11 Montero 2001

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Research

Stimulation by thimerhistamine-induced Cain intact HeLa cells seae ten m

M. M aInstitutoVallado

Summ(InsPendoon thsensiprolohistamto BAintracpreseinduclevel (Mn2[Ca2operainacti

ReceivReviseAcceptPublish

CorresMol. y FValladoe-mail:

Cell Calcium (2001) 30(3), 181190 2001 Harcourt Publishers Ltd

doi:10.10

CECA-70.QXD 8/3/01 4:16 PM Page 181quorin targeted to doplasmic reticulu

ontero, M. J. Barrero, F. Torrecilla, C. D. LobODUCTION

-release from intracellular stores is mediated by-channels belonging to two main families, inositoltrisphosphate receptors (InsP3R) and ryanodinetors [1]. Activation of phospholipase C by extracellu-onists produces InsP3, which is the main activator of

InsP3RHoweon thseveraones ([Ca2

has a trationincreabiphaand mas Ca2

have induc

de Biologa y Gentica Molecular (IBGM), Departamento de Bioqumica y Biologalid and CSIC, Valladolid, Spain

ary The oxidizing thiol reagent, thimerosal, has been shown 3) receptor in several cell types. We have studied here the effecplasmic reticulum (ER) of intact HeLa cells with targeted aequoe ER-Ca2-pump and only slightly increased the ER-Ca2-letivity to histamine of ER-Ca2-release by about two ordernged at saturating histamine concentrations and enhanced bo

ine. Moreover, inhibition of ER-Ca2 release by cytosolic [CaPTA-loading, and histamine-induced Ca2 release remaineellular BAPTA. The effects of thimerosal were reversible in thnce of a physiological redox regulatory mechanism. However, ed Ca2 release but oxidized glutathione had no effect. In addiin permeabilized cells. Thimerosal partially inhibited also plas) entry through the plasma membrane, both phenomena c]. Thimerosal-induced Ca2 entry was additive to that inducted Ca2 channels may not be involved. These results providevation of InsP3 receptors. 2001 Harcourt Publishers Ltd

ed 24 January 2001d 28 April 2001ed 4 May 2001ed online 13 July 2001

pondence to: Javier Alvarez, Departamento de Bioqumica y Biol.isiologa, Facultad de Medicina, Ramn y Cajal, 7, E-47005

lid, Spain. Tel.:34 983 423 085; Fax:34 983 423 588; [email protected] of2 releaseen withhe

tn, A. Moreno, J. Alvarez

54/ceca.2001.0224, available online at http://www.idealibrary.com on and triggers Ca2-release through these channels.ver, the activity of InsP3R is not only dependente concentration of InsP3, but can be modulated byl mechanisms [2,3]. One of the most importantis the [Ca2], particularly in the cytosolic side]c) but also in the lumenal side [4,5]. Type I InsP3R

bell-shaped dependence on [Ca2]c, so that concen-s above 300 nM stimulate Ca2-release, but furtherse to above 12M inhibits Ca2-release [6,7]. Thissic mechanism appears to be very important to startaintain regenerative Ca2-release phenomena such-waves and Ca2-oscillations [8]. In HeLa cells, weshown that this mechanism controls Ca2 releaseed by histamine in intact cells [9,10]. On the other

Molecular y Fisiologa, Facultad de Medicina, Universidad de

to activate reversibly the inositol 1,4,5-trisphosphatets of thimerosal by monitoring the [Ca2] inside therin. We show that thimerosal produced little effects

ak in intact cells. Instead, thimerosal increased thes of magnitude, made the response much moreth cytosolic and mitochondrial [Ca2] responses to2] microdomains was fully preserved and sensitived quantal in the presence of both thimerosal ande presence of dithiotreitol, suggesting the possiblein permeabilized cells thimerosal potentiated InsP3-tion, thimerosal increased the [Ca2]ER steady-statema membrane Ca2 extrusion and increased Ca2ontributing to increase the steady-state cytosoliced by emptying of the ER, suggesting that store- new insights on the mechanisms of activation and

181

182 M Montero, MJ Barrero, F Torrecilla, CD Lobatn, A Moreno, J Alvarez

Cell C

hand, the lumenal [Ca2] has also been proposed to stim-ulateactivInsP3

Actreactthimevate the ameanevencasesthe p[13,1involThe rresenInsP3activhepa

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CECA-70.QXD 8/3/01 4:16 PM Page 182alcium (2001) 30(3), 181190

Ca2 release through InsP3R [4,5]. In fact, InsP3Rate spontaneously in the presence of resting levels ofwhen the Ca2-stores are overloaded [11,12].ivation of InsP3R is potentiated by some thiol-ive oxidizing agents such as oxidized glutathione orrosal [1116]. Thimerosal has been shown to acti-

InsP3R by several mechanisms, including increase offfinity for InsP3 [12,13,16,17], and increase of the open time of the channel and the conductance,

at saturating InsP3 concentrations [15]. In many, this activation has been shown to be reversible inresence of thiol-reducing agents such as dithiotreitol6], suggesting that the mechanism of activation mayve reversible alkylation of critical sulfhydril groups.eversibility of this effect suggests that it could rep-t a physiological mechanism of redox modulation ofR. In fact, oxidized glutathione has been shown toate InsP3-induced Ca2 release in permeabilizedtocytes [11,12] and endothelial cells [14].e investigation of the mechanisms of the effects ofrosal at different points of the Ca2-homeostaticinery may provide important clues to understandmodulation under physiological conditions. It isus that, being a general thiol-oxidizing agent, thim-l is probably acting on many cysteine residues, thuscing many different effects. For example, regarding

a2-homeostasis, thimerosal has been shown toit the ER Ca2-ATPase [13,18], although the effec-oncentration required was highly variable. We havehere a new methodology that allows measuring] inside the ER ([Ca2]ER) of intact cells using ER-tar- aequorin [9,10]. This method is ideal to followfically the effect of thimerosal on InsP3-induced-release, independently of any Ca2 fluxes occurringe plasma membrane. Moreover, it allows studyingirectly in intact cells the rate of Ca2-pumping intoR, the steady-state [Ca2]ER and the rate of Ca2-rom the ER in the presence of thimerosal.

