2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca2+discharge and store-operated Ca2+influx

2-aminoethoxydiphenyl boratereveals heterogeneity inreceptor-activated Ca2� dischargeand store-operated Ca2� influx

J. P. Kukkonen, P.-E. Lund, K. E. O. Åkerman

Department of Physiology, Division of Cell Physiology, Uppsala University, Uppsala, Sweden

Summary We have investigated Ca2� release and receptor- and store-operated Ca2� influxes in Chinese hamsterovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2and patch-clamp measurements. Ca2� release and subsequent Ni2�-sensitive, store-operated influx were inducedby thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in areversible manner. It also reduced Mn2� influx in response to thapsigargin. Inhibition of Ca2� release was also seenbut this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells,ICRAC activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operatedCa2� influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca2� influxes were insensitive to 2-APB. Ca2�

release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. Theresults suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and(2) that independently of this, it also inhibits some store-operated Ca2� channels via a direct, extracellular action.© 2001 Harcourt Publishers Ltd

Cell Calcium (2001) 30(2), 117–129© 2001 Harcourt Publishers Ltd

doi: 10.1054/ceca.2001.0219, available online at http://www.idealibrary.com on

Research

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INTRODUCTION

The main pathway for Ca2� release from intracellularsites is through the action of inositol-1,4,5-trisphosphate(IP3) on its receptor on the endoplasmic reticulum (ER)membrane. The subsequent Ca2� release and store-depletion leads to the activation of so-called store-operated or capacitative Ca2� influx to replenish theCa2� stores [1,2]. So far, the nature of the signal from theER to the plasma membrane is unknown and has lead tomany models of coupling, involving soluble messengers[3–5], GTP-binding proteins [6,7] or direct coupling of

Received 21 February 2001Revised 11 April 2001Accepted 18 April 2001

Correspondence to: Jyrki Kukkonen, Department of Physiology,Division of Cell Physiology, Uppsala University, BMC, P.O. Box 572,SE-75123 Uppsala, Sweden. Tel.: �46 18 471 4171 (direct),�46 18 471 4185 (office); Fax: �46 18 471 4938;e-mail: [email protected]

the stores to the plasma membrane, the so called‘conformational coupling’ [8–10]. Apart from IP3, store-operated Ca2� influx can also be activated indepen-dently of receptor activation with compounds thatdeplete intracellular Ca2� stores, e.g. thapsigargin.Several types of store- or receptor-operated Ca2� chan-nels (SOCs and ROCs, respectively) have been phenome-nologically described, the most well-known of which isthe CRAC channel and the associated current, ICRAC [11].The CRAC channels have been identified mainly inhematopoietic cells and in other cells other types ofSOCs may be present [reviewed in 12,13]. A family ofCa2� channels, trp, has recently been identified andcloned; relationship between the members of this familyand the physiologically described receptor- or store-operated Ca2� channels is subject to active research[reviewed in 10,14].

Different activation mechanisms for store-operatedCa2� influx may be operative in different cells, but espe-cially the conformational coupling hypothesis where the

117

118 JP Kukkonen, P-E Lund, KEO Åkerman

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ER IP3 receptor is the message from ER to plasma mem-brane [reviewed in 10] has received much attention.According to this model, low lumenal Ca2� concentra-tion induces conformational change in the IP3 receptor,which in its turn activates plasma membrane Ca2� chan-nels through direct intermolecular interaction. Theinvestigation of inositol phosphate signalling has beendifficult due to the lack of selective and membrane per-meant inhibitors. This has also made the distinctionbetween store-operated and receptor-operated Ca2�

influx difficult. Recently, two membrane permeantCa2� discharge inhibitors (noncompetitive IP3 receptorantagonists) have been described, namely xestopongin-C[15] and 2-aminoethoxydiphenyl borate [2-APB; 16].Xestospongin-C has, however, suffered from some draw-backs due to its low membrane permeability and lack ofselectivity [equal inhibitory potency for the IP3 receptorand ER Ca2� pump (SERCA); 17]. On the contrary, 2-APBhas been used as a selective, easily permeating, and eco-nomical, inhibitor of the IP3 receptor. 2-APB, termed anoncompetitive inhibitor of IP3 receptor, displays a mix-ture of agonism and competitive and noncompetitiveantagonism towards the IP3-dependent Ca2� release [16].However, the mechanism of its action has not been clari-fied, and the conclusion of the inhibition of the IP3

receptor relies on the inhibition of the IP3-dependentCa2� release without any inhibition of the IP3 formation,IP3 binding or IP3-independent Ca2� release [16]. Thereceptor-dependent activation of endogenous SOCsand recombinantly expressed trp3 channels in HEK293cells has been shown to be sensitive to inhibition by2-APB [18].

The aim of this study was to investigate the diversityof Ca2� release using 2-APB as probe. CHO (Chinesehamster ovary), SH-SY5Y (human neuroblastoma) andRBL-1 (rat basophilic leukemia) cells were chosen asmodels for epithelial/fibroblast, neuronal and hemato-poietic cells, respectively. Our results prompted us toinvestigate the effect of 2-APB on the store-operatedCa2� influx. The results suggest, that 2-APB directlyinhibits capacitative Ca2� influx (including ICRAC). Thisoccurs with higher potency and thus even under condi-tions where no inhibition of Ca2� release is observed.

