KRB), suggesting that noincrease in [Ca2+]cyt (manifest as a plateau after the initialDBHQ-induced increase in [Ca2+]cyt) due to Ca2+ inflowcould be detected. To further test whether any componentof the DBHQ-induced increase in [Ca2+]cyt is due to Ca2+inflow through SOCs, the effects of Gd3+, an inhibitor ofSOCs[6], were investigated. When added in either the pres-ence or absence of Ca2+

o , Gd3+ (10�M) had no significanteffect on the shape of the curve of DBHQ-induced increasein [Ca2+]cyt (Fig. 3B and C).

3.3. Characterisation of changes in [Ca2+] cyt associatedwith refilling of the ER Ca2+ store

To measure Ca2+ in the ER, the protocol developed previ-ously by others[16,19,24]was followed. This involved treat-ing H4-IIE cells with DBHQ to release Ca2+ from the ER,

reconstituting holo-aequorin with coelenterazinen in the ab-sence of added Ca2+

o , incubating cells initially in the absenceof added Ca2+

o , then adding Ca2+o . Following Ca2+

o additionto erAEQ cells, [Ca2+]er increased and reached a plateau af-ter about 4 min (Fig. 4A). The curve stops abruptly becauseat this time point all aequorin had reacted, as indicated bythe absence of any further increase in luminescence whenthe cells are subsequently lysed in the presence of Ca2+ (re-sults not shown). This time-course is similar to that observedfor refilling of the ER in other cell types[19]. The increasein [Ca2+]er was completely inhibited by Gd3+ (Fig. 4A).

When changes in [Ca2+]cyt were monitored in cytAEQcells pre-treated with DBHQ, the addition of Ca2+

o wasfound to induce a very sharp spike of increased [Ca2+]cyt

(Ca2+o -induced [Ca2+]cyt spike) with a maximum value of

about 10�M followed by a tail (low [Ca2+]cyt plateau)in which [Ca2+]cyt remained very slightly elevated abovethe basal value for about 30 s (Fig. 4B). The duration ofthe spike was about 15 s. Subsequent addition of digitonin(to lyse the cells) and 10 mM Ca2+ (to react remaining


aequorin) caused a substantial increase in luminescence (notshown). It was calculated that about 85% of the aequorinremained unreacted after Ca2+

o addition. The duration ofthe Ca2+-induced [Ca2+]cyt spike (Fig. 4B) was noticeablyshorter than that induced by DBHQ addition in the presenceof Ca2+

o (Fig. 3A). To determine whether the characteristicsof the Ca2+

o -induced [Ca2+]cyt spike were affected by theconcentration of Ca2+

o employed, the Ca2+ add-back wasperformed with two sequential additions of Ca2+

o , 1 and10 mM (Fig. 4C). For both 1 and 10 mM Ca2+

o additions,after about 30–45 s [Ca2+]cyt returned to the same base levelas that before Ca2+

o addition. Similar results were obtainedwhen 1 mM Ca2+

o was added in place of 10 mM Ca2+o (not

shown). The spike of increased [Ca2+]cyt induced by Ca2+o

addition was substantially reduced in the presence of Gd3+(Fig. 4D).

Correlation of the increases in [Ca2+]cyt with increasesin [Ca2+]er indicates that the Ca2+

o -induced [Ca2+]cyt spikeoccurs in the early stages of ER refilling. [Ca2+]cyt returnedto a value just above the basal level when [Ca2+]er was onlyabout 20% refilled, and remained slightly elevated whilerefilling of the ER continued.

The ability of 2-APB, another inhibitor of plasma mem-brane Ca2+ channels (reviewed in[25,26]) to inhibit theCa2+

o -induced [Ca2+]cyt spike and refilling of the ER wasalso tested. While 2-APB addition did inhibit Ca2+

o -inducedincreases in aequorin luminescence, it also inhibited photonrelease from aequorin in lysed cells (results not shown).This suggests that 2-APB inhibits the reaction catalysedby aequorin which leads to photon release and hence can-not be used in experiments involving aequorin as Ca2+sensor.

