Proc. Nati. Acad. Sci. USAVol. 88, pp. 4926-4930, June 1991Cell Biology

Cytosolic Ca2' deregulation and blebbing after HgCl2 injury tocultured rabbit proximal tubule cells as determined by digitalimaging microscopy

(calcium ionophores/fura-2/kidney)

M. W. SMITH*, P. C. PHELPS*, AND B. F. TRUMP*t*Department of Pathology, University of Maryland School of Medicine, and tMaryland Institute for Emergency Medical Services Systems,Baltimore, MD 21201

Communicated by David M. Prescott, February 19, 1991 (received for review November 11, 1990)

ABSTRACT Acute injury to renal proximal tubule cellshas previously been shown to result in elevated cytosolic Ca2+([Ca2+fD, blebbing, and eventual cell death. In this study,digital imaging fluorescence microscopy was used to evaluatethese changes in response to HgCl2 treatment ofcultured rabbitproximal tubular cells. Monolayer cells loaded with fura-2 weretreated with 10, 50, or 100 ,uM HgCl2 in both 1.37 mMCaCl2-containing and nominally Ca2+-free (<5 ,uM) Hanks'balanced salt solution. [Ca2+]J was estimated by measuring theratio of fluorescent image pairs (collected at 340- and 380-nmexcitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blueexclusion. After exposure of cells to 10 ,uM HgCl2, (Ca2J1,initially increased about 2-fold by 5 min; after 50 or 100 ,uMHgCl2, [Ca2+1 rapidly rose 2- to 3-fold, peaked at 1-3 min, andthen generally decreased slightly. In nominally Ca2+-free (<5,uM) medium, [Ca2+J1 stabilized, but in 1.37 mM Ca2+-containing medium, [Ca2+] continued to slowly rise, oftenreaching levels of fura-2 saturation. The rate and extent ofblebbing and the rate of cell death were increased in thepresence of 1.37 mM Ca2+. These results show that sustainedelevations of [Ca2+]1 precede both cell blebbing and cell deathand that when these elevations are limited by removing extra-cellular Ca2+ the amount of blebbing is reduced and cellviability is prolonged.

Elevations of cellular calcium concentration have for sometime been implicated as a mediator of cell injury and death(see refs. 1-3 for recent reviews). Under normal conditionsionized cytosolic calcium serves as an intracellular messen-ger and directly regulates many cellular functions. Its con-centration is tightly maintained within a narrow range (near100 nM) by a number of mechanisms (4). When normalphysiological homeostasis is interrupted by any of a varietyof injurious conditions, the elevated concentration of cyto-solic calcium ([Ca2W]i) then becomes deleterious via thesustained activation of phospholipases, proteinases, and en-donucleases and by deregulation of a number of other Ca2+-regulated functions (4).

Several findings suggest that [Ca2+]i deregulation may playa major role in HgCl2 toxicity. In vivo treatment with HgCl2results in large increases in mitochondrial Ca2' (5) and theappearance of prominent calcium-containing precipitates inmitochondria of renal proximal tubule cells (6). HgCl2 tox-icity has also been shown to collapse the mitochondrialmembrane potential in cultured rat hepatocytes (7) and torelease Ca2' from both isolated rat renal mitochondria (8) andisolated rabbit sarcoplasmic reticulum (9). Recently thislaboratory has demonstrated that [Ca2+]J elevation occurs

when suspensions of cultured rabbit renal proximal tubuleepithelium (PTE) are treated with HgCl2 and that, whenextracellular Ca2' is omitted, the [Ca2+]i elevation is lessenedand cell death is significantly delayed (3, 10, 11).Acute cell injury in a variety of cell types is accompanied

by the formation of cytoplasmic blebs at the plasma mem-brane (10, 12, 13), and the blebs are often associated withelevated [Ca2W]i. In previous studies using cells cultured fromisolated rabbit proximal tubules, we noted that, although theaverage [Ca2+], of the total population increased after injury,the rate of bleb formation and the number and size of blebsvaried dramatically among individual cells. As a result, it hasbeen difficult to clearly establish the relationship betweenelevated [Ca2+]i and the formation of blebs. Similarly, it hasnot been possible until recently to examine the relationshipbetween the degree of [Ca2+]i elevation and the fate ofindividual cells. Now, through the use of digital imagingfluorescence microscopy (DIFM), we are able to describe thechanges in the [Ca2W]i of individual cells in response to HgCl2and report the temporal relationship between elevation of[Ca2+]i, bleb formation, and cell death.

