Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity

Brian J. Wisnoskey,2 Mark Estacion,1 and William P. Schilling1,2

1Rammelkamp Center for Education and Research, MetroHealth Medical Center, and 2Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109-1998

Submitted 31 October 2003 ; accepted in final form 18 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca2+ overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca2+-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca2+ concentration ([Ca2+]i). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140–160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca2+-binding proteins for Ca2+ and Sr2+ vs. Ba2+ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca2+ with either Sr2+ or Ba2+ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba2+ not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca2+-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba2+. Taken together, these results suggest that intracellular high-affinity Ca2+-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise.

necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2


THE ENDOTHELIUM PLAYS A CRUCIAL ROLE in normal cardiovascular function. It is involved in the regulation of blood coagulation and immune response, angiogenesis, blood-tissue permeability and transport, and vascular tone (15). Disruptions of blood flow, such as those that occur in ischemia-reperfusion injury, can decrease the availability of oxygen and compromise the metabolic state of the endothelium. These and other insults often lead to the generation of reactive oxygen species and subsequent cell death by either necrosis (i.e., oncosis) or apoptosis (2, 11, 16, 17). The common signal associated with many of these insults is an increase in intracellular free Ca2+ concentration ([Ca2+]i) (4, 14, 27). Elevations in [Ca2+]i via either Ca2+ influx across the plasma membrane or the release of Ca2+ from intracellular stores can cause disruptions in intracellular signaling and mitochondrial function; activation of endonucleases, phospholipases, and/or proteases; and ultimately oncotic cell death (29). The actual lytic event that occurs during oncosis is thought to reflect an increase in the ionic permeability of the plasma membrane, subsequent alterations in ion concentration gradients, and associated changes in cell volume, i.e., cell swelling, as a result of osmotic water flow. However, the mechanisms by which a rise in [Ca2+]i triggers changes in plasmalemmal permeability remain largely unknown.

Natural products and toxins (e.g., cholera toxin, pertussis toxin, tetrodotoxin, digitalis, ryanodine, thapsigargin) represent powerful tools for the identification and characterization of specific biochemical pathways important for cell signaling. Maitotoxin (MTX), a potent marine toxin isolated from the dinoflagellate Gambierdiscus toxicus, causes Ca2+ influx in a variety of excitable and nonexcitable cell types (10) and ultimately leads to Ca2+ overload-induced oncotic cell death (5). In particular, bovine aortic endothelial cells (BAECs) undergo a well-characterized cell death cascade when challenged with subnanomolar concentrations of MTX (5). This cascade involves three sequential changes in plasmalemmal permeability. First, MTX activates Ca2+-permeable, nonselective cation channels (CaNSC) and causes a concomitant elevation in [Ca2+]i. The rise in [Ca2+]i occurs in a graded fashion, with an EC50 for MTX of ~0.3 nM (5). Second, MTX induces the formation or activation of large endogenous pores that allow passage of low-molecular-weight molecules (<800 Da) across the plasma membrane. These large pores have been termed "cytolytic/oncotic pores" (COP) because their activation appears to be a prelude to oncotic cell death (19, 20). The activity of COP can be monitored by measuring the uptake of vital dyes such as ethidium bromide (EB). The plasma membrane is normally impermeable to EB. However, upon activation of COP, EB enters the cell, where it binds to nucleic acids and exhibits increased fluorescence. The final phase of the MTX-induced cell death cascade is the actual lytic event. Recent studies suggest that cell lysis is not associated with membrane rupture, but rather may reflect the activation of a "death channel" (7). Cell lysis can be monitored in cell populations by the release of large cytosolic proteins such as lactate dehydrogenase (LDH; 140 kDa) and at the single-cell level (i.e., in transfected cells) by the release of cell-associated green fluorescent protein (GFP; 28 kDa) (7). During each phase of the cell death cascade, MTX causes distinct morphological changes in the form of membrane blebbing (5, 7). Membrane blebs are spherical structures, 3–5 µm in diameter, that protrude from the cell surface. MTX-induced bleb formation correlates in time with COP activity. Paradoxically, during cell lysis, the blebs do not rupture or burst, but rather continue to grow in size (5, 7). The role of Ca2+ and indeed the underlying mechanisms responsible for MTX-induced bleb formation and subsequent dilation remain unknown. It is important to note that the MTX-induced cell death cascade is not specific to endothelial cells and is seen in a number of different cell types, including human embryonic kidney cells, THP-1 and BAC1 macrophages, and human skin fibroblasts (19, 20). Furthermore, the cell death cascade activated by MTX is essentially identical to that observed in a number of cell types after stimulation of purinergic receptors of the P2X7 subtype (19, 20). These findings suggest that MTX activates a ubiquitous, highly conserved biochemical cascade leading to oncotic cell death.