The Ca2+o -induced increase in luminescence of erAEQ

was imaged using a photon imaging microscope. Increasedluminescence was observed in most regions of the cell fol-lowing the addition of Ca2+

o (Fig. 5). Since the focal plane ofthe objective lens is very broad, photons arising from aboveand below the nucleus are detected in this experiment, andhence it is not possible to see the location of the nucleus.Moreover, there is not sufficient resolution to indicate fromwhich part of the ER the luminescence first originates fol-lowing Ca2+

o addition.It was noted that the shape of the spike of Ca2+

o -induced[Ca2+]cyt observed upon addition of Ca2+

o to cells previouslyincubated in the absence of added Ca2+

o (Fig. 4B) is consid-erably different to the shape of the [Ca2+]cyt spike observedin similar experiments using fura-2 and fluo-3 as a Ca2+sensor in H4-IIE cells[6,27]. In order to try to understandreasons for this difference, the profiles of Ca2+

o -induced in-creases in [Ca2+]cyt and [Ca2+]er were further investigatedusing ATP, a purinergic agonist which has been shown toinitiate the generation of InsP3 in H4-IIE cells[28]; caffeine,which activates ryanodine receptors in the ER[29]; the Ca2+ionophore ionomycin; and the Ca2+ chelators BAPTA andEGTA. Addition of ATP in the absence of Ca2+

o caused avery small increase in [Ca2+]cyt (Fig. 6A, inset). This small

Fig. 5. Images of erAEQ-generated luminescence from a single H4-IIE cellstably expressing erAEQ following the addition of extracellular Ca2+. Lu-minescence images (A–E) and corresponding bright-field images (A′–E′)of a single cell before (A), immediately after (B), and 10 s (C), 20 s (D)and 30 s (E) after the addition of 2 mM Ca2+

o . erAEQ cells were treatedwith DBHQ, and holo-AEQ was reconstituted by incubation with coe-lenterazinef in the presence of 1 mM EGTA, as described inSection 2.Cells were initially incubated in KRB which contained 100�M EGTAand no added Ca2+. Each image represents 60 s of accumulated lumi-nescence with a 10 s step between each image as indicated in (B′–E′).The colour scale indicates luminescent flux in photons per pixel and thescale bar represents 10�m. The results shown are representative of thoseobtained from five cells which gave similar images.

ATP-induced increase in [Ca2+]cyt is consistent with theprotocol employed to reconstitute aequorin (which leavesthe ER Ca2+ stores essentially depleted of Ca2+). ATP didnot substantially alter either the magnitude or the shape ofthe Ca2+

o -induced [Ca2+]cyt spike (Fig. 6A). However, ATPcaused greater than 50% inhibition of the rate and extent ofCa2+ inflow to the ER (Fig. 6B). This effect is likely to bedue to the activation of InsP3 receptors and enhancement ofCa2+ outflow from the ER.

Caffeine substantially reduced the accumulation of Ca2+in the ER (Fig. 6C), possibly due to the activation of Ca2+outflow through ryanodine receptors[29]. Ionomycin in-duced a substantial increase in [Ca2+]cyt when added in the


Fig. 6. The effects of ATP (A, B), caffeine (C) and ionomycin (D) on the [Ca2+]cyt spike and increase in [Ca2+]er induced by addition of Ca2+o . cytAEQ

or erAEQ cells were treated with DBHQ and holo-AEQ was reconstituted by incubation with coelenterazine (cytAEQ) or coelenterazinen (erAEQ) in thepresence of 1 mM EGTA, as described inSection 2. Cells were initially incubated in KRB which contained 100�M EGTA and no added Ca2+

o . (A, B)The addition of 2 mM Ca2+

o to cytAEQ (A) or erAEQ (B) cells in the presence and absence of ATP (100�M; added at the beginning of the incubation).The inset in (A) shows the trace at the addition of ATP. (C) The addition of 2 mM Ca2+

o to erAEQ cells in the presence and absence of caffeine (1 mM;added at the beginning of the incubation). (D) The addition of 2 mM Ca2+

o to cytAEQ cells in the presence and absence of ionomycin (10�M; addedat the beginning of the incubation). The inset in (D) shows the region at the addition of ionomycin. The traces shown are the means± S.E.M. of 3–11experiments. The degree of significance for comparison of the values of [Ca2+]cyt at the times shown (∗) areP ≤ 0.0001.

absence of Ca2+o (Fig. 6D, inset). The ionomycin-induced

increase in [Ca2+]cyt was much greater than that inducedby ATP (Fig. 6A, inset). It would be expected that in addi-tion to affecting Ca2+ in the ER, ionomycin would inducethe release of Ca2+ from mitochondria and other non-ERintracellular Ca2+ stores. Ionomycin did not alter the peakof the [Ca2+]cyt spike induced by Ca2+

o addition but didsubstantially extend the tail (i.e. slowed the return to basal[Ca2+]cyt) (Fig. 6D). In experiments employing erAEQ, ion-omycin (0.1�M) inhibited the uptake of Ca2+ to the ER byabout 50% (results not shown).