MATERIALS AND METHODS

Cell cultures

CHO-hOX1-C1 cells were produced as described in [19].They were grown in nutrient mixture (Ham’s F-12)medium (Life Technologies, Inc., Paisley, UK) supple-mented with 100 U/ml penicillin G (Sigma ChemicalCompany, St Louis, MO), 80 U/ml streptomycin (Sigma),400 �g/ml Geneticin (G418; Life Technologies, Inc.) and

Cell Calcium (2001) 30(2), 117–129

10% v/v fetal calf serum (Life Technologies, Inc.) at 37�Cin 5% CO2 in an air ventilated humidified incubator in260 ml plastic culture flasks (75 cm2 bottom area; NuncA/S, Roskilde, Denmark). RBL-1 and -2H3 cells weregrown in Dulbecco’s Modified Eagle Medium (DMEM;Life Technologies, Inc.) supplemented with 0.37%NaHCO3, 100 U/ml of penicillin G, 80 U/ml streptomycinand 10% (v/v) fetal calf serum at 37�C in 5% CO2 in anair ventilated humidified incubator in 260 ml plastic cul-ture flasks (75 cm2 bottom area; Nunc). SH-SY5Y cellswere grown in DMEM supplemented with 0.37%NaHCO3, 100 U/ml of penicillin G, 80 U/ml streptomycinand 7% (v/v) fetal calf serum at 37�C in 5% CO2 in an airventilated humidified incubator in 800 ml plastic cultureflasks (175 cm2 bottom area; Nunc).

For Ca2� measurements in suspension, the cells weregrown on circular plastic culture dishes (innerdiameter�94mm; Nunc). For patch-clamp and Ca2� mea-surements on coverslips, the cells were grown oncircular glass coverslips (diameters�25 and 13 mm,respectively).

Materials

ATP, carbamylcholine (CCh), EGTA (ethylene glycol-bis[�-aminoethyl ether]N,N,N�,N�-tetraacetic acid), probenecid(p-[dipropylsulfamoyl]benzoic acid) and thrombin werepurchased from Sigma and NECA (5’-[N-ethylcarboxam-ido]adenosine) and thapsigargin from RBI (Natick, MA).Fura-2 acetoxymethyl ester and Fluo-3 acetoxymethylester were purchased from Molecular Probes Inc.(Eugene, OR) and human orexin-A from PeninsulaLaboratories Europe Ltd. (St Helens, UK). 2-APB and ion-omycin were from Calbiochem (La Jolla, CA) and NiCl2and digitonin from Merck AG (Darmstadt, Germany).myo-[2-3H]inositol (1 mCi/ml) was from AmershamPharmacia Biotech (Buckinghamshire, UK).

Media

The TES Buffered Medium (TBM) consisted of 137mMNaCl, 5mM KCl, 1mM CaCl2, 1.2mM MgCl2, 0.44mMKH2PO4, 4.2mM NaHCO3, 10mM glucose, 1mM pro-benecid and 20mM 2-([2-hydroxy-1,1-bis(hydroxy-methyl)ethyl]amino) ethane sulfonic acid (TES) adjusted topH 7.4 with NaOH.

Ca2� Measurements

The cells (CHO-hOX1, RBL-1 [or -2H3], SH-SY5Y) wereharvested using phosphate buffered saline containing0.2 g/l EDTA, loaded with Fura-2 acetoxymethyl esteror Fluo-3 acetoxymethyl ester (4 �M, 20 min, 37�C) inTBM and stored on ice as pellets (medium removed).

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For CHO-hOX1 and RBL-1 cells, 10 �g/ml bovine serumalbumin (Sigma) was included in the loading buffer. Forthe measurement of intracellular free calcium, one pelletwas resuspended in TBM at 37°C. The fluorescence wasmonitored in a stirred quartz-microcuvette in the ther-mostated cell holder of either a Hitachi F-2000 or F-4000fluorescence spectrophotometer at the wavelengths340 nm or 340 nm/359 nm (excitation), 505 nm (emis-sion) for Fura-2 and at the wavelengths 480 nm (excita-tion), 540 nm (emission) for Fluo-3. Experiments werecalibrated by adding 60 �g/ml digitonin, which gives themaximum value of fluorescence, and 10 mM EGTA,which gives the minimum value of fluorescence. Theleaked Fura-2 and Fluo-3 were measured in separateexperiments by adding 10 mM EGTA which chelatesCa2� bound to extracellular indicator. The correctedfluorescence values were used to calculate [Ca2�]i (intra-cellular Ca2� concentration).

Alternatively, Ca2� measurements were performed oncoverslips. The cells grown on coverslips were loadedwith Fura-2 acetoxymethyl ester or Fluo-3 acetoxy-methyl ester (4 �M, 20 min, 37�C) in TBM, washed andplaced in a stirred quartz-cuvette in the thermostatedcell holder of either a Hitachi F-2000 or F-4000 fluores-cence spectrophotometer. The experiments were per-formed as described above, except that 1 �M ionomycinwas used instead of digitonin. The measured basal[Ca2�]i values were between 30 and 100 nM.

Inositol phosphate and IP3-measurements

Inositol phosphate and IP3-measurements were per-formed essentially as described in [19]. Briefly, for thetotal inositol phosphate measurements, the cells wereloaded with 3 �Ci/ml myo-[3H]inositol for 16-24 h. Theywere harvested as for the Ca2� measurements and pre-incubated for 10 min at 37�C in TBM devoid ofprobenecid but containing 10 mM LiCl. After 20 min ofstimulation at 37�C, the cells were rapidly spun down,the supernatants were removed and the reactions werestopped by addition of 100 �l of 0.4 M ice-cold perchloricacid. After vortex-mixing, the samples were frozen down,rethawed, vortex-mixed and the perchloric acid neutral-ized with 50 �l of ice-cold 0.36 M KOH�0.3 M KHCO4.The precipitates were spun down and the supernatantsseparated with anion exchange chromatography. Thetotal inositol phosphate fraction was isolated and theradioactivity determined by scintillation counting.