ERIAL AND METHODS

]ER measurements cell clones EM26 and EM56, producing ER-targeteda2-affinity mutated aequorin [19] were grown in

eccos modified Eagles medium supplemented withfoetal calf serum and 0.2 mg/ml G418. Cell clones

plated onto 13 mm round coverslips. Before recon-ing aequorin, [Ca2]ER was reduced by incubatingells for 510 min at 37C with the sarcoplasmic andplasmic reticulum Ca2-ATPase (SERCA) inhibitori-tert-buthyl-benzohydroquinone (BHQ) 10M inard external medium containing (in mM): NaCl,

plemfor 1ing 0The of a dardthe cellsand intra10; MpH EGTto rebuffMeaues a cocurv

Mito

HeLmedweretransmid Aftewithmenvalu

[Ca2

Cellswereincuweretropas dlated340 [Ca2

the in orsamrate a Caand Ca2

%F3obtafrom 2001 Harcourt Publishers Ltd

ented with 3 mM EGTA. Cells were then incubatedh at room temperature in standard medium contain-.5 mM EGTA, 10M BHQ and 1M coelenterazine n.overslip was then placed in the perfusion chamberurpose-built thermostatized luminometer, and stan-medium containing 1mM Ca2 was perfused to refillR with Ca2. For experiments with permeabilizedthe coverslip was placed in the perfusion chamberreated for 1 min with 100M digitonin suspended inellular medium containing (in mM): KCl, 130; NaCl,gCl2, 1; K3PO4, 1; EGTA, 0,2; ATP-Mg, 1; Hepes, 20,. Then, intracellular medium containing a Ca2- buffer providing a [Ca2] of 100 nM was perfusedill the ER. In some experiments, the 100 nM Ca2

r was included also in the permeabilization medium.urements were performed at 22C and [Ca2]ER val-ere calculated from the luminescence records usingputer algorithm [20] which follows the calibration reported before [10].

hondrial [Ca2] ([Ca2]M) measurements cells were grown in Dulbeccos modified Eaglesm supplemented with 10% foetal calf serum. Cells

plated onto 13mm round coverslips and transfectedently using Fugene (Gibco) with a pCDNA 3.1 plas-ncoding mitochondrially targeted wild-type aequorin. 1824 h, aequorin was reconstituted by incubation1M coelenterazine for 1 h prior to the measure-s. Experiments were carried out at 22C and [Ca2]Ms were calculated as described above.

]c measurementsfrom the same EM26 and EM56 HeLa cell clonesplated on coverslips and loaded with Fura-2 byation for 1 h with 4M Fura-2AM. Measurementsperformed in cell monolayers using a Cairn spec-otometer equipped with a six-filter rotating wheelscribed previously [21]. [Ca2] values were calcu-from the ratio between the fluorescence obtained atnd 380 nm excitation wavelength. Cells used for]c measurements were always depleted of Ca2 in

ame way as those used for [Ca2]ER measurements,er to allow comparison of both types of data in the

conditions. Ca2 entry was studied by following thef Fura-2 quenching induced by Mn2 entry, used as surrogate. Cells were loaded with Fura-2 as abovehe fluorescence excited at 360 nm (insensitive to) was monitored. Data were then normalized as. Coelenterazine n, Fura2-AM and BAPTA-AM were

ned from Molecular Probes. Other reagents wereSigma, Madrid or Merck, Darmstadt.

Stimulation by thimerosal of Ca2 release 183

Cell Calcium (2001) 30(3), 181190 2001

RESULTS

Reconstitution of ER-targeted aequorin with the semisyn-thetic prosthetic group, coelenterazine n, requires previ-ous depletion of Ca2 of the ER to prevent aequorinconsumption during reconstitution [9]. Once aequorinhas been reconstituted, the ER is refilled again with Ca2

by perfusing the cells with extracellular medium contain-ing 1 mM Ca2. This leads to an increase in [Ca2]ER, thatreaches a steady-state of around 600M within 35 minin HeLa cells [9,10]. Figure 1 shows a comparison of theeffects of histamine on both [Ca2]ER (upper panels) and[Ca2]c ( lower panels), either in the presence or in theabsence of thimerosal or dithiotreitol. Comparing panelsa and b, we can see that the rate of refilling of the ER wasnot significantly modified by thimerosal. In fact, Figure 1shows that even the refilling of the ER after histamineaddition, i.e. after 1520min in the presence of thimerosal100M, proceeded at a similar rate. In 15 similar experi-ments, the rate of refilling of the ER in cells treated for atleast 7 min with thimerosal 100M was 1.80.2M/s(meanSD). This value is identical to that obtained undercontrol conditions (1.80.5M/s, see [10]). The rate ineach experiment was obtained by averaging the rate ofrefilling during 100 s within the period of maximum rateof ER refilling. Another parameter that should be affectedif the ER-Ca2-pump was inhibited by thimerosal is the

steady-state [Ca2]ER. In a series of experiments per-formed in parallel in control cells or in cells treated for atleast 5 min with 100M thimerosal, the steady-state[Ca2]ER was 66080 (meanSD, n30) in control cellsand 71090 (meanSD, n36) in cells treated withthimerosal. These data suggest that thimerosal does notmodify significantly the activity of the ER-Ca2-ATPase(SERCA, from sarcoplasmic and endoplasmic reticulumCa2-ATPase) in intact HeLa cells.