In a variety of cell types, pharmacological blockade of the MTX-induced rise in [Ca2+]i blocks many of the downstream cellular events associated with MTX toxicity (3, 23). In BAECs, pharmacological blockade of MTX-induced Ca2+ influx blocks the uptake of vital dyes and rescues BAECs from oncosis (6). These results suggest that one or more steps in the cell death cascade require a rise in [Ca2+]i. However, the exact steps that are sensitive to a rise in [Ca2+]i are completely unknown. The fact that MTX-induced cell death reflects a cascade of cellular events, each of which is presumably dependent on the previous step and potentially has specific requirements for Ca2+, poses a unique challenge. For example, it is difficult to determine the effect of Ca2+ on a downstream event such as membrane blebbing or cell lysis when an upstream step in the cascade, such as COP activation, may also require a rise in [Ca2+]i. Therefore, to determine which step in the cascade is dependent on a rise in [Ca2+]i, we took advantage of the well-known differences in affinity of various Ca2+-binding proteins for Ca2+, Sr2+, and Ba2+. In particular, the affinity of Ca2+-binding proteins of the EF-hand type for Ba2+ is ~2–3 orders of magnitude less than that for Sr2+ or Ca2+ (9). Therefore, the involvement of a high-affinity Ca2+-binding protein at a specific point in the cell death cascade can be revealed by evaluation of the divalent cation selectivity of the individual steps. Experiments were performed at the population level to evaluate the average response of the cells and at the single-cell level to correlate changes in plasmalemmal permeability with changes in bleb formation and dilation. In addition, the single-cell experiments provide important information concerning the heterogeneity (i.e., cell-to-cell variability) of the response. The results show for the first time that Ca2+ affects three specific steps in the death cascade: 1) Ca2+ affects the kinetics of COP activation or formation; 2) Ca2+ dramatically affects the time to cell lysis, suggesting that a high-affinity Ca2+-binding protein may be required for the opening of the death channel; and 3) Ca2+ plays a fundamental role in bleb morphology. These results provide important clues to understanding how a rise in [Ca2+]i controls events at the plasmalemma that ultimately lead to cell demise.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Solutions and reagents. Normal HEPES-buffered saline (HBS) contained (in mM) NaCl 140, KCl 5, MgCl2 1, CaCl2 1.8, D-glucose 10, and HEPES 15, with 0.1% bovine serum albumin and pH adjusted to 7.4 at 37°C with NaOH. The composition of Ba2+-HBS and Sr2+-HBS was identical to that of HBS, with the exception that CaCl2 was isosmotically replaced with BaCl2 and SrCl2, respectively. Fura 2 acetoxymethyl ester (fura 2-AM), BAPTA acetoxymethyl ester (BAPTA-AM), EB, and propidium iodide (PrI) were obtained from Molecular Probes (Eugene, OR). Ionomycin was purchased from Calbiochem (San Diego, CA). MTX was obtained from Wako Bioproducts (Richmond, VA) and was stored as a stock solution in ethanol at –20°C. All other salts were of reagent grade.

Cell culture. BAECs were cultured as previously described (18) with Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM glutamine (complete DMEM). When grown to confluence, the cultures demonstrated a contact-inhibited cobblestone appearance typical of endothelial cells.

Measurement of apparent [Ca2+]i. [Ca2+]i was measured with the fluorescent indicator fura 2 as previously described (18). Experiments were performed with cells in the twelfth to twentieth passages 2–3 days after confluence. Briefly, cells were harvested and resuspended in HBS containing 20 µM fura 2-AM. After 30-min incubation at 37°C, the cell suspension was diluted ~10-fold with HBS, incubated for an additional 30 min, and then washed and resuspended in fresh HBS. Aliquots from this final suspension were subjected to centrifugation and washed twice immediately before fluorescence measurement. Fluorescence was recorded in a mechanically stirred cuvette with the use of an SLM 8100 spectrophotofluorometer. For measurement of Ca2+, the excitation wavelength alternated between 340 and 380 nm every second, and fluorescence intensity was monitored at an emission wavelength of 510 nm. For measurement of Sr2+ and Ba2+, excitation wavelengths of 350 and 390 nm were used. Intracellular free divalent cation concentrations were calculated as previously described (18) using Kd values of 224 nM, 780 nM, and 2.62 µM for fura 2 binding to Ca2+, Ba2+, and Sr2+, respectively. All measurements were performed at 37°C. For the measurement of [Ca2+]i or [Ba2+]i in the presence of a Ca2+ chelator, cells were first harvested and incubated in HBS containing 50 µM BAPTA-AM for 40 min at 37°C. After centrifugation, the cells were resuspended in HBS containing fura 2-AM and treated as described above.

Measurement of vital dye uptake. An aliquot (2 ml) of dispersed cells suspended in HBS at 37°C was placed in a cuvette. After addition of EB (final concentration of 2.5 µM), fluorescence was recorded at 1-s intervals as a function of time with excitation and emission wavelengths of 302 and 590 nm, respectively. EB fluorescence values were corrected for background (i.e., extracellular) dye fluorescence and expressed as a percentage of the value obtained after complete permeabilization of the cells with 50 µM digitonin. Uptake of PrI (final concentration of 2 µM) was determined as described for EB with excitation and emission wavelengths of 536 and 617 nm, respectively. MTX-induced COP activity was estimated from the maximum slope of the change in EB fluorescence as a function of time after the addition of MTX. EB influx from individual fluorescence recordings was determined before MTX-induced cell lysis and therefore reflects an accurate estimate of COP activity under each condition examined. The time to cell lysis was defined and quantified as the time until 20% PrI uptake.

Transfection of BAEC with GFP constructs. Cells were seeded onto 35-mm culture dishes and maintained until they reached 90–95% confluence. A single dish of cells was transfected with 2 µg of pEGFP-C1 cDNA as previously described (7) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, the cells were dispersed with trypsin/EDTA and reseeded onto 12-mm glass coverslips (6–9 coverslips/35-mm dish).

