BY MASAYUKI KIMURA, NORMAN LASKER AND ABRAHAM AVIV ...

Journal of Physiology (1993), 464, pp. 1-13With 6 figuresPrinted in Great Britain

THAPSIGARGIN-EVOKED CHANGES IN HUMAN PLATELET Ca2l, Na+,pH AND MEMBRANE POTENTIAL

BY MASAYUKI KIMURA, NORMAN LASKER AND ABRAHAM AVIVFrom the Hypertension Research Center, University of Medicine and Dentistry of New

Jersey, New Jersey Medical School, Newark, NJ 07103-2714, USA

(Received 21 May 1992)

SUMMARY

1. In this work we explored the effect of thapsigargin on the intracellular Ca2",pH, Na+ and membrane potential in human platelets. These parameters weremonitored using the fluorescent probes fura-2, 2',7'-bis-(2-carboxyethyl)-5,6-car-boxyfluorescein, sodium-binding benzofuran isophthalate, and 3,3'-dipropylthia-dicarbocyanine iodide.

2. Thapsigargin caused an increase in the cytosolic Ca2 , coupled with cytosolicalkalinization. Thapsigargin-induced alkalinization was Na+-dependent, indicatingthat thapsigargin stimulated the Na+-H+ exchange.

3. Using Mn2+ as a Ca2+ surrogate, we showed that thapsigargin activated Ca2+channels at relatively low levels of cytosolic Ca2 , suggesting that a rise in cytosolicfree Ca2+ is not the signal for the activation of these channels.

4. Thapsigargin-induced increase in the cytosolic free Ca2+ was greater in Na+-containing medium than in Na+-free medium, suggesting that Na+-dependentmechanisms participate in the regulation of platelet cytosolic Ca2+.

5. Thapsigargin not only increased the cytosolic Ca2+, but also elevated thecytosolic free Na+. The latter effect was more pronounced in Ca2+-free medium, afinding that may indicate that some of the Na+ enters through Ca2+ entry pathways.

6. Finally, thapsigargin evoked sustained platelet hyperpolarization which wasattenuated by charybdotoxin, indicating thapsigargin-induced stimulation of Ca2+-sensitive K+ channels.

7. Together these observations demonstrate a multifactorial effect of thapsigarginon platelets that can be utilized to further understand platelet ionic homeostasis.

INTRODUCTION

Thapsigargin elevates the cytosolic Ca2+ (Ca?+) in various cells (Takemura,Hughes, Thastrup & Putney, 1989; Gouy, Cefai, Christensen, Debre & Bismuth,1990; Thastrup, Cullen, Drobak, Hanley & Dawson, 1990) including human platelets(Thastrup, 1987; Thastrup, Linnebjerg, Bjerrum, Knudsen & Christensen, 1987;Vostal, Jackson & Shulman, 1991). Recent studies suggest that thapsigargin exertsits effect on the Ca 2+ by selective inhibition of the Ca2+-ATPase in the endoplasmicreticulum or its equivalent, the sarcoplasmic reticulum (Kijima, Ogunbunmi &Fleischer, 1991; Lytton, Westlin & Hanley, 1991; Sagara & Inesi, 1991). However,MS 1385

M. KIMURA, N. LASKER AND A. AVIV

thapsigargin effect may also involve alterations in the cellular regulation of otherions, some of which may directly relate to the status of Call. In the present work wehave further examined in human platelets the effect of thapsigargin on Ca2+regulation and other cellular ionic parameters, including the cytosolic pH (pHi), thecytosolic Na+ (Nat) and the membrane potential (Em). Moreover, the concurrentmonitoring of Ca2+ and pHi, and CaF+ and Em facilitated the evaluation of thetemporal relations of thapsigargin-evoked alterations in these platelet parameters.