Loading cytAEQ cells with dimethyl BAPTA re-sulted in a considerable broadening of the Ca2+

o -induced[Ca2+]cyt spike (Fig. 7A). In erAEQ cells loaded withdibromo-BAPTA, the rate and extent of Ca2+

o -induced fill-ing of the ER was substantially reduced (Fig. 7B). LoadingcytAEQ cells with 1 mM EGTA also resulted in considerablebroadening of the Ca2+

o -induced [Ca2+]cyt spike (Fig. 7C).

3.4. Effects of thapsigargin and DBHQ on Cao2+-induced

increases in [Ca2+] cyt and [Ca2+] er

The addition of thapsigargin to cells previously subjectedto the ER Ca2+ depletion protocol and incubated in theabsence of Ca2+

o induced a small increase in [Ca2+]cyt(Fig. 8A, inset) (cf. effect of ATP;Fig. 6A, inset), indicating

that a small amount of Ca2+ remained in the ER after theDBHQ pre-treatment. Thapsigargin had no detectable effecton either the magnitude or the shape of the Ca2+

o -induced[Ca2+]cyt spike or on the low [Ca2+]cyt plateau which fol-lowed the initial Ca2+

o -induced [Ca2+]cyt spike (Fig. 8A).Thus, in the presence of thapsigargin, [Ca2+]cyt remainedelevated slightly above base-line for about 2 min, then de-creased to the basal value. By contrast, thapsigargin causeda substantial inhibition of Ca2+

o -induced inflow of Ca2+ tothe ER (Fig. 8B). Results similar to those for thapsigarginwere obtained with DBHQ (Fig. 8C and D). When usedin combination, thapsigargin and DBHQ did not affect theCa2+

o -induced [Ca2+]cyt spike while almost completely in-hibiting Ca2+ inflow to the ER (results not shown). The lackof effect of thapsigargin or DBHQ on the Ca2+

o -induced[Ca2+]cyt spike is unlikely to be due to either saturationof aequorin with Ca2+ or exhaustion of the holo-aequorinsince subsequent addition of digitonin and 10 mM Ca2+ (toreact with remaining aequorin) caused a substantial increasein luminescence (results not shown).

The possibility that, in the presence of thapsigargin, Ca2+is taken up by mitochondria[30,31] was investigated us-ing CCCP (a mitochondrial uncoupler which releases mito-chondrial Ca2+ [30,31]). The addition of CCCP after Ca2+

oaddition induced a small increase in [Ca2+]cyt, presumablydue to the release of Ca2+ from mitochondria (Fig. 9A).


Fig. 7. The effects of loading cells with BAPTA (A, B) or EGTA (C) onthe [Ca2+]cyt spike (A, C) and increase in [Ca2+]er (B) induced by theaddition of Ca2+

o . cytAEQ or erAEQ cells were treated with DBHQ andholo-AEQ reconstituted by incubation with coelenterazine (cytAEQ) orcoelenterazinen (erAEQ) in the presence of 1 mM EGTA, as described inSection 2. Cells were loaded with dimethyl BAPTA (A), dibromo BAPTA(B) or EGTA (C) by incubation with 1 mM of the acetoxymethylesterof dimethyl BAPTA (A), dibromo BAPTA (B) or EGTA (C) for 60 minat 22◦C after holo-AEQ reconstitution. Cells were initially incubatedin KRB which contained 100�M EGTA and no added Ca2+. Ca2+

o(2 mM) was present during the period indicated. The data are expressedas means± S.E.M. of 5–12 experiments. The degrees of significance forcomparison of the values of [Ca2+]cyt at the times shown (∗) areP ≤ 0.01(A) and P ≤ 0.0001 (B and C).