Time-lapse videomicroscopy. BAECs in complete DMEM were sparsely seeded on circular glass coverslips and used within 1–3 days of seeding. The coverslips were mounted in a temperature-controlled perfusion chamber and placed onto the stage of a Leica DMIRE2 inverted microscope. The cells were illuminated with light from a 175-watt xenon lamp using filter cubes appropriate for EB and PrI (Leica N21) or GFP (Leica L5). Epifluorescence was recorded with a SPOT camera (Diagnostic Instruments, Sterling Heights, MI), and images were acquired and analyzed with SimplePCI imaging software (Compix, Cranberry Township, PA). During each experiment, phase and dual fluorescence images were sequentially collected at 30-s intervals using shutter controllers to switch between light and fluorescent illumination. The fluorescence images were used to quantify dye uptake or GFP loss. For dye uptake, a region over an individual cell was defined and the average fluorescence intensity of the region was quantified as a function of time. The kinetics of dye uptake were identical for regions chosen within the nucleus or within the cytoplasm (see Fig. 8). Phase images were digitally merged with the corresponding fluorescent images, and time-lapse videos were created with SimplePCI software. To quantify total GFP fluorescence, a region of interest was drawn around the GFP-positive cell such that it fully enclosed the cell throughout the entire experiment (i.e., including any membrane blebs). The corrected total GFP signal was obtained by subtracting the average background level, which was determined from a nearby control region lacking cells, over the entire region of interest. The background-subtracted GFP fluorescence was summed for all of the pixels within the region of interest. These data were then normalized to the baseline established during the first 5 min to enable comparison between cells.



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Fig. 8. Simultaneous measurement of MTX-induced green fluorescent protein (GFP) loss and EB uptake in single BAECs. BAECs transfected with GFP were grown on glass coverslips, mounted onto the stage of an inverted fluorescence microscope, and bathed in normal HBS containing EB at 37°C. Sequential phase and dual fluorescent images were recorded every 30 s for 40 min as described in MATERIALS AND METHODS. MTX (0.3 nM) was added to the bath at 5 min. A: each row of the montage shows 4 images (phase, GFP, EB, and merged phase/dual fluorescence) of a selected cell obtained at indicated time points. Scale bar, 10 µm. B: GFP (green line) and EB (red line) fluorescence in each image was quantified as described in MATERIALS AND METHODS and is shown as a function of time. Time-lapse videos of this experiment are shown in Videos 1 and 2 (all videos can be viewed online as Supplementary Material to this article). C: simultaneous GFP release ({bullet}) and EB uptake as function of time were quantified for 29 individual cells challenged with MTX (0.3 nM) as described in B. EB uptake was determined as the average pixel intensity in a region over the nucleus ({circ}) or over the cytoplasm ({blacktriangledown}). Symbols represent means ± SD at selected time points.

 
Statistical treatment of the data. All experiments were performed at least three times, and in most instances, the experiments shown in each panel of a single figure are paired (i.e., performed on the same day with the same batch of cells). For cuvette-based experiments, fluorescence was collected at 1-s intervals, and the curves shown in each figure are the mean values from at least three independent experiments. Unless otherwise indicated, the symbols represent means ± SE, which, for clarity, are shown only at selected time points. Where indicated, mean values were compared by performing the paired Student's t-test, with P < 0.05 considered significant. For single-cell measurements, fluorescence values were determined as described above and plotted for each cell in the field of view in a different color. Time-dependent changes in cell morphology are shown as a montage of selected images. Representative videos corresponding to the indicated experiment are available in the Supplemental Material1for this article (Videos 1–4).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MTX-induced cell death cascade. MTX initiates sequential changes in plasmalemmal permeability that culminate in oncotic cell death (5, 7). As seen in Fig. 1A, addition of MTX to a population of endothelial cells suspended in a cuvette causes a concentration-dependent increase in [Ca2+]i, as indicated by the change in the fura 2 fluorescence ratio. The initial rise in [Ca2+]i, which reflects the activation of CaNSC, is followed closely in time by the biphasic uptake of EB (Fig. 1B). The first phase of EB uptake reflects the opening of large pores in the plasmalemma (i.e., COP) and is directly proportional to the MTX concentration (Fig. 1B). The second phase of MTX-induced EB uptake is known to be associated with the release of heterologously expressed GFP (27 kDa) and with the release of the endogenous enzyme LDH (140 kDa) (5, 7). Thus the second phase of EB uptake is indicative of cell lysis. A recent study (7) suggested that PrI may exhibit low permeability via COP and that uptake of PrI may be a good index of cell lysis. To test this hypothesis, the MTX-induced uptake of PrI was examined. As seen in Fig. 1C, the rapid uptake of PrI correlated in time with the second phase of EB uptake, confirming that PrI uptake reflects the lytic phase. It should be noted that after cell lysis, fura 2 is released from the cell to the extracellular solution. Thus, at early times after MTX addition, the fura 2 signal accurately reflects [Ca2+]i, whereas at later times (i.e., during the lytic event), the fluorescence ratio reflects both intracellular and extracellular fura 2. For this reason, calibrated [Ca2+]i values are shown only for the period before cell lysis (Fig. 1A).