A very distinct effect of thimerosal was observedinstead on histamine-induced [Ca2]c peaks and [Ca2]ERdecreases. Panels a and d of Figure 1 show the effect ofhistamine on both [Ca2]ER and [Ca2]c in control cells.As we have reported previously [10], histamine producesa small and fast [Ca2]ER decrease (panel a), that trans-lates in the cytosol into the [Ca2]c peak shown in paneld. If the cells were pretreated with thimerosal for 57 min,the effects of histamine on both [Ca2]ER and [Ca2]cwere much stronger (panels b & e) and had a very pecu-liar kinetics. Histamine induced a fast drop in [Ca2]ERcoincident with the [Ca2]c peak. This was followed by ashort period of rapid refilling and then by a second andmuch slower phase of [Ca2]ER decrease. This secondphase was coincident with a persistent increase in[Ca2]c. A magnification of these experiments, shown inthe inset, reveals interesting kinetic details. The initial

Fig. 1 crecons dhistam sof the h tiv

CECA-70.QXD 8/3/01 4:16 PM Page 183 Harcourt Publishers Ltd

Effects of thimerosal and dithiotreitol on histamine-induced [Ca2]ER detituted with coelenterazine n (panels ac) or loaded with Fura-2 (panels ine (His), 100M thimerosal (Thim) or 1 mM dithiotreitol (DTT) was perfuistamine addition in panels b and e. These experiments are representarease and [Ca2]c increase in HeLa cells. Cells weref). Then, medium containing 1 mM Ca2 (Ca2), 100Med as indicated. The inset shows a magnified superimpositione of from five to 13 similar ones of each kind.


Cell C

fast [Ca2]ER decrease coincides with the [Ca2]c peak.Thenfor a backgbitionleads[Ca2

increwas wlevelsmallypreseshoutent ition. rapidbeformatcpare reducER. Oat therestineffectinduc(not reduchistamthimebeforof dimineof thepresemeanstep oprese(mean10

Thcentr10Mmine100vatioit haInsP3InsP3expecmorehistapanedetecsient

thimerosal, the same concentration of histamine inducedrong decrease in [Ca2]ER with the typical biphasictics. The [Ca2]c peak was also bigger and followed persistent increase in [Ca2]c.

he effect of histamine on [Ca2]ER in HeLa cells isngly potenciated by loading the cells with a Ca2-lator, BAPTA [9,10]. We have proposed that this effect be due to the inhibition of Ca2 release by local]c microdomains in the absence of BAPTA. Figure 3

ws that this effect was additive to that of thimerosal. left panel shows the effect of histamine on [Ca2]ER in control and in BAPTA-loaded cells. Histamine pro-

ed a very fast decrease of [Ca2]ER (about 80%) in

2 Effect of thimerosal on the [Ca2]ER decrease and [Ca2]case induced by 2.5M histamine. Cells were reconstituted with

enterazine n (lower panels) or loaded with Fura-2 (upperls). Then, medium containing 1 mM Ca2 (Ca2), 2.5Mmine (His 2.5M) or 100M thimerosal (Thim) was perfuseddicated. These experiments are representative of from five toimilar ones of each kind.

3m

coTTAMsm


, Ca2 release stops suddenly and [Ca2]ER increasesfew seconds while [Ca2]c decreases down to nearround levels. These phenomena suggest that inhi- of Ca2 release by local [Ca2]c microdomains

to rapid back-pumping of Ca2 into the ER. Finally,]ER turns to decrease again slowly while [Ca2]c

ases and stabilizes around 400 nM. Once histamineashed away, [Ca2]c suddenly dropped to resting

and the ER started to refill again with Ca2 nor-. A subsequent addition of histamine still in thence of thimerosal produced similar effects. Weld remark that thimerosal induces a slow but persis-ncrease in the resting [Ca2]c before histamine addi-However, once histamine was washed, [Ca2]c

ly dropped to resting levels for a couple of minutese starting to increase again. That period closelyhes the time required to refill the ER with Ca2 (com-panels b and e), suggesting that SERCAs are able toe [Ca2]c to resting levels while they are refilling thence the ER is full of Ca2, the pump-leak equilibrium plasma membrane appears unable to keep low theg [Ca2]c levels in the presence of thimerosal. Thes of thimerosal on resting [Ca2]c and histamine-ed [Ca2]c peaks were not reversible by washingshown). However, they could be reversed by theing agent dithiotreitol (Fig. 1, panels c & f). The firstine was added after 7 min preincubation with

rosal, and produced the same effects describede. Then, refilling of the ER took place in the presencethiotreitol. After that, the second addition of hista- produced a much smaller effect. Actually, the height histamine-induced [Ca2]c peak was smaller in the

nce of dithiotreitol (386% of the control peak,SEM, n6, compare Figs. 1d & f ). Consistently, thef decrease in [Ca2]ER induced by histamine in the

nce of dithiotreitol was only of 386MnSEM, n5) compared to 1026M (meanSEM,) in the controls (compare Figs. 1a & c).

e effects of thimerosal were dependent on the con-ation. Thimerosal was almost inactive a 1M, and thimerosal produced a clear activation of hista-

-induced Ca2-release, though smaller than withM thimerosal (data not shown). Regarding the acti-n by thimerosal of histamine-induced Ca2-release,s been reported that thimerosal does not increaseproduction [13], but increases the sensitivity of the

R to InsP3 (see Introduction). We would, therefore,t that the effects of thimerosal should be made evident by using a submaximal concentration ofmine. Figure 2 shows that this is the case. The leftl shows that 2.5M histamine produced notable effect on [Ca2]ER, and a small [Ca2]c tran-in control HeLa cells. In the presence of 100M

a stkineby a

Tstrochemay[Ca2

shoThebothduc

Fig.increcoelpanehistaas insix s

Fig.histawithBAPBAP100perfuof fro 2001 Harcourt Publishers Ltd

Effects of thimerosal and intracellular BAPTA onine-induced [Ca2]ER decrease. Cells were reconstitutedelenterazine n either in the presence (traces labeled

A) or in the absence (traces labeled Control) of 10M-AM. Then, medium containing 1 mM Ca2 (Ca2), histamine (His) or 100M thimerosal (Thim) was

ed as indicated. These experiments are representative four to 13 similar ones of each kind.