There was no difference in the CCCP-induced increase in[Ca2+]cyt between cells incubated in the presence and ab-sence of thapsigargin (Fig. 9A). The inset ofFig. 9A showsthe Ca2+

o -induced spike of [Ca2+]cyt in each experiment. Inanother series of experiments, CCCP was added before Ca2+

oin order to block any Ca2+ uptake by mitochondria duringCa2+

o add-back. For cells incubated in either the absence orpresence of thapsigargin, the magnitude of the Ca2+

o -induced[Ca2+]cyt spike was the same whether or not CCCP waspresent or absent (Fig. 9B).

3.5. Effects of TPEN on Cao2+-induced increases in

[Ca2+] cyt and [Ca2+] er

TPEN has been used to chelate Ca2+ in the ER, decrease[Ca2+]er, and activate SOCs[32]. The ability of TPEN toactivate SOCs in H4-IIE cells was tested in the followingmanner. cytAEQ cells (not pre-treated with DBHQ) were in-cubated with coelenterazine in the presence of 1 mM Ca2+

othen placed in a medium containing no added Ca2+

o . Lumi-nescence was then measured as a function of time. This pro-tocol was designed to maintain as much of the Ca2+ in theER as possible before the addition of TPEN. In the absenceof TPEN, the addition of Ca2+

o induced a spike of increased[Ca2+]cyt (Fig. 10A). The addition of TPEN to cells treatedin this way and incubated initially in the absence of addedCa2+

o did not substantially affect the Ca2+o -induced spike of

[Ca2+]cyt (Fig. 10A). Pre-treatment with TPEN almost com-pletely inhibited the signal from erAEQ (Fig. 10B), mostlikely due to the buffering of Ca2+ in the ER by the TPEN.When TPEN was added to cells incubated in the presenceof Ca2+

o , a spike of increased [Ca2+]cyt was observed and[Ca2+]cyt remained elevated for about 5 min (i.e. TPEN in-duced a low plateau of increased [Ca2+]cyt) (Fig. 10C). Thisresult suggests that reduction of [Ca2+]er by TPEN can ini-tiate the activation of SOCs.

3.6. Comparison of changes in the cytoplasmic Ca2+concentration reported by cytAEQ with those reportedby fura-2

Since the results obtained using cytAEQ as a sensor forchanges in [Ca2+]cyt appear to differ, at least quantitatively,from those reported previously for H4-IIE cells using fura-2or fluo-3 as a Ca2+ sensor[6,27], experiments were per-formed with H4-IIE cells loaded with fura-2 under condi-tions similar to those employed for the measurement of Ca2+using cytAEQ. Fura-2 has a higher affinity for Ca2+ and alower dynamic range (the concentrations of Ca2+ over whichfura-2 can be used as a Ca2+ sensor)[33] than aequorin[18,20,24,34]. The addition of DBHQ in the presence ofCa2+

o induced a prolonged increase in [Ca2+]cyt (monitoredusing the fura-2 fluorescence ratio) and this was substan-tially reduced by Gd3+ (Fig. 11A) or by removal of Ca2+

o(Fig. 11B, DBHQ addition, cf. A).

The addition of Ca2+o to fura-2 loaded cells treated with

DBHQ in the absence of added Ca2+o then washed (in the

absence of Ca2+o ) in order to remove the DBHQ caused

a transient increase in fura-2 fluorescence ratio followedby a prolonged plateau (Fig. 11B). A second additionof Ca2+

o caused no further increase in fluorescence ratio(Fig. 11B). When the experiment was repeated, but withoutremoval of DBHQ (i.e. DBHQ was present during Ca2+

oaddition), the resulting plateau was greater than that ob-served in the absence of DBHQ (Fig. 11C, cf. B). Themagnitude of the Ca2+

o -induced plateau (after subtractionof the value of fluorescence ratio immediately before Ca2+

o


Fig. 8. The effects of thapsigargin and DBHQ (present during the Ca2+o addition) on the [Ca2+]cyt spike and increase in [Ca2+]er induced by addition of

Ca2+o . cytAEQ or erAEQ cells were treated with DBHQ and holo-AEQ was reconstituted by incubation with coelenterazine (cytAEQ) or coelenterazinen