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Fig. 1. Effect of maitotoxin (MTX) on plasmalemmal permeability. A: fura 2-loaded bovine aortic endothelial cells (BAECs) were suspended in HEPES-buffered saline (HBS), and the fluorescence ratio was recorded as a function of time as described in MATERIALS AND METHODS. Two superimposed traces are shown. MTX [0.3 nM ({bullet}) or 0.05 nM ({circ})] was added to the cuvette at the time indicated (arrow). Main graph shows calibrated values for [Ca2+]i at times before cell lysis; inset shows the ratios for a longer period. In this and all subsequent figure legends, values indicate final concentrations. B: ethidium bromide (EB) uptake was determined as described in MATERIALS AND METHODS. Two superimposed traces are shown. For each trace, BAECs were suspended in HBS at 37°C. EB (2.5 µM) was added to the cuvette at 20 s, and MTX [0.3 nM ({bullet}) or 0.05 nM ({circ})] was added at 100 s. C: propidium iodide (PrI) uptake was determined as described in MATERIALS AND METHODS. PrI (2 µM) was added to the cuvette at 20 s, and MTX [0.3 nM ({bullet}) or 0.05 nM ({circ})] was added at 100 s. Curves shown are representative of at least 3 experiments. The timing of each phase of the response is indicated by dashed lines.

 
Previous studies in BAECs have shown that the MTX-induced uptake of EB is blocked by the pharmacological inhibition of Ca2+ influx (6), suggesting that a rise in [Ca2+]i is necessary for downstream events in the cell death cascade. To determine whether a rise in [Ca2+]i is necessary or sufficient, we examined the effect of the Ca2+ ionophore ionomycin on the uptake of EB (Fig. 2). The addition of ionomycin (20 µM) to BAECs produced an increase in [Ca2+]i and biphasic uptake of EB virtually identical to that observed with MTX. These results suggest that a rise in [Ca2+]i is sufficient to initiate the cell death cascade.



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Fig. 2. Effect of ionomycin on the change in [Ca2+]i and EB uptake in BAECs. [Ca2+]i (A) and EB (B) uptake were measured in BAECs as described in Fig. 1. Ionomycin (20 µM) was added at the time indicated by the arrow. Fura 2 ratios for a longer time course are shown in A (inset). Curves represent mean values of 3 independent experiments. Symbols represent means ± SE at selected time points.

 
Divalent cation selectivity. It is well known that Sr2+ substitutes for Ca2+ in a number of cellular reactions but that Ba2+ is a poor surrogate for Ca2+. In particular, the involvement of high-affinity Ca2+-binding proteins can be revealed by evaluating the divalent cation potency sequence (9). To determine the divalent cation selectivity of each step in the cell death cascade, fura 2 fluorescence, EB uptake, and PrI uptake were measured in parallel after the addition of MTX to endothelial cells suspended in solutions in which Ca2+ was replaced by either Sr2+ or Ba2+. As seen in Fig. 3A, 1 min after the addition of 0.3 nM MTX, cytosolic Sr2+, Ca2+, and Ba2+ were 9,850 ± 105, 803 ± 139, and 452 ± 60 nM, respectively. Within 3 min of MTX addition, however, [Ca2+]i and [Ba2+]i were essentially the same, whereas [Sr2+]i remained ~10-fold higher. (At times longer than ~4 min, fura 2 approaches saturation, and therefore, the values do not accurately reflect concentration.) Importantly, these results demonstrate that Ca2+, Sr2+, and Ba2+ enter the cell in response to MTX and that the presence of the surrogate cations in the extracellular solution does not prevent activation of the CaNSC.



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Fig. 3. Effect of divalent cation substitution on COP activation and cell lysis. Three superimposed traces are shown in each graph. BAECs were suspended in HBS containing 1.8 mM Ca2+ ({circ}), Sr2+ ({triangledown}), or Ba2+ ({bullet}). A: increase in divalent cation concentration was calculated from fura 2 fluorescence ratios after the addition of MTX (0.3 nM). Uptake of EB (B) and PrI (C) in each buffer were determined from the increase in fluorescence as a function of time. Note that the time frame is different in each graph. Curves represent mean values of 3 independent experiments. Symbols represent means ± SE at selected time points.

 
To determine the effect of divalent cation substitution on the activation of COP and cell lysis, the uptake of EB was examined (Fig. 3B). The EB uptake profile in the presence of Sr2+ was remarkably similar to that observed with Ca2+. There was no detectable difference in the first phase of EB uptake (i.e., activation of COP) and, on average, only a small delay in the time to cell lysis was observed when Ca2+ was replaced by Sr2+. When Ca2+ was replaced by Ba2+, the first phase of EB uptake was slightly delayed, and COP activity, as indicated by the rate of EB uptake (i.e., the slope), was slightly reduced (7.2 ± 0.5 vs. 5.9 ± 0.9%/min, n = 11; P < 0.001). The major effect of replacing Ca2+ with Ba2+, however, was the apparent lack of cell lysis, i.e., no second phase of EB uptake was observed over the course of this experiment (Fig. 3B). To determine whether the cells ultimately lyse in Ba2+-containing solutions, PrI uptake was measured over a longer time course (Fig. 3C). As expected from the EB uptake profiles, PrI uptake was delayed only slightly when Ca2+ was replaced by Sr2+. However, in the presence of Ba2+, PrI uptake was significantly delayed by ~13 min, confirming that cell survival is prolonged by substitution of Ca2+ with Ba2+. It is important to note that the basal uptake of EB and PrI in the absence of MTX was ~6% after 60 min (data not shown). Thus the uptake of EB and PrI in the presence of Ca2+, Sr2+, or Ba2+ is absolutely dependent on prior stimulation with MTX and does not reflect deleterious effects of long-term incubation of the cells with these Ca2+ surrogates.

To better evaluate the relative selectivity of each phase of the death cascade for Ca2+ vs. Sr2+, the extracellular divalent cation concentrations were varied so that the MTX-induced increase in [Ca2+]i and [Sr2+]i was essentially the same (Fig. 4A). Under these conditions, both the first and second phases of EB uptake were slightly delayed in Sr2+ relative to those phases in Ca2+-containing buffer (Fig. 4B). This result suggests that Sr2+ has a potency that is less than that of Ca2+ but greater than that of Ba2+.