Cell Calcium (2001) 30(3), 181190 200

BAPTstill incompThis [Ca2

higheabsol570100 nIn th[Ca2

was 6startiwhat buffeof thstimustate into tbut cBAPTpersisrapid

It hdepolInsP3an oxshowabilitrapidarguereleaseffectmitoction thimethimeamineinduc(meanmin oare cothe pvaluesubcesump[25]. spondmitoc(5.3ence increducedsize o

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amane

aneiTohias dmInactivation of the InsP3-gated channels, regulationa2 release by [Ca2]ER or inhibition of Ca2 releaselocal microdomains of [Ca2]c are among the pro-d mechanisms [26]. In BAPTA-loaded cells, however,

quantal effect cannot be attributed to localrodomains of high [Ca2]c. Regarding the inactivationsP3-gated Ca2 channels, we have shown here thaterosal facilitates a full and persistent activation ofe channels in BAPTA-loaded cells. We have, there-, investigated if Ca2 release induced by histamine still quantal under these conditions. Figure 5, upperel, shows that the sensitivity to histamine was dra-ically increased by thimerosal. In BAPTA-loaded cells,M histamine produced near half-maximal Ca2

ase from the ER [10], and no significant effect wasined at concentrations at or below 1M (Fig. 5, panel

n the presence of thimerosal, instead, 1M histamineuced a stronger and more persistent Ca2 release that induced by 100M histamine in the absence of

4m rmn enep

CECA-70.QXD 8/3/01 4:16 PM Page 1851 Harcourt Publishers Ltd

A-loaded cells. This was followed by a partial refilling the presence of histamine and then by a rapid and

lete refilling when the agonist was washed away.last refilling period usually led to an overshoot of]ER, reaching levels 268% (meanSEM, n8)r than those prior to stimulation. In terms ofute [Ca2]ER values, the pre-stimulation level was60 nM (meanSD, n8) and increased to 710M (meanSD, n8) after recovering of stimulation.e same series of experiments, mean steady-state]ER values obtained in cells not loaded with BAPTA6080nM (meanSD, see above). Therefore, the

ng [Ca2]ER value in BAPTA-loaded cells was some-lower than in control cells, probably because the

ring capacity of BAPTA decreases the rate of refillinge ER [22]. Agonist-induced Ca2 release may thenlate Ca2 entry and increase slightly the steady-[Ca2]c [9], stimulating SERCA to accumulate Ca2

he ER. The right panel shows the same experimentsarried out in cells pretreated with thimerosal. InA-loaded cells, histamine induced a complete andtent emptying of the stores, which was fully andly reversible after washing out the agonist.as been reported recently [23] that mitochondrialarization may modify (first enhance, then inhibit)-induced Ca2 release in HeLa cells. Thimerosal isydizing agent, and a series of oxidants have beenn to promote opening of the mitochondrial perme-y transition pore [24], a mechanism that wouldly lead to mitochondrial depolarization. It could bed, therefore, that the effects of thimerosal on Ca2

e from the ER could be indirect, mediated by itss on mitochondria. However, if that were the case,hondrial Ca2 uptake induced by histamine addi-should be strongly inhibited in the presence ofrosal. Figure 4 shows instead that treatment withrosal enhanced the peak of [Ca2]M induced by hist-. In several similar experiments, the [Ca2]M peaked by 100M histamine increased from 2.40.8MSD, n5) to 4.70.6 (meanSD, n6) after 57f incubation with 100M thimerosal. These datansistent with a larger Ca2 release from the ER in

resence of thimerosal, even though the real [Ca2]Ms in each case may be underestimated due to bothllular [Ca2]M heterogeneity and preferential con-tion of aequorin in high [Ca2]M compartmentsThe control [Ca2]M peak observed here corre-s to the nearly full consumption of aequorin in ahondrial subcompartment containing about 5%1.3%, meanSEM, n5) of aequorin. In the pres-of thimerosal, the size of this subcompartment

ased to 202% (meanSEM, n6), and this pro- the increase in the calibrated signal. Regarding thef these compartments, we should note that our

expperahist(methim(me

Wby hBAPremof relethatanisple. of Cby posethismicof Inthimthesforewaspanmat2.5releobtaa). Iprodthan

Fig. histawerehista5 mipresare riments were performed at 22C to match the tem-re used in the [Ca2]ER experiments. At 37C, theine-induced [Ca2]M peak consumed 292%SEM, n8) of aequorin (see also [25]) androsal increased that percentage to 442%SEM, n7 ).

have shown previously that Ca2-release inducedstamine in HeLa cells is quantal in the presence ofA, when the inhibition by [Ca2]c has beenved [9]. This means that submaximal concentrationsstamine produce a rapid but incomplete Ca2

e, leading to a new steady-state at a level of [Ca2]ERepends on the histamine concentration. The mech- of this quantal effect is not clear and may be multi-

Effect of thimerosal on the [Ca2]M peak induced byine. HeLa cells expressing mitochondrially targeted aequorineconstituted with wild type colenterazine. Then 100Mine was added either to control cells or to cells treated forwith 100 mM thimerosal, as indicated. Thimerosal was alsot during and after histamine addition. These experimentsresentative of from five to six experiments of each kind.