MATERIALS AND METHODSMaterials. The following reagents were used: fura 2, fura-

2/AM (acetoxymethyl ester), and ionomycin from Calbio-chem; 4-bromo-A23187 and probenecid from Sigma; andtissue culture reagents from GIBCO. Hanks' balanced saltsolution (HBSS) was modified as described previously andcontained 1.37 mM CaC12 (11). Nominally Ca2+-free HBSS,to which no CaCl2 was added, was determined to contain 2-5,uM Ca2+ by measurement with Ca2+ electrodes and withfura-2 and will be referred to as Ca2+-free or low-Ca2' HBSS.

Cell Culture. Rabbit renal PTE was cultured as describedpreviously (11). For DIFM, cells were cultured on 22-mmsquare glass (no. 1) coverslips affixed over holes drilled in thebottoms of 60-mm plastic culture dishes (14). Cells werecultured for 3-7 days.DIFM. Monolayer cells were loaded with fura 2 by expo-

sure to 3-5 ,uM fura-2/AM in HBSS for 1 hr at roomtemperature (11, 15) and then rinsed for 10 min in HBSScontaining 0.2% bovine serum albumin. All assays wereperformed at room temperature to retard the loss of fura-2.Fluorescent images of fura-2-loaded cells were acquired byusing a Nikon inverted microscope, a chopper-based TracorNorthern FluoroPlex III (Tracor Northern, Middleton, WI),which has a xenon lamp and two adjustable monochrometers,

Abbreviations: [Ca2+], concentration of cytosolic Ca2+; [Ca2+]e,concentration of extracellular Ca2+; PTE, proximal tubular epithe-lium; DIFM, digital imaging fluorescence microscopy; R, Rmin, andRma,,, the ratios of fura-2 fluorescence at 340-nm excitation to that at380-nm excitation in routine measurements, in the absence of Ca2+,and in strinCfrespectivelygand in saturating Ca +, respectively.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991) 4927

an intensified high-resolution Newvicon video camera (Dage-MTI, Michigan City, IN), and a Tracor Northern TN-8502image analysis system for digitizing, storing, and processing.Fura-2-loaded cells were exposed to rapidly alternating 340-and 380-nm excitation, and the emitted light was transmittedthrough a filter cube containing a dichroic beam splitter anda barrier filter to exclude wavelengths below 400 nm andabove 515 nm. Pairs of fluorescent images, typically theaverage of 15 frames each and captured at a rate of 5 pairs offrames per sec, were acquired at 0 time and at 0.5- to 5-minintervals after treatment. The same field of cells was followedfor the duration of a treatment. Each treatment was repeatedon at least three different cell preparations. Backgroundfluorescence was subtracted and the ratio of the 340-nmimage to the 380-nm image was determined (the pairs were"ratioed") on a pixel-by-pixel basis. The average pixel valueof each ratioed image was obtained by histogram analysis.

Calibration of Ratioed Images. Conversion of ratios andcalibration of the pseudocolor scale for ratioed images to[Ca2+]j (in nM) was performed by using the formula [Ca2+],= Kd(R - Rmin)/(Rmax - R), where Kd = 225 nM (15). Rm.,the ratio of Ca2+-saturated fura-2, was obtained by treatingcells with S ,M ionomycin or 5 ,uM 4-bromo-A23187. Wemade the assumption that [Ca2+], for untreated monolayercells was similar to the [Ca2+], value of 100 nM obtained fromuntreated cells in suspension (11) and that the ratio (R) valueobtained from the untreated monolayer cells corresponded tothis value. The ratio of fura-2 in the absence of Ca2+ (Rmi)was calculated by substitution of R, Rmax, and the 100 nMvalue for [Ca2+]J into the equation. Attempts to obtain Rm-inby lowering [Ca2+], in situ by methods suggested by others(16) were not successful. In vitro calibration using solutionsof 5 AM fura-2 gave a Rmin close to the calculated values;however, Rmax was higher than that seen with ionomycin.