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Fig. 4. Comparison of Ca2+ with Sr2+. Two superimposed traces are shown in each graph. BAECs were suspended in HBS containing either 10 mM Ca2+ ({circ}) or 1.25 mM Sr2+ ({triangledown}). A: increase in divalent cation concentration was calculated from fura 2 fluorescence ratios after the addition of MTX (0.3 nM). Uptake of EB (B) in each buffer was determined from the increase in fluorescence as a function of time. Curves represent mean values of 3 independent experiments. Symbols represent means ± SE at selected time points.

 
To determine whether the effect of Ba2+ on the MTX-induced cell death cascade reflects an alteration in MTX binding, and to further explore the effects of divalent cation substitution, changes in plasmalemmal permeability were examined at concentrations of MTX (0.05–1.0 nM) both above and below the apparent EC50 (determined previously to be ~0.3 nM). The activity of the CaNSC was estimated in a pairwise fashion from the initial rate of change in [Ca2+]i or [Ba2+]i (determined before activation of COP). As seen in Fig. 5A, MTX-induced divalent cation influx was affected little by replacing Ca2+ with Ba2+; i.e., there is little or no shift in the MTX concentration-response curve. We next examined COP activity in Ca2+- vs. Ba2+-containing solutions, (estimated from the maximum slope of EB uptake before cell lysis). At 0.05 and 0.1 nM MTX, COP activity was, on average, 1.8- and 1.4-fold greater, respectively, in Ca2+ than in Ba2+ (Fig. 5B). At 0.3 nM MTX, the difference was only 1.1-fold, and at 1.0 nM MTX, COP activity in Ca2+ was slightly reduced to 0.82 of that seen in the presence of Ba2+, i.e., the Ca2+ curve appears to cross over the Ba2+ curve. A similar crossover phenomenon was observed previously for EB uptake in THP-1 monocytes when stimulated by high concentrations of MTX (20), suggesting that a rapid, large increase in [Ca2+]i produced by high MTX causes feedback inhibition of COP activity. These results suggest that there may be a change in COP activity in Ba2+- vs. Ca2+-containing solutions, but the MTX concentration required is similar for both divalent cations tested. We next examined, in a pairwise fashion, the time to cell lysis as indicated by PrI uptake (Fig. 5C). Clearly, the time to cell lysis was significantly longer at each concentration of MTX examined when Ca2+ was replaced by Ba2+. These results provide further support for the conclusion that Ba2+ has little effect on MTX binding or on its ability to activate CaNSC, but that some intracellular steps involved in COP activation and the kinetics of cell lysis are sensitive to the replacement of Ca2+ with Ba2+. If in fact the effect of Ba2+ on the cell death cascade reflected interaction with intracellular proteins, we reasoned that simply chelating cytosolic divalent cation should produce a similar delay in the activation of COP and a delay in cell lysis. To test this hypothesis, the effect of a low concentration of MTX was examined in cells loaded with the Ca2+ chelator BAPTA. As seen in Fig. 6, A and B, loading the cells with BAPTA significantly delayed the MTX-induced rise in [Ca2+]i and delayed both phases of EB uptake, again suggesting that a rise in [Ca2+]i is necessary for both COP activation and cell lysis. The MTX-induced increase in [Ba2+]i and EB uptake via COP were likewise significantly delayed (Fig. 6, C and D). These results provide additional evidence that activation of COP is sensitive to intracellular Ca2+ and that the delay observed in Ba2+-containing solutions likewise reflects an intracellular event.



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Fig. 5. Effect of Ca2+ replacement by Ba2+ on the MTX concentration-response curve. The [Ca2+]i, [Ba2+]i, EB uptake, and PrI uptake were determined as described in Fig. 3 at MTX concentrations of 0.05, 0.1, 0.3, and 1.0 nM in the presence of either Ca2+ ({circ}) or Ba2+ ({bullet}). The d[divalent]/dt was determined from the maximum slope of calibrated fura 2 traces before activation of COP (i.e., before EB uptake). The activity of COP was quantified as the slope of the first phase of EB uptake before cell lysis (i.e., before PrI uptake). The time to cell lysis was quantified as the time to 20% PrI uptake. Values were determined on the basis of individual fluorescence traces. Symbols shown represent means ± SE from 3 experiments.

 


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Fig. 6. Effect of BAPTA loading on MTX-induced changes in [Ca2+]i and [Ba2+]i and associated EB uptake. Two superimposed traces are shown in each graph. BAECs were loaded with fura 2 and BAPTA as described in MATERIALS AND METHODS and suspended in HBS containing 1.8 mM Ca2+ (A and B) or Ba2+ (C and D). A and C: net change in divalent cation concentration was calculated from fura 2 fluorescence ratios after addition of MTX (0.05 nM) in control ({bullet}) or BAPTA-loaded cells ({circ}). B and D: uptake of EB was determined after addition of MTX (0.05 nM) in control ({bullet}) or BAPTA-loaded cells ({circ}). Curves represent mean values of 3 independent experiments. Symbols represent means ± SE at selected time points. Note that y-axis in D is expanded to highlight the first phase of EB uptake.