METHODS

Platelet preparationPlatelets were isolated as described (Kimura, Gardner & Aviv, 1990; Kimura, Lasker & Aviv,

1992). Briefly, 50 ml of blood was drawn from healthy volunteers into acid citrate-dextrose buffer(20/1, v/v) consisting of (mM): sodium citrate, 14; citric acid, 11.8, dextrose, 18 (final pH 6 5).Platelet-rich plasma was obtained by centrifugation at 200 g for 10 min at room temperature.Platelet-rich plasma was then centrifuged at 1000 g for 10 min and cells were. washed 3 times (bycentrifugation at 1000 g for 10 min) in buffer consisting of (mM): NaCl, 140; KCl, 5; glucose, 10;ethylene-bis-(,/-aminoethyl ether)-N, N'-tetracetic acid (EGTA), 0-2; N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid (Hepes), 10; aspirin, 0-1. Fatty acid-free bovine serumalbumin (final concentration 0 1 %) was added to the third washing and EGTA was deleted.

SolutionsMost experiments were performed using Hepes-buffered solution (HBS) consisting of (mm):

NaCl, 140; KCl, 5; MgCl2, 1; CaCl2, 1; glucose, 10; Hepes, 10; with 0 1% bovine serum albumin(pH 7 4). Several experiments were performed in Na+-free HBS, where Na+ was isosmoticallyreplaced by N-methyl-D-glucamine. In a number of other experiments Ca2+ was omitted from theHBS and 0 3 mm EGTA was added.

Fluorescent dye loadingFor measurements of pH,, Ca2 and Nat platelets were incubated in HBS plus 5 /M 2',7'-bis-(2-

carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF AM), fura-2 AM and sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI AM), respectively. In preparation forSBFI AM loading, 1 mm SBFI AM and a half-volume of 20% (w/v) pluronic F-127 were mixedbefore the addition into the buffer. Platelet loading with the fluorescent dyes was performed at37 0C (60 min for BCECF AM and SBFI AM, and 30 min for fura-2 AM). In several experimentsplatelets were co-loaded with both fura-2 AM and BCECF AM for the concurrent measurements ofCa2+ and pHi (i.e. fura-2 AM was added 30 min after the initiation of incubation withBCECF AM). There was no interaction of the fluorescence between the two dyes.

Monitoring of pHi, Ca 2+ and Na+Aliquots (100 1ul) of 1-3 x 106 dye-loaded platelets were centrifuged to remove the extracellular

dye. In some experiments platelets were resuspended in HBS (pH 7 4) for measurements of basalpH,, Ca2+ and Na+. These and other measurements were performed at 37 °C under constant stirringin SPEX Fluoromax (model DM300OF; SPEX Industries, Edison, NJ, USA). This instrument canrapidly shift among different wavelengths of excitation/emission. Excitation and emissionwavelengths for pHi measurements were 440/503 and 530 nm, respectively. For Ca 2+ mea-surements, excitation and emission were set for 340/380 and 505 nm, respectively. The nigericin-K+method was used for calibration of pH, (Thomas, Buchsbaum, Zimniak & Racker, 1979). For thecalibration of Ca 2+ the ratio of maximum and minimum fluorescence was determined by theaddition of 20 fIM digitonin followed by 10 mm EGTA. Autofluorescence was determined at the endof each experiment by the addition of 1 mm MnCl2 and 20 /M digitonin. Monitoring of Nai wasperformed at excitation wavelength of 340/385 nm and at emission wavelength of 505 nm.Calibration of Nat was accomplished by the gramicidin method (Minta & Tsien, 1989). GramicidinD (2 /SM) was added to Nat calibration solutions consisting of (mM): sodium gluconate, 0-115;potassium gluconate, 0-1 15 (where the sum of sodium gluconate plus potassium gluconate = 115);KCl, 30; CaCl2, 1; MgCl2, 1; glucose, 10; Hepes, 10 (pH 7 4). The ratio of fluorescence intensities ateight different concentrations of Nat was used to obtain the standard equation parameters.

2

THAPSIGARGIN AND PLATELET Ca2+ 3

Autofluorescence was obtained using unloaded platelets. The Ca2+ and pH, signal output alternatedat 2-5 s intervals.

Monitoring ofMn2+ uptakeTo measure the rate of Mn2+ entry, MnCl2 (0 5 mm in 1 mm Ca2+ medium and 0 3 mm in Ca2+-free

medium) was added to HBS containing fura-2-loaded platelets. Excitation wavelength was set at360 nm (the isosbestic wavelength) and emission at 505 nm. Mn2+ quenching of cellular fura-2 wasmonitored for 10 min.