(erAEQ) in the presence of 1 mM EGTA, as described inSection 2. Cells were initially incubated in KRB which contained 100�M EGTA and no addedCa2+. (A, B) cytAEQ (A) or erAEQ (B) cells incubated in the presence or absence of thapsigargin (10�M; added at the beginning of the incubation).(C, D) cytAEQ (C) or erAEQ (D) cells were incubated in the presence or absence of DBHQ (10�M) (added at the beginning of the incubation). Ca2+(2 mM) was added. The data are expressed as means± S.E.M. of 4–13 experiments. In (B), the degrees of significance (Student’st-test for unpairedsamples) for comparison of the values of [Ca2+]er (presence or absence of thapsigargin) at the time points indicated (∗) are P ≤ 0.0001. In (A) and(C), for results in either the presence or absence of thapsigargin or DBHQ, the degrees of significance for comparison of the value of [Ca2+]cyt at 15,30 or 45 s after Ca2+

o addition with the value of [Ca2+]cyt immediately before Ca2+o addition were eachP ≤ 0.05 to P ≤ 0.001. In (B) (for results in

the presence of thapsigargin) and (D) (for results in the presence of DBHQ), the degrees of significance for comparison of the values of [Ca2+]er at 1,2, or 3 min after Ca2+

o addition with the value of [Ca2+]er immediately before Ca2+o addition were eachP ≤ 0.05 to P ≤ 0.001.

addition) was 0.15 ± 0.01 and 0.07 ± 0.02 (n = 4) forcells loaded with fura-2 and incubated in the presence orabsence (after washing) of DBHQ, respectively (P ≤ 0.05,Student’t-test for unpaired samples). A second addition ofCa2+

o caused a further small increase in fluorescence ratio(Fig. 11C).

4. Discussion

4.1. The short transient Cao2+-induced increase in

[Ca2+] cyt

Three interesting findings reported here are (i) the shortduration of the transient increase in [Ca2+]cyt reported bycytAEQ, (ii) the apparent difference between the changesin [Ca2+]cyt sensed by cytAEQ compared with the changessensed by the fluorescent Ca2+ reporter fura-2 followingCa2+ inflow induced by re-addition of Ca2+

o , and (iii) theobservation that SERCA inhibitors can substantially inhibitthe inflow of Ca2+ to the bulk of the ER yet the activation ofSOCs (assessed by the Ca2+

o -induced increase in [Ca2+]cyt)is not sensitive to these inhibitors. A particularly striking

feature of the Ca2+ signals reported by cytAEQ is the shortduration (about 15 s) of the spike of increased [Ca2+]cyt

observed on adding Ca2+o to cytAEQ cells in which the ER

has previously been depleted of Ca2+. [Ca2+]cyt remainedelevated during the period of refilling of the ER (monitoredby erAEQ) but this elevation in [Ca2+]cyt was very small,especially when compared with the value of [Ca2+]cyt atthe peak of the spike. The Ca2+

o -induced spike of [Ca2+]cytand low [Ca2+]cyt plateau were substantially reduced byGd3+, indicating that the Ca2+

o -induced spike of [Ca2+]cytrepresents Ca2+ inflow through open SOCs.

One explanation for these observations is that, in the ab-sence of SERCA inhibitors, when Ca2+

o is added to cells inwhich the ER Ca2+ has been depleted, Ca2+ flows throughSOCs and is then very rapidly taken up by the ER (shownschematically by the solid line and arrows inFig. 1). Apseudo steady-state is then reached during refilling of theER in which there is a small elevation of [Ca2+]cyt (the low[Ca2+]cyt plateau) in all or some of the cytoplasmic space (cf.[10,35]). The initial sharp Ca2+

o -induced spike of increased[Ca2+]cyt may represent the initial inflow of Ca2+ throughopen SOCs (the cells were previously incubated with DBHQto decrease [Ca2+]er and the DBHQ subsequently removed)


Fig. 9. The effect of thapsigargin on the increase in [Ca2+]cyt induced byCCCP added after Ca2+

o addition to cytAEQ cells (A), and the effect ofCCCP, added before Ca2+

o , on the Ca2+o -induced spike of [Ca2+]cyt in the

presence and absence of thapsigargin (B). cytAEQ cells were treated withDBHQ and holo-AEQ reconstituted by incubation with coelenterazine inthe presence of 1 mM EGTA, as described inSection 2. Cells were initiallyincubated in KRB which contained 100�M EGTA and no added Ca2+.(A) CCCP (10�M) was added after 2 mM Ca2+

o . The inset shows theincrease in [Ca2+]cyt following the addition of Ca2+

o . (B) CCCP (10�M)was added before 2 mM Ca2+

o . The inset shows the effects of the additionof CCCP. The results are expressed as the means± S.E.M. of three toseven experiments.

resulting in a rapid increase in [Ca2+]cyt (possibly in thevicinity of SOCs) then Ca2+-dependent feed-back inhibitionof SOCs[4,14], thus creating an overshoot in [Ca2+]cyt. An-other possible contributor to the decline in [Ca2+]cyt whichoccurs after the initial increase is the activity of the plasmamembrane (Ca2+ + Mg2+)ATP-ase[36–38].