 
Although all three divalent cations tested support MTX-induced cell death, the results suggest that the proteins and/or enzymes involved in the activation of COP and subsequent cell lysis exhibit either a lower affinity for Ba2+ than for Ca2+ or Sr2+ or a lower efficacy with Ba2+ bound. Alternatively, Ba2+ may actually inhibit various steps in the cell death cascade. To distinguish between these possibilities, low concentrations of Ca2+ were readmitted to the Ba2+-containing extracellular buffer, and the response to MTX was examined (Fig. 7). Readdition of 0.1 to 0.5 mM Ca2+ to the Ba2+-containing buffer produced a graded leftward shift in the first phase of EB uptake (Fig. 7, A and A') and in the time to cell lysis, as indicated by the leftward shift in both the second phase of EB and PrI uptake (Fig. 7B). These results are consistent with the conclusions that Ba2+ does not inhibit the cell death cascade and that the affinity of the various steps for Ca2+ is relatively high and much greater than that for Ba2+.



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Fig. 7. Effect of Ca2+ readmission on COP activation and cell lysis. Five superimposed traces are shown in each graph. BAECs were suspended in HBS containing 1.8 mM Ca2+ ({circ}) or Ba2+ ({bullet}). Where indicated, Ba2+-HBS was supplemented with Ca2+ to obtain a final concentration of 0.1 mM [Ca2+]e ({blacktriangledown}), 0.2 mM [Ca2+]e ({blacklozenge}), or 0.5 mM [Ca2+]e ({blacksquare}). A: EB uptake was recorded in normal Ca2+-HBS, Ba2+-HBS, or Ca2+-supplemented Ba2+-HBS. A': data from A are replotted on expanded scale to highlight first phase of EB uptake. B: PrI uptake was recorded in normal Ca2+-HBS, Ba2+-HBS, or Ca2+-supplemented Ba2+-HBS (note expanded time scale). Curves represent mean values of 3 independent experiments. Symbols represent means ± SE at selected time points.

 
Effect of divalent cation substitution on cell morphology. Previous experiments revealed that BAECs challenged with MTX exhibit dramatic changes in cell morphology (5, 7). To determine the effect of Ba2+ substitution, each phase of the MTX-induced cell death cascade was compared with time-dependent changes in cell morphology by using simultaneous phase and fluorescence videomicroscopy. For these studies, BAECs were transfected with a GFP construct and seeded onto glass coverslips. The cells were bathed in either Ca2+- or Ba2+-containing solution along with the vital dye EB. The first phase of EB uptake at the single-cell level was used as an index of COP activity. The second phase of EB uptake and the loss of GFP were used as indexes of single-cell lysis. In parallel experiments, the loss of GFP and the uptake of PrI were simultaneously monitored as a third index of cell lysis. Phase and fluorescence image pairs were recorded every 30 s and quantified at the single-cell level as described in MATERIALS AND METHODS. The representative montage in Fig. 8 shows phase, GFP, EB, and merged images of an endothelial cell before and at various times after the addition of MTX. As previously reported (5), the addition of MTX (at 5 min) caused the formation of membrane blebs that develop within 5–10 min of MTX addition and appear as balloon-like structures on the surface of the cell (Fig. 8A). The formation of membrane blebs occurred during the first phase of EB uptake (Fig. 8B) and therefore was correlated with the activation of COP. At 18 min, GFP began to leave the cell, and by 23 min, cell-associated GFP was undetectable. The second phase of EB uptake correlates in time with the loss of GFP (Fig. 8B). The EB uptake profile was the same whether quantified in a region within the nucleus or within the cytoplasm (Fig. 8C), confirming that the biphasic profile observed does not reflect limited access of the dye to the nucleus. Note from the images in Fig. 8A that blebs did not rupture during and after cell lysis, but rather continued to appear as smooth spherical structures without obvious tears or discontinuities. In fact, during and after the loss of GFP, the blebs continued to grow in size. This is most evident in two of the time-lapse videos (Videos 1 and 2) that accompany this article. The same experiment was repeated using solutions in which Ca2+ was replaced by Ba2+. A representative montage with three cells in the field of view is shown in Fig. 9. All of the cells exhibited the first phase of EB uptake, indicative of COP, but the time to activation of COP was delayed relative to that observed in Ca2+-containing solution. Over the time frame shown, only one of the GFP cells exhibited the second phase of EB uptake and rapid loss of GFP. Furthermore, the time to lysis for the cell shown was 40 min after MTX addition, much longer than the time observed in Ca2+-containing solutions. Thus, as suggested by the cuvette studies, Ba2+ 1) delays activation of COP, 2) has relatively little effect on MTX-induced COP activity once activated, and 3) greatly delays cell lysis. Importantly, blebbing is either not observed or greatly attenuated when the cells are challenged with MTX in Ba2+-containing solution. In particular, when Ba2+ replaces Ca2+, the cells tended to exhibit a more general enlargement or swelling, with little or no bleb formation. Furthermore, if blebs do form in the presence of Ba2+, they generally lack the defined neck region needed to give the distinct spherical appearance typical of dilated blebs seen in Ca2+-containing solutions. Many times, the cells fail to bleb at all or exhibit only a single bleb in the presence of Ba2+ (Videos 3 and 4).



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Fig. 9. Effect of Ba2+ on MTX-induced cell death cascade in single BAECs. The experiment was performed and fluorescence was quantified exactly as described in Fig. 8, with BAEC bathed in Ba2+-HBS. Time-lapse videos of this experiment can be viewed in Videos 3 and 4.