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Fig. 1. Dose-response effect of thapsigargin on Cai+ and pH, profiles in 1 mm Ca2+. Ca2+and pH, were concurrently monitored. Thapsigargin was added at time = 0. Theconcentrations of thapsigargin were (nM): 10 (U), 50 (EA), 100 (A), 250 (0) and 1000 (@).For simplicity, the Ca2+ and pH, levels are depicted at 30 s intervals. The absence ofhorizontal bar indicates that the S.E.M. is within the margin of the symbol. The sameapplies for all subsequent figures.

Monitoring of membrane potential (Em)The Em of platelets was measured by the membrane permeant, cationic fluorescent probe 3,3'-

dipropylthiadicarbocyanine iodide (DiSC3(5)). Fura-2-loaded platelets were added to HBS with250 nm DiSC3(5), and Ca2+ and Em were monitored concurrently. For Em measurements, excitationwas set at 643 and emission at 666 nm. The signal was calibrated at the end of each experimentusing 2 /tM valinomycin and a stepwise increase in the concentration of KCl. Em was calculatedusing the relation Em = 61-5 x log ([K+]O/[K+],), where [K+]O is the K+ concentration in the mediumand [K+]i is the cytosolic K+ concentration which was presumed to be 145 mm (the [K+], was not


corrected for cell shrinkage). There was no interaction between fura-2 and DiSC3(5). The Em andCa 2+ signal output alternated at 5 s intervals.

Data analysisAll computations were performed with an IBM-compatible PC (SAS REG and GLM program,

SAS Institute, Cary, NC, USA). Data are presented as means+ S.E.M. Unless indicated, eachexperiment was performed using platelets from at least four subjects.

RESULTS

The effect of thapsigargin on Ca2+ and pHiBasal CaF+ and pHi in 1 mm Ca2+ medium were 37-3 + 3-6 nM (n = 26) and

7*18 + 0004 (n = 20). In Ca2+-free medium basal CaO+ and pHi were 24-9+ 1-7 nM (n= 23) and 7-20+0-013 (n = 23). In 1 mm Ca2 , thapsigargin caused an increase inCa2+, coupled with cytosolic alkalinization (Fig. 1). The thapsigargin-evoked Ca2+and pHi profiles demonstrated modest changes within 100 s after exposure andsubsequent, rapid alterations in Ca2+ and pHi (at thapsigargin concentrations of100 nM or above). Thapsigargin-induced rise in Ca2+ was dependent on extracellularCa2+ concentration, whereas the alkalinization was dependent on extracellular Na+(Fig. 4). Thapsigargin did not influence the cellular buffering power and thethapsigargin-evoked alkalinization was blocked by the amiloride analogue, 5-(N-ethyl-N-isopropyl) amiloride (not shown). In Ca2+-free medium thapsigargin causeda small increase in Ca2+; maximal effect was already observed at 10 nm (Fig. 2).As a Ca2+ surrogate Mn2+ can serve to examine mechanisms of Ca2+ entry (Sage,

Merritt, Hallam & Rink, 1989; Missiaen et al. 1990; Sage, Reast & Rink, 1990;Gardner, Tokudome, Tomonari, Maher, Hollander & Aviv, 1992) and thus can beused to further explore the thapsigargin-evoked CaF+ response. Thapsigargin

100

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Fig. 2. The effect of thapsigargin on Ca 2+ profile in Ca2+-free medium. Thapsigargin wasadded at time = 0. The concentrations of thapsigargin were (nM): 10 (O), 50 (A), 100(E), 250 (0) and 1000 (-).

4

THAPSIGARGIN AND PLATELET Ca2+

accelerated the rate ofMn2+ entry (as shown by the quenching of cellular fura-2) bothin 1 mM Ca2+ and in Ca2+-free media (Fig. 3). Hence, thapsigargin opens Ca2+channels,irrespective of the extracellular Ca2+ concentration. The thapsigargin-inducedopening of these channels was observed at concentrations of thapsigargin as low as

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Time (s)Fig. 3. Dose-response effect of thapsigargin on the rate of Mn2+ entry in 1 mm Ca21 (upperpanel) and in Ca2+-free medium (lower panel). Thapsigargin and MnCl2 (0 5 mm in 1 mmCa2+ and 0 3 mm in Ca2+-free medium) were added at time 0. The effect of thapsigarginon Mn2+ quenching of cellular fura-2 was expressed as the percentage of fluorescentintensities at time 0. The concentrations of thapsigargin were (nm): 0 (EI), 10 (-), 50(A), 100 (A), 250 (0) and 1000 (@).