For the IP3 measurements, the cells were not loadedwith myo-[3H]inositol but instead treated as for Ca2�

measurements but without probenecid. They weredetached, washed and resuspended at 37�C degrees. Thestimulations were performed at 37�C for 5–15 seconds,and the reactions were stopped by addition of ice-cold


perchloric acid to a final concentrations of 4% (v/v). Thesamples were neutralized with 1.5 M KOH and �60 mMHepes and Tris–HCl buffer (pH 8.6) was added to a finalconcentration of 0.1 M. The precipitates were removedand the IP3 content of the samples was analyzed with[3H]IP3 radioreceptor assay kit (NEN Life ScienceProducts, Boston, MA). The radioactivity was determinedby scintillation counting.

Patch-clamp experiments with ICRAC in RBL-1 cells

Patch-clamp experiments were performed in the tight-seal, whole-cell configuration at room temperature(24�2�C) in a standard bath solution containing 140 mMNaCl, 2.8 mM KCl, 10 mM CaCl2, 2mM MgCl2, 5 mMCsCl, 3 mM glucose, and 10 mM Hepes (pH 7.4. withNaOH). CsCl was included to inhibit potassium currents.Patch pipettes were pulled from borosilicate glass(Kimax-51, Kimble, Vineland, NJ), Sylgard coated and firepolished. They had resistance of 2–4 M� when filledwith a standard pipette buffer containing 145 mM Cs-glutamate, 8 mM NaCl, 10 mM EGTA, 3 mM Mg-ATP, and10 mM Hepes (pH 7.2 with CsOH). The osmolarity of thesolutions was 295�5 mosM. Current recordings wereperformed with a computer-based patch-clamp amplifiersystem (EPC-9, HEKA Elektronik GmbH, Lambrecht,Germany) controlled by Pulse software (HEKA). ICRAC

was measured using voltage ramps (�100 mV to�100 mV in 50 ms) applied once in every 2 seconds fromthe holding potential of 0mV, and the Ca2� currentwas analyzed at the ramp segment corresponding to�80 mV. All currents were leak-subtracted by averagingthe first 2 to 6 ramps after breaking in and then sub-tracting this from all subsequent traces. Capacitative cur-rents were cancelled before each voltage ramp using theautomatic compensation of the EPC-9 amplifier. Seriesresistance was in the range of 3–12 M�.

Data analysis

In the Results section, mean � standard error is given.n refers to the number of the measurements; even whennot indicated, the experiments have been performed inat least triplicate with similar results, and with at leasttwo separate batches of cells. Student’s two-tailed non-paired t -test was used for evaluation of statisticalsignificance.

RESULTS

The effect of 2-APB on the IP3-dependent Ca2� releasein CHO-hOX1 cells

2-APB was introduced as a cell-permeant IP3 receptorinhibitor (see Introduction). We thus wanted to test itseffect on IP3-dependent Ca2� responses via endogenous

Cell Calcium (2001) 30(2), 117–129


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and heterologous receptors (P2Y purinoceptors and OX1

orexin receptors, respectively) in CHO-hOX1 cell. Tolargely exclude the effect of any known or putativereceptor-operated Ca2� influx pathway the experimentswere performed in 140 nM Ca2�

e (extracellular Ca2�

concentration). Under these conditions, both receptorscause a moderate elevation (� 2–3 times) in the IP3 pro-duction in 140 nM Ca2�

e [data not shown; see also19–21]. Stimulation of either receptor (10 �M ATP, Fig. 1Aand 100 nM orexin-A, Fig. 1B) caused a marked Ca2�

release. The decaying Ca2� response shot under thebaseline in both cases, and recovered slowly. This base-line ‘overshoot’ is suggested to be due to sustained over-activity of the Ca2� pumps [22,23; see also 19]. A 5-minpreincubation with 100 �M 2-APB caused a significantthough small reduction in the maximum peak amplitudeof the Ca2� response to 10 �M ATP (Fig. 1A; see alsoFig. 3G, light gray bars and white bars with black stripes,respectively). In contrast, the response to 100nM orexin-Awas barely affected by 2-APB (Fig. 1B). This suggests that2-APB inefficiently inhibits IP3-dependent Ca2� mobi-lization in CHO-hOX1 cells.

Receptor-independent Ca2� release and influxresponses in CHO-hOX1 cells

2-APB has previously been reported to affect store-operated Ca2� influx [18]. We thus studied its effecton thapsigargin-induced Ca2� elevations. In 1 mMCa2�

e, thapsigargin induced a peak Ca2� elevation,which decayed to a slight sustained elevation (Fig. 2A).Preincubation for 5 min with 100 �M 2-APB and reduc-tion of Ca2�

e to 140 nM had an identical effect on thethapsigargin response: the peak elevation was left intact

Cell Calcium (2001) 30(2), 117–129

Fig. 1 The effect of 2-APB on Ca2� release in CHO-hOX1 cells. The ceATP (A) or 100 nM orexin-A (B) in 140 nM Ca2�

e was added.

but the sustained phase was eliminated (Figs 2A–C).Thus, there was no additional effect of 2-APB in 140 nMCa2�

e. (Fig. 2B). In some batches of cells a non-completeinhibition of the sustained phase was seen. 5 mM NiCl2also eliminated the influx component of the thapsigarginresponse (data not shown). When Ca2�

e was increasedfrom 140 nM to 1 mM after thapsigargin, 2-APB inhibitedthe appearing Ca2� influx (Fig. 2B). In some batches ofcells a full inhibition was seen whereas in other batchesonly a partial inhibition was obtained. Addition of differ-ent concentrations of 2-APB after thapsigargin caused arapid reduction in the intracellular Ca2� level with abaseline ‘overshoot’ at 100 �M 2-APB (Fig. 2D).