 
The effect of MTX in either Ca2+- or Ba2+-containing solution was examined on several coverslips, and the loss of GFP was correlated with either EB or PrI uptake. The individual responses for each cell examined are shown in Fig. 10, and the means ± SD are summarized in Fig. 11. In Ca2+-containing buffer, the uptake of EB was biphasic at the single-cell level. In addition, the rapid loss of GFP correlated closely in time with both the second phase of EB uptake and the rapid phase of PrI uptake, i.e., between 15 and 25 min, the majority of cells release GFP and rapidly stain with both EB and PrI (Fig. 10, B and C). Thus MTX-induced EB and PrI uptake observed in the population-based cuvette assays were qualitatively recapitulated at the single-cell level. In Ba2+-containing solution, it is clear that MTX-induced activation of COP is delayed, as indicated by the time lag before the first phase of EB uptake (Fig. 11B). Furthermore, once activated, COP activity (i.e., the slope of the first phase of EB uptake) is the same in both Ca2+- and Ba2+-containing solutions (Fig. 11B). However, both the second phase of EB uptake and the rapid phase of PrI uptake are significantly delayed. Furthermore, there is substantial cell-to-cell variability in the time to cell lysis in Ba2+. In Ca2+, the majority of cells lyse within a narrow 10- to 15-min window, whereas in Ba2+, lysis occurs between 30 and 60 min, and a number of cells (26 of 37) failed to lyse over the course of these experiments (Fig. 10, C and F). Thus there appears to be substantial heterogeneity in the time to cell lysis when Ba2+ replaces Ca2+. In Ba2+ solution, only 6 of 15 cells challenged with MTX showed a rapid phase of GFP release (Fig. 10D). The majority of cells exhibited only a slow decline in GFP fluorescence over the course of these experiments. However, the rapid loss of GFP in the six cells was correlated in time with the second phase of EB uptake. The slow decrease in GFP fluorescence in these long time course studies may reflect either photobleaching or, more likely, the movement of GFP out of the focal plane as the cells swelled and enlarged in response to MTX.



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Fig. 10. Composite single-cell fluorescence data. Experiments were performed and fluorescence was quantified as described in Figs. 8 and 9 in the presence of Ca2+-HBS (A–C) or Ba2+-HBS (D–F). Each line represents a single cell from several (n = 3–5) independent experiments. In all graphs, MTX (0.3 nM) was added at time 5 min. The total numbers of cells evaluated under each condition are shown in parentheses. Note that the loss of GFP fluorescence was monitored simultaneously in parallel experiments with either EB or PrI uptake.

 


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Fig. 11. Average single-cell responses. The average single-cell GFP (A), EB (B), and PrI (C) fluorescence values were calculated from the data shown in Fig. 10 in either Ca2+-HBS ({bullet}) or Ba2+-HBS ({circ}). The symbols shown at selected time points represent means ± SD.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Stimulation of endothelial cells with MTX leads to a well-defined sequence of permeability changes that culminate in oncotic cell death. First, MTX causes a large increase in [Ca2+]i via the activation of CaNSC. Second, there is an opening of large endogenous pores (i.e., COP) that allows for the uptake of EB. Finally, the cells undergo lysis, an all-or-nothing event evidenced by the release of large macromolecules from the cell and the rapid uptake of PrI. During each phase of the response, there is a dramatic change in cellular morphology, characterized by the appearance of membrane protrusions or blebs that form concurrently with the activation of COP and dilate as the cell progresses through the lytic phase. As the concentration of MTX increases, there is an increase in [Ca2+]i, an increase in COP activity, and a decrease in the time to cell lysis. Although these events appear to be sequential and to depend on the rise in [Ca2+]i, the biochemical mechanisms linking each step in the cascade and the specific role of [Ca2+]i in each phase of the response remain unknown. Similar changes in plasmalemmal permeability have been observed after the stimulation of purinergic receptors of the P2X7 subtype (20), suggesting commonality in the cell death cascades.

To evaluate the role of Ca2+ as either a trigger or a modulator of COP activity and/or cell lysis, we took advantage of the well-known differences in affinity of various Ca2+-binding proteins for Ba2+ (9). Although Ba2+ and Sr2+ are permeable through many types of voltage-gated and receptor-operated cation channels, Ba2+ is a poor substrate for sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and plasma membrane Ca2+-ATPase (PMCA) pumps (8, 21, 24, 28) and in general has much lower affinity for Ca2+-binding proteins of the EF-hand and C2 type than does Ca2+ (9). The results of the present study show that the kinetics and the magnitude of each step in the cell death cascade were similar when Ca2+ was isosmotically replaced by Sr2+, with only a slight delay in the average time to cell lysis. A delay in the time to COP activation was revealed in Sr2+, however, when the extracellular divalent cation concentrations were adjusted to produce comparable increases in cytosolic concentrations. When Ca2+ was isosmotically replaced by Ba2+, COP activation was again slightly delayed and the time to cell lysis was greatly prolonged. These changes noted in Ba2+-containing solution were observed at both the population and the single-cell level. Thus proteins that couple channel activation with downstream events observed in the cell death cascade appear to have a lower affinity for Ba2+. Furthermore, in the presence of Ba2+, the addition of 0.1–0.5 mM Ca2+ to the extracellular buffer produced a concentration-dependent decrease in the time to COP activation and cell lysis. Overall, these results provide strong support for the conclusion that proteins with high-affinity Ca2+-binding sites are critically involved in the sequential changes in plasmalemmal permeability that accompany MTX-induced cell death.