[Ca2+]J Measurements. Intracellular noncytosolic localiza-tion of fura-2 was evaluated by permeabilizing cells with0.01% saponin in 20 mM NaCl/100 mM KCl/2 mM MgSO4/0.96mM NaH2PO4/10 mM Hepes/1 mM EGTA, pH 7.1 (17).Relative amounts of mitochondrial and nonmitochondrialintracellular Ca2+ pools were determined by treating cells inlow-Ca2' HBSS + 20 ,M EGTA with 5 mM KCN for 5-10min and then adding S ,uM ionomycin for 5-10 min. Proben-ecid (2.5 mM) was used to evaluate the mechanism of fura-2loss from treated cells.

Cells were treated with 10, 50, and 100 ,M HgCl2 in thepresence of normal or low [Ca2+]. Images were collected atfrequent intervals and the time at which blebs were first seenon the cells was noted. HgCl2, as previously shown, does notinterfere with fura-2 measurements (7, 11).

Viability. Cell viability was determined by adding 2-3 dropsof0.4% trypan blue to the dish after the fluorescence droppedto very low levels or when cells appeared very granular byphase-contrast microscopy.

RESULTSValidation of Fura-2 Technique. Cultured rabbit PTE

readily loaded fura-2/AM. As in previous studies usingcultured rabbit PTE cells suspended by trypsinization (11),fura-2/AM appeared to be completely deesterified by cyto-solic esterases under the loading conditions employed.Fura-2 loading appeared homogeneous in the cytoplasm, andmost (>95%) of the fura-2 was released by treatment with0.01% saponin as indicated by decreased emission intensityat 340-, 365-, or 380-nm excitation. Fluorescence at 365-nmexcitation is insensitive to changes in [Ca2+]i and is thereforeuseful in evaluating incomplete deesterification, interferencewith fluorescence by NADH, or loss of fura-2 from the cell.When 340/380-nm and 340/365-nm image pairs were alter-nately collected and subsequently ratioed, the same patterns

of change after HgCl2 (Fig. 1) or ionomycin treatment wereseen in both ratioed pairs. This further authenticates our useof 340/380 nm for Ca2+ determinations.The loss of fura-2 from HgCl2-treated cells was rapid, as

shown by decreased fluorescence at 340-, 365-, or 380-nmexcitation (Fig. 1). The loss was only slightly retarded by theaddition of 2.5 mM probenecid, an inhibitor of organic acidtransport frequently used to block transport of fura-2 fromcells (18). Data collected at low fura-2 fluorescence are notincluded because R values were not reproducible.

[Ca21]J Controls. Little or no variation in [Ca2+], wasdetected among untreated PTE cells on the same dish, amongcells on different dishes cultured the same amount of time, oramong cells of different ages (3-7 days). When untreated cellswere measured repeatedly for up to an hour, fura-2 fluores-cence measured at 340-, 365-, and 380-nm excitation de-creased about 10% but the estimated [Ca2+]i did not change(Fig. 2A).HgCl2-Induced Changes in [Ca21],. Typical changes in

[Ca2+]1 in response to HgCl2 are shown in Figs. 2 and 3. With10 ,tM HgCl2, [Ca2+] increased 2-fold, stabilized at about 5min, and, in normal [Ca2+]e, underwent a secondary increase(Figs. 2 B and C, and 3A). After 50 ,uM HgCl2, [Ca2+] roserapidly, peaked at about 3 min, generally decreased slightly,and then, in normal [Ca2+Ie, slowly increased (Figs. 2D and3B). With 100 ,uM HgCl2, the [Ca2+], increased rapidly,peaked at about 1 min, recovered slightly, and then, in normal[Ca2+], underwent a second increase, often reaching fura-2saturation (Figs. 2 E and F and 3C). The initial transientincrease was greater with 100 ,uM than with 50 ,uM HgCl2. Ineach case, when cells were treated with HgC12 in Ca2+-freeHBSS, only the initial [Ca2+ i increases were seen and littlefurther change was noted (Fig. 2F). Data are summarized inTable 1.

Evaluation of Intracellular Ca21 Pools. When cells in low-[Ca2'+e + 50 ,uM EGTA were treated with 5 mM KCN for5-10 min to release mitochondrial Ca2+, [Ca2+]i routinelyshowed little or no change. Subsequent treatment with 5 ,uMionomycin + 5 mM KCN to release Ca2+ from nonmitochon-drial pools resulted in a 2-fold increase in [Ca2+]1, whichrapidly returned to near control levels within 10 min.