The Ca2+o -induced spike of [Ca2+]cyt was observed when

(i) Ca2+o was added to cells pre-treated with DBHQ to re-

lease Ca2+ from the ER, (ii) a second addition of Ca2+o

was made 5 min after the first Ca2+o addition to cells in

which ER Ca2+ stores had previously been depleted bytreatment with DBHQ, and (iii) Ca2+

o was added to cells inwhich holo-aequorin had been reconstituted in the presenceof 1 mM Ca2+

o in the absence of pre-depletion of the storeswith DBHQ. Under conditions (ii) and (iii), the ER Ca2+stores should be essentially replete. But since there is no evi-dence that other plasma membrane Ca2+ channels are open,it is likely that the observed increase in [Ca2+]cyt representsCa2+ inflow through SOCs under conditions (ii) and (iii) aswell as condition (i).

Caffeine, an activator of ryanodine receptors[29], sub-stantially inhibited refilling of the ER. While there is some

Fig. 10. Effect of TPEN on Ca2+o -induced increases in [Ca2+]cyt and

[Ca2+]er. (A) Holo cytAEQ was reconstituted by incubation of cytAEQcells with coelenterazine in the presence of 1 mM Ca2+

o , as describedin Section 2. The cells were initially incubated in KRB in the absenceof added Ca2+. TPEN (400�M) was added to cytAEQ cells before theaddition of Ca2+

o . (B) erAEQ cells were treated with DBHQ and holo-AEQreconstituted by incubation with coelenterazinen in the presence of 1 mMEGTA, as described inSection 2. Cells were initially incubated in KRBwhich contained 100�M EGTA and no added Ca2+. TPEN (400�M)was added before the addition of 2 mM Ca2+

o . (C) In cytAEQ cells notsubjected to DBHQ pre-treatment, holo cytAEQ was reconstituted byincubation with coelenterazine in the presence of 1 mM EGTA. Afterwashing, the cells were incubated in KRB in the presence of 2 mM Ca2+

o .TPEN (400�M) was added after the addition of 2 mM Ca2+

o . The resultsare expressed as the means± S.E.M. of 4–11 experiments. The degreesof significance for comparison of the value of [Ca2+]cyt in (B) at thetime indicated (∗) is P ≤ 0.0001.

evidence for the presence of ryanodine receptors in livercells [39–41], not all workers agree that the hepatocyte ERpossesses these receptors[42]. Our results obtained withcaffeine provide additional functional evidence for the pres-ence of ryanodine receptors in the ER of liver cells, althoughthe possibility that caffeine activates another type of Ca2+channel in the ER[42] cannot be excluded.


Fig. 11. Changes in [Ca2+]cyt measured using fura-2 under conditions similar to those employed using aequorin as a sensor for [Ca2+]cyt. (A) DBHQwas added in the presence of Ca2+

o , and in the presence and absence of Gd3+ (10�M). (B) DBHQ was added in the absence of Ca2+o , cells were washed

to remove DBHQ, then Ca2+ was added. (C) DBHQ was added in the absence of Ca2+o followed by addition of Ca2+ (while DBHQ was still present).

The additions of DBHQ (20�M) and Ca2+o (2 mM) are shown by the horizontal bars. Cells were loaded with fura-2 and fluorescence ratios measured

as described inSection 2. Each trace represents one of two to four traces which each gave similar results.