Cell C

thimconcsizeaBAPTquan

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CECA-70.QXD 8/3/01 4:16 PM Page 186Effect of thimerosal on the sensitivity to histamine of Ca2se. Cells were reconstituted with coelenterazine n in thence of 10M BAPTA-AM. Then, medium containing 1M(Ca2), 100M thimerosal (Thim), and either 10 nM, 1M orM histamine was perfused as indicated. These experimentspresentative of 1214 similar ones of each kind.

Fig.InsPrecoperfuER (2Moxidjust Theskind100Thenindicsimilalcium (2001) 30(3), 181190

erosal. The lower panel shows that even histamineentrations as low as 10 nM were able to produce able Ca2 release in the presence of thimerosal andA. The kinetics of this response was still typicallytal.e possible role of GSSG as a physiological counter-of thimerosal was studied by measuring the release2 of the ER induced by InsP3 in permeabilized cells.

re 6a shows that InsP3 released Ca2 from the ER inantal manner, and its potency was not modified byious incubation with GSSG. The percentage of Ca2

sed by 100 nM InsP3 with respect to that released by3 2M was 3217% (meanSD, n10) in controlitions and 2914% (meanSD, n6) in the pres- of 4mM GSSG. In permeabilized cells, instead, 10Merosal strongly potentiated the effect of 100nM InsP36b), but it also increased considerably the steady-state of [Ca2]ER. In parallel experiments, the steady-state]ER was 83060M (meanSEM, n6) in the con- and 145070M (meanSEM, n9) in the pres- of 10M thimerosal. Higher concentrations oferosal (100M) increased still further the refilling ofR, leading to a very fast consumption of aequorin,

precluded studying the effect of InsP3 (not shown).e have investigated also the effect of thimerosal on as of systems related to cell Ca2 homeostasis,

nameCa2

ily estadditisuch Figure[Ca2

withinbut siER (trempty111signiftrace lbut n[Ca2

that tafter Additthat rcells BHQ

Thetreatecells, braneEffect of oxidized glutathione and thimerosal onnduced Ca2 release in permeabilized HeLa cells. Cellstituted with coelenterazine n were permeabilized and thened with an ATP-containing 100 nM [Ca2] buffer to refill thee Experimental). Panel a: medium containing 100 nM orsP3 (IP3) was perfused as indicated. In the right panel, 4 mMd glutathione (GSSG) was included in the perfusion mediumer permeabilization (7 min before the first InsP3 addition). experiments are representative of 610 similar ones of eachanel b: cell permeabilization is started by perfusion of digitonin (dig) in medium containing 100 nM [Ca2] buffer.

InsP3 100nM or thimerosal 10M were perfused ased. These experiments are representative of from five to nine ones of each kind carried out in parallel. 2001 Harcourt Publishers Ltd

ly ER Ca2 leak, plasma membrane Ca2 pump andentry. The rate of Ca2-leak from the ER can be eas-imated from the rate of [Ca2]ER decrease after theon of a maximal concentration of a SERCA inhibitoras 2,5-di-tert-buthyl-benzohydroquinone (BHQ ). 7a shows that BHQ induces a rapid decrease of]ER, leading to complete emptying of Ca2 of the ER 510 min (trace B). Thimerosal produced a small

gnificant increase in the rate of Ca2-leak from theace C). In five similar experiments, the half time foring of the ER was 1303s in control conditions and13 s in cells treated with thimerosal (meansSD,icantly different at P0.05, independent t-test). Theabeled A corresponds to cells treated with thimerosalot with BHQ. Figure 7b shows the behaviour of]c in a similar series of experiments. Trace A showshimerosal alone induces a slow increase in [Ca2]ca delay that was variable between 5 and 10 min.ion of BHQ induced a transient increase in [Ca2]ceturned to resting levels within 10 min in control(trace B). Instead, in cells treated with thimerosal,produced a persistent increase in [Ca2]c (trace C). persistent elevation of [Ca2]c observed in cellsd with thimerosal (Figs 1 & 7) suggests that, in thesethe pump-leak equilibrium at the plasma mem- is unable to keep the resting [Ca2]c levels. This


20

meathe an iinvebranfreecondleveonlybranlittlerate300absen6not thatthatthe presconthighing mem

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CECA-70.QXD 8/3/01 4:16 PM Page 1877 Effects of thimerosal and BHQ on [Ca2]ER and [Ca2]c. Cells reconstituted with coelenterazine n (panel a) or loaded with-2 (panel b). In the curves labeled A and C, cells were treated100M thimerosal for 7min before the arrow. The arrow indicatesddition of 10M BHQ to curves B and C. These experiments aresentative of four to five similar ones of each kind.