10 nm when the Ca2+ response was only very modest (Figs 1 and 2). It is noteworthythat the increase in the thapsigargin-evoked cellular fura-2 quenching by Mn2"demonstrated a lag in onset compared with the rise in Ca 2+

Since the alterations in Cai2 and pHi were extremely large and rapid atthapsigargin concentrations > 50 nm, all further experiments were performed using50 nm of thapsigargin. This approach permitted concise monitoring of permutationsof Ca 2+, Na+, pHi and Em evoked by thapsigargin.

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The experiments depicted in Fig. 4 were designed to explore the nature of thethapsigargin-evoked changes in Ca 2+ and pHi. All these experiments were performedin the presence of thapsigargin (added at time 0). Incubation of platelets in Ca2+-free medium for 300 s resulted in a very modest increase in Ca 2+ and a slight decline

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Fig. 4. The effect of extracellular Ca2+ and Na+ on thapsigargin-induced changes in Ca2+and pH,. Ca2+ and pH, were concurrently monitored. Platelets were suspended in Ca2+-freeand 140 mm Na+ medium (0) or in Ca2+-free and Na+-free medium (@). Thapsigargin(50 nM) was added at time 0. CaCl2 (1-3 mM) and 20 mm NaCl were added at the timeindicated by arrows.

in the pHi. These changes were the same with or without Na+ in the medium. Theaddition of Ca2+ to the medium resulted in rapid (and undelayed) changes of bothCa 2+ and pHi. In Na+-containing, Ca2+-free medium the addition of Ca2+ resulted insustained Ca 2+ response. However, the addition of Ca2+ to Na+-free, Ca2+-freemedium resulted in the formation of a peak in the thapsigargin-induced Ca2+ profile,namely an initial and rapid increase in the Ca?+ followed by its decline (Fig. 4). Thesubsequent addition of Na+ to the medium resulted in a further increase in the Ca2+.The observation that the thapsigargin-evoked CaW+ response is lower in Na+-free

6


medium was substantiated by another set of experiments, designed to measure thethapsigargin-evoked changes in the Em (Fig. 6). The addition of Ca2+ to Ca2+-free,Na+-containing medium produced rapid and undelayed alkalinization. In Ca2+-free,Na+-medium, the addition of Ca2+ provoked rapid acidification. This process was

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Fig. 5. The effect of thapsigargin and ouabain on Nat in 1 mm Ca2+ medium (upper panel)and in Ca2+-free medium (lower panel). Thapsigargin (50 nM; circles), ouabain 0 1 mM;triangles) or thapsigargin + ouabain (squares) were added at time 0. Data are expressedas the difference (A) from basal Nat concentration.

reversed by the subsequent addition of Na+ (Fig. 4). Ouabain (0-1 mM) had noapparent effect on the thapsigargin-evoked CaF+ response (not shown).

The effect of thapsigargin on NatiBasal Na+ in 1 mm Ca2+ and in Ca2+-free media were 7-06 + 050 and 6-08 + 037 mM,

respectively. In 1 mM Ca2 , thapsigargin caused a gradual and slight increase in Naiduring 300 s of monitoring (Fig. 5). The effect of ouabain on Na+ was smaller thanthat of thapsigargin. The combination of both thapsigargin and ouabain was notmuch different in the first 180 s after exposure to thapsigargin than the effect of

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Time (s)Fig. 6. The effect of thapsigargin and charybdotoxin on Ca2+ and Em in 140 miv Na+medium and in Na+-free medium. Ca2+ and Em were concurrently monitored. Plateletswere incubated with or without 20 nM charybdotoxin from 180 s and thapsigargin (T) wassubsequently added. 0, 140 mMi Na+ medium without charybdotoxin; @,140 mM Na+medium with charybdotoxin; A, Na+-free medium without charybdotoxin; A, Na+-freemedium with charybdotoxin. Three subjects were used for this experiment. Ten minutesafter the addition of thapsigargin, the Cai2+ levels were significantly higher in Na+-containing HBS than in Na+-free HBS at P=0011 (using a two-way ANOVA bycharybdotoxin and Na+). There was no effect of charybdotoxin on the Cai2+ proffle.


thapsigargin alone. Higher levels of Na' were observed after 180 s of combinedtreatment with thapsigargin and ouabain than treatment with thapsigargin alone.However, these differences were not statistically significant. In Ca2+-free medium theeffect of thapsigargin on Nat was greater than that in 1 mm Ca21 medium. As in Ca2+-containing medium, thapsigargin raised the Na+ to a higher level than ouabain. Thecombination of thapsigargin plus ouabain had no further effect than thapsigarginalone.