4.2. Comparison of aequorin with fura-2 as a reporter of[Ca2+] cyt

Under conditions in which DBHQ was added to cells in-cubated in the presence or absence of Ca2+

o and in the pres-ence or absence of Gd3+, the results obtained using cytAEQas a sensor for changes in [Ca2+]cyt were quantitatively dif-ferent from those obtained using fura-2. Following the ad-dition of DBHQ in the presence of Ca2+

o fura-2 reported atransient increase in fluorescence followed by a substantialsustained increase (cf.[6,25,43]). The latter was inhibited byremoval of Ca2+

o or by addition of Gd3+ in the presence ofCa2+

o . The transient increase in fluorescence observed usingfura-based Ca2+ sensors in liver cells and other cell typesis attributed to the release of Ca2+ from the ER while thesustained increase is attributable to the activation of Ca2+inflow across the plasma membrane[6,25,43]. Thus, it ap-pears that, in contrast to fura-2, cytAEQ reports a lower in-crease of [Ca2+]cyt as assessed by the reduced plateau afterthe Ca2+

o -induced [Ca2+]cyt spike.There are several possible reasons for the differences in

changes in [Ca2+]cyt reported by cytAEQ and fura-2. Thesecan be briefly summarised as follows. (i) Loading cells withdimethyl BAPTA or EGTA broadened the Ca2+

o -inducedspike of [Ca2+]cyt sensed by cytAEQ so that the generalshape of the curve describing increases in [Ca2+]cyt as afunction of time is more like the plots observed using fura-2.These results suggest that one of the differences may be theadditional buffering capacity introduced by fura-2[44,45].Such a buffering by fura-2 of the [Ca2+]cyt signal in cerebel-lar granule neurones has been proposed to be one explana-tion for the observation that fura-2 masks a caffeine-inducedrelease of Ca2+ from the ER[46]. (ii) The presence of somefura-2 in the ER may distort the ability of fura-2 to sense[Ca2+]cyt, although it is considered this is unlikely to be amajor reason for differences in [Ca2+]cyt reported by cy-tAEQ and fura-2. (iii) Differences in affinity for Ca2+ (theapparent dissociation constants for the binding of Ca2+ toaequorin and fura-2 are about 30�M [34] and 0.2�M [33],

respectively) and dynamic range (fura-2[33] has a lower dy-namic range than aequorin[18,20,24,34]) may account forsome of the difference observed with cytAEQ and fura-2.(iv) A likely and significant reason is the possibility that un-der the conditions in which [Ca2+]cyt was measured thereare localised regions of high Ca2+ concentration in the cyto-plasmic space. (For example, near the mouth of SOCs dur-ing Ca2+ inflow when [Ca2+]o is added to cells previouslyincubated in the absence of added [Ca2+]o.) Aequorin candetect localised regions of high Ca2+ concentration whichare not, or may not be, detected by fura-2. Thus, the largedynamic range for Ca2+ allows aequorin to detect regionsof high Ca2+ concentration (which may be localised andwould not be observed with fura-2) but the aequorin in theseregions of putative high Ca2+ concentration is quickly con-sumed and further aequorin luminescence from those regionsdepends on the diffusion of fresh aequorin into the regions[37,47–49]. The steep response curve for the aequorin sig-nal and the differences between aequorin and fura-2 in de-tecting changes in [Ca2+]cyt reported in the present studiesare similar to those described using these two Ca2+ sensorsin studies of arterial smooth muscle cells[48] and for theuse of cytAEQ as a Ca2+ sensor in CHO cells[37]. Theresults described here using cytAEQ to measure [Ca2+]cyt

(i.e. the Ca2+o -induced [Ca2+]cyt spike of a short duration)

are similar to those obtained by Woods et al.[50] who mi-croinjected aequorin into freshly-isolated rat hepatocytes inorder to monitor changes in [Ca2+]cyt. The present resultsobtained with aequorin may point to a local region of highCa2+ concentration in the cytoplasmic space of H4-IIE cellsduring Ca2+ inflow through SOCs.

4.3. Effects of SERCA inhibitors on the Cao2+-induced

increases in [Ca2+] cyt and [Ca2+] er

Within the limits of detection, thapsigargin or DBHQ (ora combination of both inhibitors) did not affect either theCa2+

o -induced [Ca2+]cyt spike or the low [Ca2+]cyt plateauwhich followed the spike. However, thapsigargin and DBHQ


each separately, or in combination, substantially inhibitedthe flow of Ca2+ to the ER as detected by erAEQ. The inhi-bition was not complete, and there was a small refilling ofthe ER Ca2+ store (or of any related intracellular organelleswhich express and retain erAEQ) in the presence of theSERCA inhibitor. Not only was there no inhibition by thap-sigargin or DBHQ of the Ca2+