In contrast to the shorter preincubation time (5 min), a1 h incubation with 100 �M 2-APB significantly inhibitedeven the thapsigargin-induced peak Ca2� elevationboth in 1 mM and 140 nM Ca2�

e (Fig. 2E). In differentbatches of cells an inhibition between 25% and 50% wasseen.

Store-operated Ca2� influx was also assessed using the‘Mn2�-quench’ method [24]. 2-APB partially inhibitedthapsigargin-stimulated Mn2� influx (Fig. 2F).

Effect of 2-APB on the different components of theATP-induced Ca2� responses in CHO-hOX1 cells

In 1 mM Ca2�e, 100 �M 2-APB (5-min preincubation) still

caused a small inhibition of the peak Ca2� elevation to10 �M ATP (Figs 3A, B, D & G [black and dark gray bars,respectively]; compare to Fig. 1A). On the contrary, thesustained Ca2� elevation was completely abolished asseen in comparison with the ATP response in 140 nMCa2�

e (Fig. 3A). 5 mM NiCl2 also caused a similar inhibi-tion (data not shown). 2-APB caused a similar ‘baseline


lls were preincubated with 100 �M 2-APB for 5 min whereafter 10 �M


Fig. 2 The effect of 2-APB on non-receptor-mediated Ca2� responses in CHO-hOX1 cells. Control thapsigargin (2 �M) response (thicktraces) and the response to 2 �M thapsigargin after 5 min preincubation with 100 �M 2-APB (thin traces) in 1 mM (A) and 140 nM (B) Ca2�

e.Also shown is the restoration of 1 mM Ca2�

e after thapsigargin (B). (C) The average responses in 1 mM Ca2�e (n�3–4) with the maximum

peak response and the response 1 and 2 min after the addition of thapsigargin for cells under control conditions (black bars) and cellstreated with 100�M 2-APB for 5 min (white bars). �[Ca2�]i�change in [Ca2�]i (stimulated–basal). Comparisons are to the correspondingtime points under control conditions: ns, not significant, P�0.05; *, P�0.05; **, P�0.01. (D) Addition of different concentrations of 2-APB(thin trace) after 2 �M thapsigargin in 1 mM Ca2�

e. (E) The average maximum peak responses to thapsigargin in 1 mM and 140 nM Ca2�e

(n�4–6) after long-term (1 h) 2-APB (100 �M) preincubation (white bars) as compared to ctrl cells (black bars). **, P�0.01; ***, P�0.001.(F) Normalized Fura-2 fluorescence traces at the excitation wavelength 359 nm. The Fura-2 fluorescence of the cells was monitored in TBMwith no added Ca2� (Ca2�

e�1 �M) at the excitation wavelengths 340 and 359 nm, and 100 �M 2-APB and 2 �M thapsigargin were addedsequentially. No responses were seen at the 359 nm but thapsigargin caused a fluorescence increase at 340 nm due to the Ca2� release(data not shown). Subsequent addition of 30 �M MnCl2 caused a quenching of the Fura-2 fluorescence at both excitation wavelengths. Mn2�

quench response in the presence of 2-APB and thapsigargin (dotted trace) is compared to the Mn2� quench response in the cells not treatedwith 2-APB (thin solid trace) and to the Mn2� quench in the cells in the absence of 2-APB and thapsigargin (thick solid trace).

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overshoot’ as reduction of Ca2�e to 140 nM. The ‘over-

shoot’ was not caused by the reduction in the peakamplitude since 3.5 �M ATP, which caused a substan-tially lower response than 10 �M ATP, did not cause any‘overshoot’ (Fig. 3B). The inhibition of the sustainedCa2� influx was also seen when the cells were stimu-lated with 10 �M ATP in the presence of 140 nM Ca2�

e

followed by restoration of 1 mM Ca2�e (Fig. 3C).

The inhibition of the Ca2� influx by 2-APB was con-centration-dependent (Fig. 3D). 2-APB caused a rapid andconcentration-dependent reduction in [Ca2�]i also whenadded during the sustained phase of the ATP-inducedCa2� response, 100 �M producing the typical baseline‘overshoot’ (Fig. 3D).


Further experiments with 100 �M 2-APB for 30 secondsbefore the addition of 10 �M ATP (Fig. 3E), showed inagreement with the experiments above, that a full inhi-bition of the influx response is obtained almost imme-diately since no additional inhibition of the influx phaseis seen after a longer preincubation (Fig. 3G, whitebars and dark gray bars, respectively). In fact, the inhibi-tions produced by reduction of Ca2�

e to 140 nM and30 seconds incubation with 100 �M 2-APB are indis-tinguishable (Fig. 3G, light gray and white bars,respectively).