The effect of thapsigargin on the EmThe resting Em in platelets was - 65-9 + 2-1 mV in HBS containing 140 mm Na+.

In Na+-free HBS the resting Em was - 66-8 + 0-8 mV. Thapsigargin caused sustainedhyperpolarization and charybdotoxin, an inhibitor of Ca2+-dependent K+ channels,inhibited this hyperpolarization (Fig. 6). In itself, charybdotoxin caused a slightdepolarization of the resting Em and it had no effect on thapsigargin-induced increasein Ca0+ in Na+-containing or Na+-free medium.

DISCUSSION

The effect of thapsigargin on Ca2+Previous studies have shown that thapsigargin raises Ca 2+ by redistributing Ca 2+

between the intracellular storage sites and the cytosol and by increased Ca2+ entrythrough Ca2+ channels in the plasma membrane (Takemura et al. 1989; Gouy et al.1990; Thastrup et al. 1990; Vostal et al. 1991). In platelets, Thastrup et at. (1987)reported that removal of extracellular Ca2+ by EGTA had only an insignificant effecton the thapsigargin-induced rise in Cai . In our study a major portion of thethapsigargin-induced increase in Ca 2+ was dependent on extracellular Ca2+. This wasalso supported by the observation of thapsigargin increased Mn2+ entry. The reasonfor the difference between our study and that of Thastrup et al. (1987) is not clear.However, in a subsequent work, Thastrup et at. (1989) documented thapsigargin-evoked increase in Mn2+ entry, consistent with the findings of the present work.Moreover, our study used aspirin-treated platelets. It is thus possible that variationsin the thapsigargin response also relate to the status of the cyclo-oxygenase system(Brune & Ullrich, 1991). The main target of thapsigargin appears to be the Ca2 -ATPase in non-mitochondrial Ca2+ storage organelles (Kijima et al. 1991; Lytton etal. 1991; Sagara & Inesi, 1991). In platelets the main storage site for rapidlyexchangeable Ca2+ is the dense tubular membrane system. The mechanisms by whichthapsigargin promotes Ca2+ influx from the extracellular medium are not fullyunderstood. It is unlikely that the cellular signal for accelerated Ca2+ entry simplyrelates to elevated C Using Mn2+ as a Ca2+ surrogate, our experimentsdemonstrated that thapsigargin accelerated Ca2+ entry across the plasma membranewhen the Ca 2+ levels were only minimally increased (i.e. Fig. 2 and the lower panelof Fig. 3). It is thus likely that the signal for enhanced Ca2+ entry depends on thestatus of the intracellular storage sites. Vostal et al. (1991) have recently proposedthat in human platelets such a signal is relayed by the interplay between Ca2+-sensitive tyrosine kinase and phosphatase that are associated with the Ca2+ storagesites. Others have proposed that such a signal is mediated through microsomal

9


cytochrome P-450 (Alonso, Alvarez, Montero, Sanchez & Garcia-Sancho, 1991).Together these observations may explain the delay in both the rapid increase in Ca2+and the rapid acceleration of Mn2+ entry after treatment with thapsigargin.Our experiments demonstrated that the presence of extracellular Na+ promoted a

greater increase of the thapsigargin-evoked rise in Ca2+. Two possibilities should beconsidered to account for this observation. Firstly, thapsigargin-evoked increase inNa+ entry is likely to stimulate the Na+ pump. In turn, the activated Na+ pump candeplete cellular ATP, thereby slowing the activity of the plasma membrane Ca2+-ATPase. This was ruled out, as the inhibition of the Na+ pump by ouabain did notmodify the thapsigargin-evoked Ca2+ response. Secondly, thapsigargin-evoked rise inNa+ in Na+-containing medium may enhance Ca2+ entry through the Na+-Ca2+exchange, acting in a reverse mode (Leblanc & Hume, 1990; Rodrigo & Chapman,1991; Sage, Breemen & Cannell, 1991).