[Ca2+], Distribution. Often the changes in concentration ofCa2` did not appear homogeneous throughout the cytosol(Fig. 2 B-F). In untreated cells, the ratio of fura-2 in the

0

atIX1.1

0 10 20Time, min

(.)

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a)C.)Ca)C.)0)

CFCua)

30 40

FIG. 1. Effect of 100 ,uM HgCI2 on the fluorescence intensity offura-2 from image pairs acquired at 340 and 365 nm and at 340 and380 nm. Fluorescence declines rapidly at all three wavelengths. Theratio of image pairs at 340/365 nm follows the same pattern as thatat 340/380 nm. Note that the working range from Rmin to Rmax of the340/365 curve is less than half that of the 340/380 curve.

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FIG. 2. Digital fluorescent and ratioed images ofcontrol and HgClrtreated cells. (x 1100.) The grey tone fluorescent images, acquired at 340-nmexcitation, are included to demonstrate fura-2 distribution. The ratioed images were generated by obtaining the ratios ofpairs offluorescent imagesacquired at 340- and 380-nm excitation. The ratioed images are displayed in pseudocolor to map [Ca2+]i levels corresponding tonM Ca2+ as indicatedon the calibrated color scale. (A) Untreated control cells in normal (1.37 mM) [Ca2"]. (Al = 0 min; A2 = 60 min). Little or no change in [Ca2Wlcan be seen. (B) Addition of 10 pM HgCl2 in normal [Ca2+1] (B) = 0 min; B2 = 7.5 min; B3 = 20 min; B4 = 24 min; BS = 30 min; and B6 = 31min). There is a gradual increase in [Ca2+]i as indicated by changes in color (see curve marked * in Fig. 3A). Tiny blebs, not obvious in the ratioedimages, were seen in fluorescent images at 24 min on the upper right of the cell. This cell was dead by 40 min. (C) Addition of 10 pM HgCl2 inlow (<5 ,uM) [Ca2+]. (Cl = 0 min; C2 = 30 min). The cells were still alive at 50 min but the cytosolic fura-2 was too low to continue analysis (seecurve marked ** in Fig. 3A). (D) Addition of 50 1AM HgCl2 in normal [Ca2+], (Dl, D2 = 0 min; D3 = 5 min; D4 = 10 min; D5 = 12 min; D6, D7= 14.75 min; D8, D9 = 16 min; and D10 = 33 min). D2, D7, and D9 are fluorescent images. [Ca2+]i increased rapidly during the first few minutes(see curve marked with * in Fig. 3B.) Blebs appeared at about 14 min, after [Ca2+]i was >300 nM (D6, D7). Note the high levels ofCa2+ in blebs.(D9) A fluorescent image (340-nm excitation) acquired at 16 min. By focusing on the upper plane of the cells, three large blebs can be seen. (DJO)Cells at 33 min, just before cellular fluorescence vanished. Nuclei stained with trypan blue by 35 min, indicating cell death. (E) Addition of 1001.LM HgCl2 in normal [Ca2+] (El = 0 min; E2 = 5 min; E3, E4 = 10 min; E5, E6 = 13 min; and E7 = 19.5 min). E4 and E7 are fluorescent images.[Ca2+]i was significantly elevated by 5 min and continued to increase rapidly (see curve marked * in Fig. 3C). Many tiny blebs formed at about

4928 Cell Biology: Smith et al.

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10 AtM HgC12 .--- Low [Ca2+]100 7 50 ,uM HgC12 - Normal [Ca2+].