A longer preincubation (5 min and 1 h) with 100 �M2-APB caused a progressive inhibition of the peakresponse to ATP (Figs 3E & G). However, the inhibition

Cell Calcium (2001) 30(2), 117–129

Cell Calcium (2001) 30(2), 117–129 © 2001 Harcourt Publishers Ltd

Fig. 3 The effect of 2-APB on P2Y-purinoceptor-mediated Ca2� responses in CHO-hOX1 cells. (A) Control response to 10 �M ATP (thicksolid trace) and the response after 5 min preincubation with 100 �M 2-APB (thin solid trace) in 1 mM Ca2�

e. Also overlaid for comparison isthe ATP response in 140 nM Ca2]

e (dotted trace). (B) The response to 10 �M ATP after 5 min preincubation with 2-APB (thin solid trace) iscompared to the response to 10 �M (thick solid trace) and 3.5 �M ATP (dotted trace) under control conditions in 1 mM Ca2�

e. (C) Theresponse to 10 �M ATP in 140 nM Ca2�

e in control cells (thick trace) and in cells pretreated with 100 �M 2-APB for 5 min (thin trace). 1 mMCa2�

e is restored where indicated. (D) The response to 10 �M ATP after 5 min preincubation with different concentrations of 2-APB (0 �M,thick solid trace; 10 �M thin solid trace; 100 �M, dotted trace) in 1 mM Ca2�

e. For the trace in the presence of 10 �M 2-APB, 100 �M 2-APB isadded after the ATP-stimulation where indicated. (E) The response to 10 �M ATP in cells treated with 100 �M 2-APB for 30 s (thin solid trace)and for 1 h before stimulation (dotted trace) as compared to the nontreated cells (thick solid trace). (F) The effect of wash of 1 min (thin solidtrace) or 15 min (dotted trace) of the 2-APB-pretreated cells (100 �M, 1 h) on the inhibition of ATP (10 �M) response as compared to controlcells (thick solid trace) and cells treated with 100 �M 2-APB for 30 seconds (dashed trace). (G) the statistics (n�3–17) for the ATP (10 �M)responses under different conditions: 1 mM and 140 nM Ca2�

e under control conditions (black solid bars and light gray bars, respectively),after 30 seconds, 5 min and 1 h pretreatment with 100 �M 2-APB in 1 mM Ca2�

e (white solid bars, dark gray bars and black bars with whitestripes, respectively) and after 5 min preincubation with 100 �M 2-APB in 140 nM Ca2�

e (white bars with black stripes). The maximum peakresponse and the response 1 and 2 min after the addition of receptor agonist are presented. The first comparisons are to the responseunder control conditions in 1 mM Ca2�

e (black solid bars). ns, not significant, P�0.05; *P�0.05; **, P�0.01; ***, P�0.001. Also indicated isa second comparison of 2-APB for 1 h to 2-APB for 30 seconds or 5 min (††, P�0.01). A third comparison of non-treated cells in 140 nM to2-APB-pretreated cells (30 s) in 1 mM Ca2� gives P�0.05 (not significant).

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of the sustained Ca2� response was not furtherenhanced (Fig. 3E). Thus, while the onset of the 2-APBeffect on ATP-induced Ca2� influx is rapid (seconds) andcan be seen even at lower 2-APB concentrations, theeffect on the Ca2� release requires minutes or even tensof minutes of incubation with maximum 2-APB concen-tration. Actually, the inhibition of the sustained Ca2�

influx by 2-APB was apparently reduced (P�0.01) after1 h preincubation as compared to 30 seconds or 5 minincubation (Figs 3E & G). This is likely to reflect the radi-cally reduced Ca2� release, which reduces the activationof Ca2� pumps, which in its turn results in a smaller‘overshoot’. After 1 h preincubation, the sustainedresponse was rapidly restored after wash (1 min) whereasa complete restoration of the peak response required amore long-lasting wash (15 min; Fig. 3F).


Fig. 4 The effect of 2-APB on OX1 receptor-mediated Ca2� responsesorexin-A in 1 mM (thick traces) and 140 nM (thin traces) Ca2�

e. In (B) 1orexin-A after 5 min preincubation with different concentrations of 2-APB100 �M, dashed trace) in 1 mM Ca2�

e. (D) Addition of 100 �M (thick tracorexin-A in 1 mM Ca2�

e. (E) The statistics (n�5–9) for the orexin-A resp5 min preincubation with 100 �M 2-APB (white bars). ns, not significant,ATP and 30 nM orexin-A to test whether the block by 2-APB would be IPpretreated with 100 �M 2-APB for 5 min.

The effect of 2-APB on the different components of theorexin-A-induced Ca2� responses in CHO-hOX1 cells

Stimulation of CHO-hOX1 cells with 100 nM orexin-Aresulted in release from the intracellular stores followedby Ca2� influx, as indicated by the disappearance of thesustained Ca2� response when the Ca2�

e was reduced to140 nM (Fig. 4A). The influx-dependent minor part inorexin-responses may be due to the receptor-activatedchannel [19].