Fig.and medperf100the ato si01 Harcourt Publishers Ltd

ns that thimerosal must produce either an increase ofrate of Ca2-entry through the plasma membrane ornhibition of the plasma membrane Ca2-pump. Tostigate the effects of thimerosal on the plasma mem-e Ca2-pump, we treated the cells with BHQ in Ca2-

medium, as shown in Figure 8a. Under theseitions, release of Ca2 from the ER increases [Ca2]c

ls. Subsequent return to resting conditions depends of Ca2-pumping mediated by the plasma mem-e Ca2-pump. Treatment with thimerosal modified the kinetics of return of [Ca2]c to resting levels. The

of [Ca2]c decrease, measured at a [Ca2]c of350 nM, was 1.70.6 nM/s (meanSD, n5) in thence of thimerosal and 1.40.5 nM/s (meanSD,) in the presence of thimerosal. The difference wassignificant (P0.1, independent t-test), indicating

the activity of the plasma membrane Ca2-pump at [Ca2] was scarcely affected by thimerosal. However,final steady-state [Ca2]c reached was higher in theence of thimerosal (18941nM vs 11124nM inrol cells, meanSD, n6). The difference here wasly significant (P0.005, independent t-test), suggest-

that thimerosal may reduce the activity of the plasmabrane Ca2 pump particularly at low [Ca2]c. measure the effect of thimerosal on Ca2-entryugh the plasma membrane, we have used Mn2 as a

Ca2

entrythimeBHQ.with n4)Ca2

Mn2

BHQ-tivelymay of the

DISC

We hhomeand [cally InsP3smallhomeentryactiviing [Ca2Effects of thimerosal on the plasma membrane Ca2 pump Ca2 (Mn2) entry. Cells were loaded with Fura-2. Then,

m containing 0.5 mM EGTA, 10M BHQ or 1 mM Mn2 wased as indicated. In the curves labeled Thimerosal or BHQ, thimerosal or 10M BHQ were perfused for 7 min before

ditions. These experiments are representative of from threesimilar ones of each kind.Cell Calcium (2001) 30(3), 181190

-surrogate. Figure 8b compares the rates of Mn2

obtained in control cells and in cells treated withrosal, both in the presence and in the absence of

Mn2 entry was faster in cells treated with BHQrespect to the controls (2.20.4 fold, meanSD, due to the activation of the store-operated-channels. In the presence of thimerosal, the rate of

entry increased similarly both in controls and intreated cells (1.50.1 and 1.40.3 fold, respec-; meanSD, n3). This suggests that thimerosal

activate Ca2-entry through a pathway independent store-operated channels.

USSION

ave investigated the effects of thimerosal on Ca2

ostasis in HeLa cells by monitoring [Ca2]ER, [Ca2]cCa2]M. Our results show that thimerosal dramati-increases the sensitivity and the maximum rate of-induced Ca2-release. In addition, it produced alsoer modifications in other parameters related to Ca2

ostasis in intact cells. Thimerosal activated Ca2- through the plasma membrane and decreased thety of the plasma membrane Ca2 pump under rest-Ca2]c conditions. Instead, the activity of the ER--pump in intact cells was not affected and the rate of


Cell C

the ER-Ca2-leak was only slightly increased. In perme-abilizwas iwere becaufully

ThInsP3typestionsto protherstronshowtaminmechabouminepreteInsP3ductithimeinducconceabsenThis thimechanstatesduratcompBAPTwas nstill ithat Ition elocal tion i

ThinhibproviCa2

that biphaby a releasnomeics (soriginof resInsP3Ca2

tion ostops

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CECA-70.QXD 8/3/01 4:16 PM Page 188ed cells, however, the steady-state [Ca2]ER levelncreased by thimerosal. The effects of thimerosalnot mediated by mitochondrial depolarization,se the ability of mitochondria to take up Ca2 was

preserved in the presence of thimerosal.e effects of thimerosal increasing the sensitivity to

of the InsP3R have been reported in many cell, including HeLa cells [13]. In these cells, concentra- of thimerosal as high as 100M have been shownoduce still activation of Ca2-release, contrarily to cell types where this concentration produced ag inhibition [16,17,26,27]. By looking at [Ca2]ER, we here that 100M thimerosal strongly activates his-e-induced Ca2-release. Activation involved severalanisms. On the first place, thimerosal increased byt two orders of magnitude the sensitivity to hista- of Ca2 release (Fig. 5). This effect can only be inter-d as an increase in the sensitivity to InsP3 of theR, because thimerosal does not increase InsP3 pro-on in these cells [13]. On the second place,rosal increased the magnitude of histamine-ed Ca2 release, even in the presence of maximumntrations of histamine. This is evident both in thece (Fig. 1) and in the presence (Fig. 5a) of BAPTA.effect is consistent with the reported ability ofrosal to increase the mean open times of the InsP3R

nels and to shift them to higher subconductance [15]. On the third place, thimerosal increased theion of histamine-induced Ca2 release, leading tolete and persistent Ca2-depletion of the ER inA-loaded cells. In control cells, instead, depletionot complete and the ER started to refill with Ca2

n the presence of histamine (Fig. 3). This suggestsnsP3R channels undergo a slow developing inactiva-ven in BAPTA-loaded cells, when the generation of[Ca2]c microdomains is prevented. This inactiva-s abolished by thimerosal.e interplay among the effects of thimerosal and theition of InsP3R by local [Ca2]c microdomainsded a very peculiar kinetics to histamine-induced-release, revealing interesting kinetic details aboutprocess of inhibition. The effect of histamine wassic, composed of a fast initial release (10s) followedshort period (20s) of refilling and then again bye, but at a much slower rate. These [Ca2]ER phe-na closely correlate with the biphasic [Ca2]c dynam-ee Figs 1df and inset) induced by histamine,ally described by Bootman et al. [28]. This patternponse can be easily explained by the inhibition ofR by microdomains of high [Ca2]c. After the initialrelease, InsP3R are rapidly blocked by the accumula-f Ca2 in microdomains around the channels. This

Ca2 release and leads to the rapid dissipation of the

longthe becInsPondpersparton (seehistatte[Ca2

mayphalibrianc[Ca2

releing ishecomthimthe the presby adizethimwithandobsdevstill

OCa2

chamicsitivthimdelaonlying encinacpresandmicthisdeptioninduencquaalcium (2001) 30(3), 181190 2001 Harcourt Publishers Ltd

r that the microdomains, leading to rapid refilling ofR through the SERCAs, that are strongly stimulatedse [Ca2]c is then at the peak. After about 20 s, the