0 10 20 30 0 10 20 30 0 10 20 30 40Time, min

FIG. 3. Effect ofHgCl2 on [Ca2+]i offura-2-loaded rabbit PTE. (A) Changes in [Ca2+]i of cells treated with 10 ,AM HgC12 in normal or Ca2+-freeHBSS. [See Fig. 2B (*) and Fig. 2C (**) for corresponding ratioed images.] (B) Results of 50 /LM HgCl2 treatment in normal [Ca2+k. After arapid increase in [Ca2+]i, one curve shows some recovery, whereas the other two show a short pause before the [Ca2+] rises dramatically. [SeeFig. 2D (*) for corresponding ratioed images.] (C) Treatment with 100 /AM HgCl2 produced an immediate increase in [Ca2+]i followed by slightrecovery. [Ca2+]i underwent a secondary increase in normal [Ca2+]r but stabilized in low [Ca2+k. [See corresponding ratioed images in Fig.2E (*) and Fig. 2F (**).]

nucleus was usually much lower than in the surroundingcytosol (see Fig. 2 A, BR, C1, Dl, El, and Fl). In the first fewminutes after addition of 50 A&M or 100 ,M HgCl2 the ratiowas generally higher in and around the nucleus; after about5 min, the [Ca2+] near the cell periphery often appeared todecrease, and occasionally distinct bands of different Ca2+concentrations were seen.

Blebs. Blebs were not normally found on untreated cells oron cells with [Ca2+]i estimated to be less than 300 nM.Blebbing was always preceded by an elevation of [Ca2+],,although we observed a few cells with elevated [Ca2+]i thatdid not appear to bleb. Blebbing occurred most rapidly inresponse to treatments that resulted in the highest [Ca2+]i;omitting [Ca2+]e delayed and reduced the amount of blebbing(Table 1). The series of ratioed images of cells treated with 50,uM (Fig. 2D) or 100 ,M (Fig. 2E) HgCl2 in the presence of1.37 mM [Ca2+]e show the temporal relationship of [Ca2+], tobleb formation. Some [Ca2+]i elevation occurs very rapidly,but blebs do not appear until after [Ca2+li has risen to overapproximately 300 nM.

Viability. Cells treated with 10, 50, or 100 ,M HgCl2 innormal HBSS died only after [Ca2+]h had increased several-fold and at dose-dependent rates (Table 1). Most cells treatedwith 10 ,uM HgCl2 survived for up to 40 min. With 50 ,uM,most cell killing occurred between 25 and 35 min, and with100 AM nearly all cells were dead by 25 min. In Ca2+-freeHBSS, little cell killing was seen with 10uM HgCl2 during thefirst hour of treatment; with higher doses, cell killing wassignificantly delayed.

DISCUSSIONElevations of [Ca2+] in response to injury have previouslybeen measured on various cell populations, and this increasehas been linked to the formation of blebs and cell death (10,11). However, since changes in [Ca2+], of individual cellscould not be measured, the relationship of [Ca2+], to blebformation and cell death could not be definitely established.Although the technique is not without drawbacks (e.g., seerefs. 16 and 18-20), we are now able to use DIFM coupled

with the Ca2"-sensitive fura-2 to observe spatial and temporalchanges of [Ca2+]i concurrent with morphological alterationsof individual cells.The effect of HgCl2-induced injury on [Ca2+]i and bleb

formation in cultured rabbit PTE cells is illustrated in Fig. 2.Treatment of cells with HgCl2 resulted in an initial rapid 2- to3-fold elevation of [Ca2+]i due to redistribution of intracel-lular Ca2+ (Fig. 2B-F). In Ca2+-free HBSS, the elevated[Ca2+], stabilized (Fig. 2 C and F); whereas in normal HBSS,there was a slower, dose-dependent, increase (Fig. 2 B, D,and E), which is attributed to influx from the extracellularmedium. Blebs were not seen to form unless [Ca2+] had risento over 300 nM (Fig. 2 B, D, and E), and cell killing wasclearly accelerated by elevation of [Ca2+]. This differs fromthe study of Nieminen et al. (7), who found increases in[Ca2+], in rat hepatocytes treated with HgCl2 only after blebshad formed and immediately prior to cell death.HgCl2 exerts numerous deleterious effects on a cell, many

attributable to its affinity for thiol groups. In addition, Hg2+competes with Ca2+ for binding sites, such as with calmod-ulin (21), and perhaps for membrane receptors. Mercury andother divalent ions have been shown to trigger release ofCa2+from the sarcoplasmic reticulum of isolated skeletal muscle(9) and to stimulate ATP-dependent release of Ca2+ fromhuman platelet membranes (22). The initial [Ca2 ke-independent [Ca2+]i elevation we and others (23) have ob-served may be related to these effects. The mechanism ofCa2+ influx after HgCl2 treatment is unknown. An earlierstudy found that channel blockers, calmodulin inhibitors, ormitochondrial inhibitors do not alter patterns of [Ca2+],changes or cell killing after HgCl2 treatments (11). Alterationof permeability could possibly be due to modification ofprotein thiol groups or to lipid peroxidation and subsequentactivation of phospholipases.