3 nM orexin-A also caused a peak and sustained Ca2�

response in 1 mM Ca2�e (Figs 4B–D); however, neither

response was seen in 140 nM Ca2�e (Fig. 4B) since the

maximum peak Ca2� response to low concentrations oforexin-A is dependent on Ca2� influx and even the IP3

production requires Ca2� influx [19]. The massive IP3

production brought about secondarily to the receptor

Cell Calcium (2001) 30(2), 117–129

in CHO-hOX1 cells. The response to 100 nM (A) and 3 nM (B)mM Ca2�

e is restored where indicated. (C) The response to 3 nM (0 �M, thick solid trace; 1 �M, thin solid trace; 10 �M, dotted trace;e) or different concentrations of 2-APB (thin trace) after 3 nMonse in 1 mM Ca2�

e under control conditions (black bars) and after P�0.05; **, P�0.01. (F) cumulative additions of 100 nM and 100 �M3 receptor use-dependent. Thick trace, control cells; thin trace, cells


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activation and Ca2� influx in 1 mM Ca2� is yet likely tocause some release and store-operated Ca2� influx.Restoration of 1 mM Ca2�

e after 3 nM orexin-A in140 nM Ca2�

e caused appearance of the Ca2� influx-dependent components (Fig. 4B).

The peak Ca2� response to 3 nM orexin-A was notaffected by 2-APB preincubation (100 �M, 5min) (Figs 4C& E). However, the sustained influx was concentration-dependently inhibited by 2-APB, both when preincubatedand when added after orexin-A (Figs 4C–E). 100 �M 2-APBcaused ‘overshoot’ of the baseline (Fig 4C–E).

We also used cumulative additions of different con-centrations of ATP and orexin-A to test whether theblock of the IP3-dependent Ca2� release could be IP3

receptor use-dependent. However, this does not seem tobe the case, as seen in Figure 4F. In inositol phosphatemeasurements, 100 �M 2-APB preincubated for 10 mindid not inhibit the inositol phosphate production by anyof the orexin-A concentrations tested (0, 0.1, 1, 10 and100 nM; data not shown).

Patch-clamp and Ca2� measurements with RBL cells

The results with CHO-hOX1 cells suggest that 2-APBinhibits (some) store-operated Ca2� influx pathways (i)independently of its effect on IP3 receptor, and (ii) fromthe extracellular side. We thus wanted to test thishypothesis by investigating its effect on the most well-characterized store-operated Ca2� influx pathway, theCRAC channel. RBL-1 cells are a widely used modelsystem for ICRAC, which was specifically investigated byusing patch-clamp technique. Inclusion of 10 mM EGTAin the pipette solution activated ICRAC (Figs 5A & B) aspreviously described in, for example [25]. Whenincluded in the extracellular solution, 100 �M 2-APB eli-minated ICRAC completely (Fig. 5A). On the contrary, whenincluded in the pipette solution, only a non-completeinhibition was seen (Fig. 5A). This may suggest that theblocking action of 2-APB on the ICRAC occurs from theextracellular side, or at another site close to the plasmamembrane with better access from the extracellular side.When added from the extracellular side after the activa-tion of ICRAC, 2-APB blocked the major part of the cur-rent within seconds (Fig. 5B).

In Ca2� measurements, 100 �M 2-APB (5 min)markedly but non-completely reduced the ATP- (10 �M;data not shown), NECA- (40 �M; data not shown) andthrombin-induced (2 U/ml; Fig. 5C) peak Ca2� eleva-tions. The sustained Ca2� elevation appeared to be com-pletely inhibited when 2-APB was added after thereceptor-response but a less than complete effect wasseen with 2-APB preincubation. A more marked effecton the Ca2� influx was seen with thapsigargin (2 �M) asthe thapsigargin response was apparently much more

Cell Calcium (2001) 30(2), 117–129

dependent on Ca2� influx (Fig. 5D). However, even herea minor part of the response was not blocked by 2-APBpreincubation. This puzzling effect may be explained bythat even after an apparently complete attenuation ofthe Ca2� influx by 2-APB addition some influx may per-sist, but that this influx is balanced by the Ca2� pumpoveractivity under particular conditions. Similar receptor-responses to 2-APB were seen in RBL-2H3 cells (data notshown).

Ca2� measurements with SH-SY5Y cells

Very little is known about SOCs in neuronal cells. Forcomparison with CHO and RBL cells, we tested the effectof 2-APB both on IP3-dependent Ca2� release and store-operated Ca2� influx in a human neuronal cell line, SH-SY5Y, which expresses muscarinic cholinoceptorscoupled to IP3 elevation, Ca2� release and store-operatedinflux [data not shown for inositol phosphates and IP3;see also 26–29]. Here no inhibition of the carbamyl-choline-induced (10 �M or 1 mM) peak or sustainedCa2� response by 2-APB could be seen in 1 mM or140 nM Ca2�

e (Figs 6A–E). Reduction of Ca2�e to 140 nM

(Figs 6B & D) obliterated the sustained influx present in1 mM Ca2�

e (Figs 6A, C & E). To investigate the possibil-ity of use-dependent block of the IP3 receptor by 2-APBin SH-SY5Y, we performed cumulative additions of10 �M and 1 mM carbamylcholine (Figs 6A & B). Theonly effect seen was an insignificant reduction in the10 �M peak and a corresponding increase in 1 mM peakindicative of a slight shift in the carbamylcholineconcentration-response curve (Fig. 6E). The peak andsustained Ca2� responses to 2 �M thapsigargin were alsoinvestigated. A 5 min preincubation with 100 �M 2-APBleft the thapsigargin response intact (Fig. 6F). Altogether,the Ca2� responses in SH-SY5Y cells seem to be com-pletely resistant to 2-APB.

DISCUSSION

The results of the present study show that 2-APB causesa time-dependent inhibition of Ca2� release in some butnot in other cells. In addition, it causes an instantaneousblock of some Ca2� influx pathways.