The effect of thapsigargin on pHiIn previous studies we demonstrated the central role of CaF+ in regulating the

Na+-H+ exchange in human platelets (Kimura et al. 1990; Kimura et al. 1992). Thisconcept is further supported by the present findings. Gouy et al. (1990) could notdemonstrate a thapsigargin-evoked increase in pHi in T lymphocytes. The reason forthis observation is not clear. Our studies (M. Balasubramanyam, M. Kimura, J. P.Gardner & A. Aviv, unpublished observations) showed that activation of theNa+-H+ antiport by thapsigargin in peripheral lymphocytes was not alwaysexpressed by cytosolic alkalinization. However, when subjected to thapsigargin, inthe presence of an amiloride analogue, lymphocytes with inhibited Na+-H+ antiportdemonstrated profound acidosis compared with lymphocytes with intact Na+-H+antiport. Thus, as suggested previously (Kimura et al. 1990; Sage, Jobson & Rink,1990), the function of the Na+-H+ antiport in some cells is primarily aimed atattenuating the tendency for acidification associated with a rise in Ca2+, rather thanthe promotion of cytosolic alkalinization.

The effect of thapsigargin on NatiMonovalent ions can enter the cytosol through Ca2+ channels in several cell types,including platelets (Mahaut-Smith, Sage & Rink, 1990; Borin & Siffert, 1991),myocytes (Hess & Tsien, 1984; Hess, Lansman & Tsien, 1986) and skeletal musclecells (Almers, McCleskey & Palade, 1984). In this study thapsigargin evoked agreater rise in Na+ in Ca2+-free medium than in Ca2+_containing medium and theeffect of thapsigargin on Nat was greater than that of ouabain alone. These resultssuggest that thapsigargin increased Na+ not only by stimulating the Na+-H+antiport but also by augmenting its entry through Ca2+ channels. The fact thatthapsigargin caused a greater rise in Na+ than the inhibition of the Na+ pump byouabain suggests, but does not exclude, the possibility that the thapsigargin-evokedrise in Na+ is not mediated through the inhibition of the Na+ pump. Moreover, othershave shown that thapsigargin has no effect on the enzymatic analogue of the Na+pump, namely the Na+-K+-ATPase (Lytton et al. 1991).

10


The effect of thapsigargin on the EmOur group has shown that platelets have Ca2+-activated K+ channels (Fine,

Hansen, Salcedo & Aviv, 1989). Values for platelet resting Em in this study arecompatible with previous observations (Fine et al. 1989; Mahaut-Smith, Rink,Collins & Sage, 1990). Charybdotoxin, a purified protein of Leiurux quinquestriatusvenom, can block certain classes of K+ channels in many cell types, includingplatelets (Mahaut-Smith et al. 1990). Mahaut-Smith et al. (1990) reported thatcharybdotoxin-sensitive K+ channels are major contributors to the high resting Emin unstimulated platelets. In our study, charybdotoxin caused a small depolarizationof the resting Em, probably because of blocking some basal activity of K+ channels.The thapsigargin-evoked hyperpolarization was inhibited by charybdotoxin with noeffect of the K+ channel inhibitor on Ca2+. These data suggest that thapsigargininduced an increase in Ca2+-activated, charybdotoxin-sensitive K+ channels. Changesin Nat appear to play no apparent role in the thapsigargin-induced hyperpolarizationbecause such a process was also observed in Na+-free medium.

ConclusionsThe present work demonstrates that the effect of thapsigargin in human platelets

is not limited to elevation of the CaF+. Rather, thapsigargin produces substantialalterations in the overall cellular ionic homeostasis, including profound changes inthe pHi, the Nat and the Em; some of these changes may relate to the Ca2+ status.Thapsigargin may thus serve as a powerful probe to understand the complexinteractions among mechanisms that regulate cellular ionic composition.

This work was supported by grants from the National Heart, Lung, and Blood Institute(HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoralresearch fellow of the American Heart Association, New Jersey Affiliate.

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