o -induced spike of increased[Ca2+]cyt and the low [Ca2+]cyt plateau, but the SERCA in-hibitors did not increase the magnitude of these parameters.It might have been expected that, in the presence of thap-sigargin or DBHQ, Ca2+ that was prevented from enteringthe ER would be re-directed to the cytoplasmic space asshown schematically inFig. 1(broken lines and arrows). In-deed, when fura-2 was used as the Ca2+sensor, the plateauof the Ca2+

o -induced increase in [Ca2+]cyt was higher in thepresence of DBHQ than in the absence of this SERCA in-hibitor. Similar differences in changes in Ca2+ concentrationreported by cytoplasmic aequorin and fura-2 have been de-scribed for arterial smooth muscle cells upon re-addition ofCa2+

o to cells previously incubated in the absence of addedCa2+

o in the presence or absence of cyclopiazonic acid[48].In experiments using cytAEQ, the existence of the

Ca2+o -induced [Ca2+]cyt spike and the low [Ca2+]cyt plateau

in the presence of thapsigargin or DBHQ indicates that Ca2+can still flow into the cell through open SOCs under theseconditions. The results of previous patch clamp recordingexperiments conducted with H4-IIE cells[7] also clearlyindicate that in the presence of thapsigargin (and an intra-cellular Ca2+ buffer to prevent Ca2+-dependent feedbackinhibition) the SOCs remain open and convey Ca2+. Theobservation that SOCs are active in the presence of thap-sigargin is also consistent with results obtained previouslyusing fluorescent dyes as Ca2+ sensors in liver cells[6,27](and present results) and in many other cell types[1,3,14].

Thus in the present experiments, while thapsigargin andDBHQ have inhibited most of the inflow of Ca2+ to the bulkof the ER, they do not seem to have inhibited the activationof SOCs. But it appears that, after an initial flow of someCa2+ through SOCs into the cytoplasmic space, the flow ofCa2+ stops (most likely due to Ca2+-dependent feedbackinhibition of the SOCs). Ca2+ does not flow into the ER(there is no significant refilling of the ER), the mitochondria(there is no increase in CCCP-releasable Ca2+) or the cyto-plasmic space (there is no large increase in [Ca2+]cyt). Theobservation that Ca2+ inflow to the bulk of the ER can beinhibited while the activation of SOCs is not affected sug-gests (as one of several possible explanations) that a smallintracellular Ca2+ store (rather than the bulk of the ER) isresponsible for the activation of SOCs in H4-IIE liver cells.This suggestion is consistent with the results of other ex-perimental approaches in other cell types which have beeninterpreted to indicate that a “small ER Ca2+ store”, whichcan be functionally distinguished from the bulk of the ER,is required for the activation of SOCs (cf.[51–53] and ref-erences therein). Since the ER is heterogenous[53], andin the present experiments which employ ER-targeted ae-

quorin is defined by the peptide sequence on erAEQ whichtargets aequorin to the ER, it is possible that the small ERCa2+ store hypothesised to be involved in the activation ofSOCs is another type of intracellular organelle, for examplea Golgi-derived vesicle (cf.[54]).

Thus, one explanation for the present results is that (i)the activation of SOCs and the maintenance of Ca2+ inflowthrough SOCs is achieved by the hypothesised small ERCa2+ store[51–54](ii) (Ca2++Mg2+)ATP-ases are requiredto draw Ca2+ into this small ER Ca2+ store and hence tomaintain the flow of Ca2+ through SOCs[10–13], and (iii)the (Ca2++Mg2+)ATP-ases involved are insensitive to thap-sigargin and DBHQ[14]. These ideas, which are consistentwith the proposals of others on the mechanism of activa-tion of SOCs[51–53], are summarised inFig. 1. However,other explanations are possible, and further experiments arerequired to test this working hypothesis. These include theuse of more refined imaging of changes in [Ca2+]er and theuse of patch clamp recording techniques.

Acknowledgements

This work was supported by CERG-RGC grant HKUST6131/99M, RGC-DAG 98/99.SC30, and an Australian Re-search Council grant awarded to Dr. Andrew L. Miller andProfessor Greg J. Barritt. We gratefully acknowledge Mrs.Diana Kassos and Miss Lee-Anne Addis for preparationof the typescript, and advice provided by Dr. Roland Gre-gory, Department of Medical Biochemistry, Flinders Uni-versity, and Professor Peter Cobbold, Department of HumanAnatomy and Cell Biology, University of Liverpool.

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