Ca2� release

2-APB has been widely used as a selective blocker ofIP3 receptors in a variety of tissues and a few cellslines. Only in HEK293, DDT1-MF2, A7r5 and rat pan-creatic acinar cells, in human neutrophils and platelets,rabbit aorta and rat cerebellar microsomes have IP3-dependent responses been implicated as a target for itsaction [16,18,30]. The inhibition produced by 2-APB at



Fig. 5 The effect of 2-APB on Ca2� release and influx responses in RBL-1 cells. (A) and (B) Patch-clamp measurements of ICRAC as repre-sented by the current at �80 mV. (A) average currents (�SEM) of 13 control cells (�2-APB), seven cells with 100 �M 2-APB included in theextracellular solution (�100 �M 2-APB (extracellular)) and 10 cells with 100 �M 2-APB included in the pipette solution (�100 �M 2-APB[intracellular]). (B) A representative trace of addition of 100 �M 2-APB from the extracellular side after the activation of ICRAC. The dashedtrace indicates the extrapolated baseline. Observe, that the fast component of the inhibition, comprising to 90% of the total inhibition, reachesits maximum within 10 s of the 2-APB application. For the Ca2� measurements (C and D), the cells were either preincubated with 100 �M 2-APB for 5 min after which 2 U/ml thrombin (C) or 2 �M thapsigargin (D) were added (thin traces). Alternatively, 100 �M 2-APB was addedafter the stimulation of the non-treated cells where indicated (thick traces).

CECA-65.QXD 6/30/01 12:45 AM Page 125

physiologically relevant concentrations of IP3 is ratherinsignificant in rat cerebellar microsomes [16]. On theother hand, the block of IP3 dependent responses isprominent in HEK293 and DDT1-MF2 cells [18]. In thepresent study, there was a noncomplete inhibition of theputatively IP3-dependent Ca2� release from the CHO-hOX1 and RBL-1 cells after a 5 min preincubation with100 �M 2-APB, whereas the IP3-independent Ca2� peakresponse to 3 nM orexin-A in CHO cells was unaffected.In SH-SY5Y cells, the IP3-dependent muscarinic receptorresponse was not inhibited by 2-APB.

Why is the block of the IP3-dependent Ca2� release by2-APB so different in different cell types? One interesting


possibility is offered by the IP3 receptor subtype expres-sion: SH-SY5Y cells: only type 1 receptor; rat cerebellum:type 1 (and 2); CHO-K1 cells: all the subtypes equally;HEK293 and rat pancreatic acinar cells: almost equalamounts of type 2 and 3 receptors (or possibly domi-nantly type 2 for the latter); A7r5 cells: mainly type 1 butalso �25% type 3; rat hepatocytes: mainly type 2 butalso �20% type 1; DTT1-MF2 cells: at least type 1 and 3.mRNA measurements indicate expression of all the sub-types in RBL-2H3 cells [31,32, reviewed in 33]. Thus itappears as the preparations that rely mainly on type 1IP3 receptor (SH-SY5Y cells and rat cerebellar micro-somes) are essentially resistant to 2-APB, whereas the

Cell Calcium (2001) 30(2), 117–129


Fig. 6 The effect of 2-APB on muscarinic receptor-mediated Ca2� responses in SH-SY5Y cells. (A) Responses to cumulative addition of10 �M and 1 mM carbamylcholine (CCh) in control cells (thick solid traces) and in the cells treated with 100 �M 2-APB for 5 min (thin solidtraces). The effect of similar treatments on carbamycholine (1 mM) response in 1 mM (C) and 140 nM Ca2�

e (D). (E) The statistics of theresponses in (A) with the maximum peak response to 10 �M carbamylcholine, the response 2 min after the addition of 10 �M carbamyl-choline and the maximum peak response to 1 mM carbamylcholine after 10 �M carbamylcholine. Black bars, control cells; white bars, 2-APB-pretreated cells (100 �M, 5 min). ns, not significant, P�0.05; *, P�0.05; **, P�0.01. (F) Thapsigargin (2 �M) response in control cells (thicktrace) and in cells treated with 2-APB for 5 min (thin trace).

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preparations that contain type 3 IP3 receptors in signifi-cant amounts (CHO-K1, HEK293, RBL, DTT1-MF2 and ratpancreatic acinar cells) show at least some sensitivity to2-APB. A7r5 cells appear somewhat odd since they con-tain mainly type 1 receptors but the inhibition couldcome through the yet expressed type 3 receptors. Type 2receptors may also be insensitive to 2-APB since IP3

responses in rat hepatocytes are not affected [34]; how-ever, Ca2� spikes in rat pancreatic acinar cells areaffected [16,35], although the latter could result from theeffects on type 3 receptors and/or SOCs. Putting thescheme in other words, the expression of type 1 IP3

receptor may correlate negatively and the expression oftype 3 IP3 receptor may correlate positively with the sen-sitivity to 2-APB. This is an attractive hypothesis sincethe sensitivity of the different subtypes to 2-APB is not

Cell Calcium (2001) 30(2), 117–129

known, but cannot yet be backed up by other thanphenomenological evidence. Whether this or any otherexplanation is correct has to be settled by furtherexperimentation.

Long incubation times with 2-APB may cause unspe-cific effects on the Ca2� release, as suggested by themarked decrease in the thapsigargin peak response inCHO-hOX1 cells after 1 h preincubation with 100 �M2-APB. This may be due to depletion of the intracellularCa2� stores, e.g. via inhibition of SERCAs, as shown forA7r5 cells [30]. The inhibition produced by 2-APB on theATP response after 1 h preicubation could well beexplained by an additive effect of the faster inhibition ofthe Ca2� response (�35%) and a long-term pooldepletion (25–50%). This reasoning is further supportedby the time scale for the wash away of this effect of



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2-APB: the time of full restoration of the Ca2� responseto ATP (15 min) may (in part) correspond to the timerequired for refilling of the Ca2� stores.