Several differences were noted between the results ob-tained by using monolayer cells in the present study and thecells suspended by trypsinization in an earlier study (11). Intrypsinized cells, the initial increase in [Ca2+]i due to redis-tribution was much higher, the recovery seen with 10 and 100

10 min (E3, E4) and subsequently enlarged (E5-E7). This cell was dead by 25 min. (F) Addition of 100 AM HgCl2 in low [Ca2+le (F) = 0 min;F2 = 0.5 min; and F3 = 30 min). After an initial increase (F2), there was some recovery of [Ca2+]i but no further elevation (F3) (see curve marked** in Fig. 3B). The cell did not bleb or die during the 40 min that images were collected.

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Table 1. Blebbing (time and [Ca2+]f) and cell death with HgCl2Time of blebbing,* min [Ca2+]i at blebbing,t nM Time of cell death,* min

HgCl2, Normal Low Normal Low Normal LowAuM [Ca2+l [Ca2+l [Ca2+] [Ca2+] [Ca2+l [Ca2+]I10 21 ± 3 (6) 30(1) 300-620 300 42 ± 4(3) -50

None (2)50 15 ± 0.5 (5) None (2) 300-700 33 ± 3 (4) .40

26 (1) 300100 9 ± 3 (13) None (1) 300-620 24 ± 3 (13) .30

21,40 (2) 300The results are mean ± SD (range where indicated), with the number of observations for each data point given in

parentheses.*The time at which blebs were first seen on the imaged cells.tThe range of [Ca2+], seen when blebs were first noted.tEstimated time of death of the cells being imaged.

AM HgCl2 was more dramatic and complete, and the increasedue to influx of Ca2' was slower than in monolayers. Whenthe amount of Ca2+ sequestered intracellularly was evaluatedin monolayer cells, we noted that Ca2+ stores were consid-erably smaller in monolayer cells than in trypsinized cells.Little or no increase in [Ca2+]i was associated with mito-chondrial Ca2" release, and only a 2-fold increase in [Ca2+]iwas attributable to nonmitochondrial pools (Fig. 4). In con-trast, in trypsinized cells, mitochondrial Ca2' release re-sulted in 1.5- to 2-fold increases in [Ca2"]i, while release fromnonmitochondrial pools resulted in 6- to 8-fold increases (11).Despite the larger increase in [Ca2W]' due to intracellular Ca2'redistribution after Hg2' treatment of trypsinized cells,[Ca2+], levels recover much more completely and remainlower in trypsinized cells. The reason for the recovery andlower sustained [Ca2+]i is suggested by reports that trypsinactivates a plasma membrane Ca2+-ATPase (4). Activation ofa Ca2+-ATPase could help control the [Ca2+], increases intrypsinized cells. In trypsinized cells we also noted thatHgCI2-treated monolayer cells died more rapidly thantrypsinized cells (10, 11). One explanation for the differencein the rate of cell killing between monolayer and trypsinizedcells is that hardier cells may be selected by trypsinization;another possibility is that protection may be afforded bylower sustained increases in [Ca2li.

In the present study blebbing was observed only in cellswith elevated [Ca2e]i. Blebs appeared prior to cell death(Table 1) and, once formed, generally enlarged and oftendetached and became free floating. The Ca2+ concentrationin blebs was generally similar to that of adjacent cytoplasm.However, blebs often showed more intense fluorescence (at340-, 365-, and 380-nm excitation) than the rest of the cellwhich frequently was very faded prior to cell death (e.g., seeFig. 2D9). Omitting Ca2+ clearly lowered the sustained[Ca2+]i elevation after HgCl2 injury and also diminished therate and quantity of blebbing.Although the cause of blebbing is unknown, it may be

influenced by the elevation of [Ca2e], through a number ofpathways, including activation of Ca2+-dependent phospho-lipases to modify cell membranes (13); activation of Ca2+-dependent proteinases to alter actin filament attachment tomembranes (24); modification of actin polymerization, in-cluding the interaction ofactin with calmodulin (21) and otherCa2+-binding proteins (25, 26); and interaction of Ca2+ withacidic membrane phospholipids (27). Differences in one ormore of these systems may account for the variation inblebbing activity seen among various cell types.