R channels reactivate partially and we observe a sec-phase of slow release. This phase coincides with astent increase of [Ca2]c, which can be attributed ino Ca2 release but also to other effects of thimerosala2 entry and extrusion at the plasma membraneelow). In the absence of thimerosal, the response to

mine at the [Ca2]c level follows a similar, althoughuated, pattern (Fig. 1d). Instead, only a single step in]ER is observed (Fig. 1a). This apparent discrepancy

be explained by the generation, in the last sustained of the [Ca2]c transient, of a new pump-leak equi-m in which increased Ca2 release is exactly bal- by stimulated Ca2 pumping due to the high]c. Then, as soon as histamine is washed, Ca2

se stops and both [Ca2]ER and [Ca2]c return to rest-alues. This pump-leak equilibrium phase was abol- by dithiotreitol (Fig. 1f ), suggesting that thisound may not only revert the stimulation by

erosal but also inhibit Ca2 release with respect toontrol condition. In fact, both the [Ca2]c peak andtep of Ca2 release from the ER were reduced in thence of dithiotreitol. This effect could be interpretedsuming the presence of a certain resting level of oxi-/activated InsP3R, which could be increased by

erosal or decreased by dithiotreitol. In cells loadedBAPTA, the [Ca2]c microdomains are not generatedonly the initial rapid phase of Ca2 release wasved. Then, a slow inactivation of the channels

lops, Ca2 release stops and the ER starts refillingn the presence of the agonist.r results suggest, therefore, that InsP3-inducedrelease is limited by two types of inactivation of the

nels, one dependent of the inhibition by [Ca2]cdomains and sensitive to BAPTA, and the other sen- to thimerosal. In the presence of BAPTA, only theerosal-sensitive mechanism is operative, leading to aed slow inactivation. In the presence of thimerosal,the [Ca2]c-dependent mechanism is operative, lead- the biphasic kinetics described above. In the pres-of both BAPTA and thimerosal, the channels do notivate and the ER remains empty while the agonist isnt. [Ca2]c-dependent inactivation was long-lastingpersisted for at least 20 s in the absence of [Ca2]cdomains. This suggests that the mechanism of

inhibition may include phenomena such as Ca2-ndent phosphorylation of the channels or interac-with Ca2-sensitive proteins [29]. Ca2 releaseed by histamine remained quantal even in the pres-of both BAPTA and thimerosal, indicating that thetal nature of Ca2 release under these conditions


200

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CECA-70.QXD 8/3/01 4:16 PM Page 189t be attributed to inactivation of the channels. Theprobable alternative explanation appears to be thedence of the rate of Ca2 release on [Ca2]ER. lack of effect of oxidized glutathione on InsP3-ed Ca2 release in permeabilized HeLa cells wasising. This compound has been shown to activate-induced Ca2 release in hepatocytes [11,12], andtion of intracellular reduced glutathione sensitized cells to the effect of thimerosal [13]. We have nonation for the discrepancy, but it may be possible cytosolic factor required for this effect is lost after

eabilization of HeLa cells. In permeabilized cells,rosal was still able to stimulate InsP3-induced Ca2

e from the ER. However, the effect of thimerosal ineabilized cells was more complex, as it stronglyased also Ca2 uptake by the ER and the steady-[Ca2]ER levels. This made more difficult comparingfects of InsP3 in control and thimerosal-treated cells,se differences in the [Ca2]ER level may also modify

esponse to InsP3. The effects of thimerosal on the]ER level are discrepant with those obtained byan et al. [13], that showed a substantial reduction in

a2 content of the ER in permeabilized cells treated10M thimerosal. The reasons for the discrepancynclear, and may rely on the different experimentaltions, in particular the permeabilization procedure. we measure the rate of ER refilling immediatelypermeabilization (on line 1-min permeabilization,ig. 6b), the protocol of Bootman et al. was muchr, including a 10-min permeabilization at 37C, cen-ation and resuspension, treatment with mitochon-inhibitors and storage on ice for up to 2 h. Theanism of the increased [Ca2]ER level induced byrosal is unknown. As far as we know, redox modu- of SERCA has not been described, and this phe-non deserves further study.arding to other components of the Ca2 homeostaticinery, we show here that thimerosal activates Ca2

through a pathway independent and additive to thated by emptying of the intracellular Ca2 stores. In

ion, thimerosal reduced the activity of the plasmabrane Ca2 pump at resting [Ca2]c but not at higher]c. Both effects contribute to the slow-developingse in the mean steady-state [Ca2]c levels induced

imerosal, as reported previously [13]. Thimerosal hadeffects on the ER Ca2 pump in intact cells and pro- only a small, although significant, increase in thef ER Ca2 leak. This effect, however, was unable to

fy the steady-state [Ca2]ER level. In permeabilizedinstead, thimerosal strongly increased Ca2 uptakee ER and the steady-state [Ca2]ER level.onclusion, we show here a direct view of the effects

imerosal on InsP3-induced Ca2 release. Activation2-release by thimerosal revealed interesting kinetic

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els and on the mechanism of quantal Ca2 release.ition, the effects of thimerosal were reversible inesence of dithiotreitol, suggesting that they couldelevance from a physiological point of view.

OWLEDGEMENTS

cial support from Direccin General de Enseanzaior (PM 98-0142) and Junta de Castilla y Len (VA) are gratefully acknowledged. We thank Jessndez for technical assistance.