Different properties of the inhibition of store-operatedCa2� influx and Ca2� release

The results show, that the inhibition of the store-operated Ca2� influx and Ca2� release by 2-APB are twoseparate phenomena: for the first, inhibition of ICRAC israpid, apparently almost full inhibition can be reachedwithin 10 seconds. In Ca2� measurements, full inhibi-tion can be reached within similar time scale, especiallywhen the time required for the out-pumping of Ca2� istaken into consideration. On the contrary, inhibition ofthe Ca2� release is non-complete even after 1 h preincu-bation. For the second, inhibition of these two pathwaysshows different concentrations requirements. 10 �M of2-APB produces a noncomplete inhibition of the influxwhereas no inhibition of the release is seen. Thus, inhi-bition of the influx can be seen under conditions whereno inhibition of the release is seen. For the third, ICRAC ismore readily inhibited by extracellularly than intracellu-larly applied 2-APB. Similarly, ICRAC in isolated inside-out patches from RBL-2H3 cells has been shown to beinhibited only by intra-pipette 2-APB [36]. This stronglysuggests that the site of action for 2-APB is on the out-side of the plasma membrane (see also below). Thus, thedifferent concentration and time requirements for theinhibition of Ca2� influx and release could, at least inpart, correspond to a more difficult access to the intracel-lular site. However, the overall low degree of inhibitionof the Ca2� release as compared to, e.g. HEK293 cells,and especially the completely absent inhibition in SH-SY5Y cells, is difficult to understand except in the lightof different expression of components of Ca2� releasecomplex.

Store-operated Ca2� influx

Interestingly, essentially no inhibition of influx was seenin SH-SY5Y cells. Similarly, experiments with CHO-hOX1

and RBL-1 cells suggest, that certain components of thestore-operated Ca2� and Mn2� influx are insensitive to2-APB. It is plausible that 2-APB displays some specificityfor a particular type of Ca2� influx, probably similar tothe CRAC channel. CRAC channel has a low single-channel conductance for Ca2�, compared to the otherchannels activated by store-depletion [reviewed in12–14]. These channels also display different ionic selec-tivity than CRAC channel. Mn2� is more slowly permeat-ing than Ca2� and works effectively as a blocker of ICRAC,which would exaggerate the non-CRAC dependent partof divalent cation influx when monitored using Mn2�.


ICRAC – as also the other SOCs – has been shown in avery few cell types, mainly of hematopoietic origin.Among the cells used in the present study, the expres-sion of SOCs has only been investigated in RBL cells,which may express both ICRAC and other SOCs [37,reviewed in 12]. Especially prominent is the lack ofpatch-clamp data from excitable cells, where mainlyFura-2 measurements show prominent store-operatedCa2� influxes. Thus, the expression of different types ofSOCs in excitable cells is largely unknown. In conclu-sion, any differences in the ability of 2-APB to blockSOCs probably depends on the expression of particulartypes of SOCs in the cell. It is thus possible that 2-APB-sensitive part of the Ca2� (and Mn2�) influx occursthrough ICRAC-like channels and the 2-APB-insensitivepart through some of the other types of channels.

The results of this study show that 2-APB is a blockerof store-operated Ca2� influx. This occurs more potentlythan the block of the release, and even under conditionswhere no block of release can be seen. This suggests adistinct mechanism for these processes. Our results alsoindicate that the block of the Ca2� influx occurs fromthe extracellular side [see also 36]. Why 2-APB woulddisplay block of both IP3 receptor and CRAC channel/SOCs remains elusive. Mozhayeva and coworkers [38,39]have shown that the IP3-activated miniature Ca2� chan-nels in the plasma membrane share many of the proper-ties of the IP3 receptor channel in the ER. Although it isunlikely that CRAC channel with minute single-channelconductance would be identical to the IP3 receptor witha large single-channel conductance, it is possible thatthey would share common components or similar chan-nel motives. 2-APB displays inhibition of both Ca2�

release and influx, yet it is not completely non-selective.It has previously been shown not to block L-type voltage-gated Ca2� channels [16], and we could in the presentstudy show that it did not affect certain the store-operated Ca2� fluxes and it also left the OX1 receptor-operated Ca2� influx unchanged. Development ofanalogues of 2-APB would offer an interesting tool forfurther investigations, if they were (1) cell-impermeantfor studies on SOCs, and (2) otherwise modified to abol-ish the SOC and SERCA but not IP3 receptor block.

ACKNOWLEDGMENTS

Dr Martin Bootman (Laboratory of Molecular Signalling,The Babraham Institute, Cambridge, UK) is gratefullyacknowledged for comments on the manuscript and onthe experimentation. This study was supported by theEuropean Union (contract N° ERBBIO4CT960699, BMH4-CT98-2343; KÅ), the Swedish Medical Research Council(KÅ), the Cancer Research Fund of Sweden (KÅ, JPK), the

Cell Calcium (2001) 30(2), 117–129


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Swedish Society for Medical research (P-EL), Åke Wibergfoundation (JPK) and Lars Hierta Foundation (JPK).

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Documents

2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca2+discharge and store-operated Ca2+influx