This study using DIFM and fura-2-loaded monolayers ofrabbit PTE supports and extends our previous findings con-cerning the role of [Ca2+]i in HgCl2-induced cell injury (3, 10,11). HgCl2 treatment first results in [Ca2+]e-independent and

then [Ca2+]e-dependent increases in [Ca2+]i. [Ca2+1] elevationprecedes bleb formation and cell death. Our results clearlyshow that limiting the increase in [Ca2]1j by omitting Ca2+significantly retards blebbing and delays cell death.This work was supported in part by Navy Contract N00014-88-

K-0427 and National Institutes of Health Grant DK15440.

1. Pounds, J. G. (1990) Environ. Health Perspect. 84, 7-15.2. Thomas, C. E. & Reed, D. J. (1989) Hepatology 10, 375-384.3. Trump, B. F., Berezesky, I. K., Smith, M. W., Phelps, P. C.

& Elliget, K. A. (1989) Toxicol. Appl. Pharmacol. 97, 6-22.4. Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433.5. Trump, B. F., Croker, B. P., Jr., & Mergner, W. J. (1971) in

Cell Membranes: Biological and Pathological Aspects, eds.Richter, G. W. & Scarpelli, D. G. (Williams & Wilkins, Balti-more), pp. 84-128.

6. Gritzka, T. L. & Trump, B. F. (1968) Am. J. Pathol. 52,1225-1277.

7. Nieminen, A. L., Gores, G. J., Dawson, T. L., Herman, B. &Lemasters, J. J. (1990) J. Biol. Chem. 265, 2399-2408.

8. Chavez, E. & Holguin, J. A. (1988) J. Biol. Chem. 263, 3582-3587.

9. Abramson, J. J., Trimm, J. L., Weden, L. & Salama, G. (1983)Proc. Natl. Acad. Sci. USA 80, 1526-1530.

10. Phelps, P. C., Smith, M. W. & Trump, B. F. (1989) Lab.Invest. 60, 630-642.

11. Smith, M. W., Ambudkar, I. S., Phelps, P. C., Regec, A. L. &Trump, B. F. (1987) Biochim. Biophys. Acta 931, 130-142.

12. Lemasters, J. J., Diguiseppi, J., Nieminen, A. L. & Herman,B. (1987) Nature (London) 325, 78-81.

13. Troyer, D. A., Kreisberg, J. I. & Venkatachalam, M. A. (1986)Kidney Int. 29, 530-538.

14. Hennings, H., Kruszewski, F. H., Yuspa, S. H. & Tucker,R. W. (1989) Carcinogenesis 10, 777-780.

15. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol.Chem. 260, 3440-3450.

16. Cobbold, P. H. & Rink, T. J. (1987) Biochem. J. 248, 313-328.17. Burgess, G. M., McKinney, J. S., Fabiato, A., Leslie, B. A. &

Putney, J. W., Jr. (1983) J. Biol. Chem. 258, 15336-15345.18. McDonough, P. M. & Button, D. C. (1989) Cell Calcium 10,

171-180.19. Al-Mohanna, F. A. & Hallett, M. B. (1988) Cell Calcium 9,

17-26.20. Becker, P. L. & Fay, F. S. (1987) Am. J. Physiol. 253, C613-

C618.21. Mills, J. S. & Johnson, J. D. (1985) J. Biol. Chem. 260, 15100-

15105.22. Adunyah, S. E. & Dean, W. L. (1986) J. Biol. Chem. 261,

13071-13075.23. Nemeth, E. F. & Scarpa, A. (1987) J. Biol. Chem. 262, 5188-

51%.24. Nicotera, P., Hartzell, P., Davis, G. & Orrenius, S. (1986)

FEBS Lett. 209, 1392-1394.25. Bennett, J. & Weeds, A. (1986) Br. Med. Bull. 42, 385-390.26. Pollard, T. D. & Cooper, J. A. (1986) Annu. Rev. Biochem. 55,

987-1035.27. Verkleij, A. J. & Post, J. A. (1987) Basic Res. Cardiol. 82,

85-91.

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