RENCES

ridge, MJ. Elementary and global aspects of calciumnalling. J Physiol 1997; 499.2: 291306.lich, BE. Functional properties of intracellular calcium-releasennels. Curr Op Neurobiol 1995; 5: 304309.oshiba K. The InsP3 receptor and intracellular Ca2

naling. Curr Op Neurobiol 1997; 7: 339-345.lor CW. Inositol trisphosphate receptors: Ca2-modulated

racellular Ca2 channels. Biochim Biophys Acta 1998; 1436:33.siaen L, De Smedt H, Droogmans G, Casteels R. Ca2 releaseuced by inositol 1,4,5-trisphosphate is a steady-statenomenon controlled by luminal Ca2 in permeabilized cells.

ture 1992; 357: 599602.prozvanny I, Watras J, Ehrlich BE. Bell-shaped calcium-ponse curves of Ins(1,4,5)P3- and calcium-gated channelsm endoplasmic reticulum of cerebellum. Nature 1991; 351:754.tan EJ, Ehrlich BE, Watras J. Inositol 1,4,5-trisphosphatesP3) and calcium interact to increase the dynamic range ofP3 receptor-dependent calcium signalling. J Gen Physiol 1997;: 529538.

gar, RE, Burgstahler AD, Nathanson MH, Ehrlich BE. Type IIIP3 receptor channel stays open in the presence of increasedcium. Nature 1998; 396: 8184.ntero M, Barrero MJ, Alvarez J. [Ca2] microdomains controlnist-induced Ca2 release in intact HeLa cells. FASEB J 1997; 881885.rero MJ, Montero M, Alvarez J. Dynamics of [Ca2] in theoplasmic reticulum and cytoplasm of intact HeLa cells.

iol Chem 1997; 272: 2769427699.ssiaen L, Taylor CW, Berridge MJ. Spontaneous calciumease from inositol trisphosphate-sensitive calcium stores.ture 1991; 352: 241244.ssiaen L, Taylor CW, Berridge MJ. Luminal Ca2 promotingntaneous Ca2 release from inositol trisphosphate-sensitiveres in rat hepatocytes. J Physiol 1992; 455: 623640.otman MD, Taylor CW, Berridge MJ. The thiol reagent,merosal, evokes Ca2 spikes in HeLa cells by sensitizing thesitol 1,4,5-trisphosphate receptor. J Biol Chem 1992; 267:1325119.

nschke PN, Elliott SJ. Oxidized glutathione decreases luminal2 content of the endothelial cell Ins(1,4,5)P3-sensitive Ca2

re. 1995. Biochem J 1995; 312: 485489.rower EC, Duclohier H, Lea EJ, Molle G, Dawson AP. Thesitol 1,4,5,-trisphosphate-gated Ca2 channel: effect of thetein thiol reagent thimerosal on channel activity. Biochem J6; 318: 6166.Cell Calcium (2001) 30(3), 181190


Cell C 2001 Harcourt Publishers Ltd

16. Karhapaa L, Titievsky A, Kaila K, Tornquist K. Redox modulationof calcium entry and release of intracellular calcium bythimerosal in GH4C1 pituitary cells. Cell Calcium 1996; 20:447457.

17. Missiaen L, De Smedt H, Parys JB, Sienaert I, Valingen S, CasteelsR. Threshold for inositol 1,4,5-trisphosphate action.J Biol Chem 1996; 271: 1228712293.

18. Parys JB, Missiaen L, De Smedt H, Droogmans G, Casteels R. Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2

release by thimerosal in permeabilized A7r5 smooth-musclecells. Pflugers Arch 1993; 424: 516522.

19. Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T,Rizzuto R. Monitoring dynamic changes in free Ca2

concentration in the endoplasmic reticulum of intact cells.EMBO J 1995; 14: 54675475.

20. Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R.Transfected aequorin in the measurement of cytosolic Ca2

concentration ([Ca2]c). A critical evaluation. J Biol Chem 1995;270: 98969903.

21. Villalobos C, Garca-Sancho J. Glutamate increases cytosoliccalcium in GH3 pituitary cells acting via a high affinityglutamate transporter. FASEB J. 1995; 9: 815819.

22. Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I,Garca AG, Garca-Sancho J, Alvarez J. Ca2-induced Ca2

release in chromaffin cells seen from inside the ER with targetedaequorin. J. Cell Biol. 1999; 144: 241254.

23. Collins TJ, Lipp P, Berridge MJ, Li W, Bootman MD. Inositol 1,4,5-trisphosphate-induced Ca2 release is inhibited bymitochondrial depolarization. Biochem J 2000; 347: 593600.

24. Crompton M. The mitochondrial permeability transition poreand its role in cell death. Biochem J 1999; 341: 233249.

25. Rizzuto R, Bastianutto C, Brini M, Murgia M, Pozzan T.Mitochondrial Ca2 homeostasis in intact cells. J Cell Biol1994; 126: 11831194.

26. Parys JB, Missiaen L, De Smedt H, Sienaert I, Casteels R.Mechanisms responsible for quantal Ca2 release from inositoltrisphosphate-sensitive calcium stores. Pflugers Arch 1996;432: 359367.

27. Mezna M, Michelangeli F. Effects of thimerosal on the transientkinetics of inositol 1,4,5-trisphosphate-induced Ca2 releasefrom cerebellar microsomes. Biochem J 1997; 325: 177182.

28. Bootman MD. Berridge MJ, Taylor CW. All-or-nothing Ca2

mobilization from the intracellular stores of single histamine-stimulated HeLa cells. J Physiol 1992; 450: 163178.

29. Mackrill JJ. Protein-protein interactions in intracellular Ca2

release channel function. Biochem J 1999; 337: 345361.


INTRODUCTIONMATERIAL AND METHODS[Ca 2+] ER measurementsMitochondrial [Ca 2+] ( [Ca 2+]M ) measurements[Ca 2+] c measurements

RESULTSFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8

DISCUSSIONACKNOWLEDGEMENTSREFERENCES

Documents

11 Montero 2001