The kinetics of COP formation or activation were delayed by the replacement of Ca2+ with either Sr2+ or Ba2+ or by the chelation of intracellular Ca2+ or Ba2+ by BAPTA. Furthermore, simply increasing [Ca2+]i by adding ionomycin produced biphasic uptake of EB similar to that seen with the addition of MTX. These results suggest that an intracellular step between the activation of CaNSC and COP is affected by Ca2+. The actual mechanism by which MTX activates COP is unknown. Likewise, the mechanism by which the activation of P2X7 purinergic receptors is linked to the formation of COP is unclear. It was originally proposed that P2X7 receptors might aggregate to form increasingly larger pore structures (13, 25). However, studies with GFP-tagged receptors have shown that EB uptake is not associated with changes in receptor distribution or in changes in receptor density (22). In addition, we have shown that although the permeability of COP is inversely proportional to dye molecular weight, MTX-induced dye uptake is linear during the COP phase, which is inconsistent with a dilation model (5). The MTX-activated channel and the channel formed by P2X7 receptor protein are clearly distinct, but the COP activated by both MTX and purinergic agonists are virtually indistinguishable (20). This has led to speculation that COP is a separate, membrane-associated pore structure that can be activated by a variety of Ca2+-permeable cation channels. In this regard, we have shown that overexpression of P2X7 receptors produces a shift in the MTX concentration-response curve, such that higher concentrations of the toxin are required to activate COP (20). This result suggests that the MTX channel and the purinergic channels compete for a common pool of COP and that the channels may be associated physically with the COP structure. Indeed, rapid pharmacological inhibition of MTX-induced Ca2+ influx during the first phase of EB uptake produces an immediate blockade of COP activity, consistent with tight functional coupling between CaNSC activity and COP (5). A rise in [Ca2+]i may therefore be necessary for physical interaction or coupling between CaNSC and COP.

The single-cell experiments revealed additional differences in the cell death cascade in the presence of Ba2+. Ba2+ caused a desynchronization of cell lysis, as indicated by the dispersion in PrI uptake. In the presence of Ca2+, all of the cells rapidly accumulated PrI within a narrow 10-min window, whereas in Ba2+-containing solution, cell lysis was delayed and occurred during a 35- to 60-min span. This heterogeneity with respect to cell lysis was also evident in the time to the second, rapid phase of EB uptake. The biochemical reason for the difference in cell synchronization between Ca2+ and Ba2+ is unclear. One possibility is that a critical cellular factor must be exhausted before the cell lyses and that the degradation or metabolism of this factor is slower in Ba2+ than in Ca2+. Because Ba2+ is a poor substrate for Ca2+ pumps and will not be sequestered within the endoplasmic reticulum, a possible candidate for this protective factor is ATP. During Ca2+ overload conditions, Ca2+ pumps are expected to operate at near-maximum level, consuming large amounts of ATP while mitochondrial function is probably seriously compromised. Under Ba2+ overload conditions, however, pump activity, and hence ATP consumption, is reduced. The hypothesis that MTX-induced cell lysis occurs when ATP is reduced to a critical level is currently under investigation.

The single-cell experiments also showed that Ba2+ substitution dramatically altered the membrane blebbing profile. In Ca2+-containing solution, blebs initially form during and after COP activation. Once formed, blebs remain relatively constant in size until the lytic event, at which time dramatic bleb dilation is observed. Interestingly, the cell morphology remains relatively intact even during massive bleb dilation, consistent with a previous suggestion that membrane blebbing may be a mechanism by which the cell regulates or relieves large increases in intracellular pressure (1). The blebs seen in Ca2+ solutions also have a narrow neck at the point of attachment to the cell, which contributes to the balloon-like appearance. However, the MTX-induced bleb formation is dramatically attenuated when Ca2+ is replaced by Ba2+, and a more generalized cell swelling is often observed. If blebs do form in the presence of Ba2+, they lack a defined neck structure, consistent with the hypothesis that a protein with high affinity for Ca2+ is needed to maintain the integrity of the neck complex. Although the mechanisms responsible for necrotic and apoptotic membrane blebbing appear to be different (1), previous studies of membrane blebbing have shown that the actin-myosin cytoskeleton forms a ring centered at the neck of membrane blebs and that myosin light-chain phosphorylation via myosin light-chain kinase (MLCK) plays an important role in bleb formation (1, 12, 26). MLCK is a Ca2+/calmodulin-dependent enzyme and is therefore expected to have a low affinity for Ba2+.

In conclusion, the results of the present study show for the first time that high-affinity Ca2+-binding proteins are involved in regulating changes in plasmalemmal permeability in response to MTX. Specifically, high-affinity Ca2+-binding proteins appear to play a critical role in the transition from CaNSC to COP and in the transition from COP to lytic pore. Although Ca2+-sensitive phosphatases (e.g., calcineurin), proteases (e.g., calpain), and/or lipases (e.g., phospholipase A2) may be indirectly involved, a more direct effect of Ca2+ on the formation of COP and the assembly or activation of a lytic death channel cannot be excluded. Additional studies are necessary to determine the identity of the Ca2+-binding proteins involved in each step and to understand their mechanism of action at the plasma membrane.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-65323 (to W. P. Schilling). B. J. Wisnoskey was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant DK-07678.


    ACKNOWLEDGMENTS
 
We thank Milana Belich for excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. P. Schilling, Rammelkamp Center for Education and Research, Rm. R322, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland, OH 44109-1998 (E-mail: wschilling{at}metrohealth.org).

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

1 Supplementary Material to this article (Videos 1–4) is available online at http://ajpcell.physiology.org/cgi/content/full/00473.2003/DC1. Back


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