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Am J Physiol Cell Physiol 291: C657-C667, 2006. First published May 3, 2006; doi:10.1152/ajpcell.00063.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

William P. Schilling,1,2 Deborah Snyder,2 William G. Sinkins,2 and Mark Estacion1

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

Submitted 9 February 2006 ; accepted in final form 27 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The plasmalemmal Na+-K+-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca2+. However, the ability of PTX to directly increase cytosolic free Ca2+ concentration ([Ca2+]i) via Na+ pump channels and to initiate Ca2+ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca2+]i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca2+]i that was dependent on extracellular Ca2+. The increase in [Ca2+]i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC50 < 1 µM. The elevation in [Ca2+]i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca2+]i occurs via the Na+ pump. Subsequent to the rise in [Ca2+]i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca2+ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca2+ overload.

necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2

NATURAL TOXINS AND POISONS (e.g., cholera toxin, pertussis toxin, tetrodotoxin, conotoxin, digitalis, ryanodine, and thapsigargin) have proved useful in the identification and functional characterization of specific proteins in biochemical pathways critical for cell homeostasis and signaling. Palytoxin (PTX), originally isolated from sea corals of the genera Palythoa (24), is one of the most potent toxins known. PTX causes membrane depolarization, loss of cellular K+, and a dramatic increase in cytosolic Na+. PTX causes contractions of all muscles, release of neurotransmitters, hemolysis of red blood cells, and, ultimately, oncotic cell death (for review, see Ref. 38). It is now clear that the molecular receptor for PTX is the plasmalemmal Na+-K+-ATPase (NKA) pump. Early studies showed that the cardiac glycoside ouabain could effectively antagonize the actions of PTX, and it was suggested that PTX might convert the NKA into a channel (14, 15). Indeed, a variety of investigators showed that PTX activates a relatively nonselective cation channel with conductance in the range of 8–14 pS (16, 17, 20, 25, 27, 28, 39, 43). PTX-induced cation fluxes were activated when the NKA was heterologously expressed in yeast that lack an endogenous NKA activity (30, 31). Furthermore, PTX-induced single channels were observed after reconstitution of the NKA in planar lipid bilayers following in vitro expression (16). These results provide strong evidence that the NKA is the receptor for PTX and that PTX converts the NKA pump into a channel. As mentioned above, ouabain blocks the effect of PTX on the NKA pump. Although ouabain and PTX binding are not mutually exclusive, ouabain greatly increases PTX dissociation rate, suggesting that the binding of either PTX or ouabain destabilizes the binding of the other, presumably via an allosteric mechanism (4).

Recent elegant experiments by Artigas and Gadsby (2–4) provide a detailed biophysical picture of the mechanism of PTX action on the NKA. The NKA pump is thought to have two gates that control the access of ions to their binding sites within the protein. These gates normally open and close as the pump changes between two major conformational states designated E1 and E2. When the pump is in the E1 form, the inner gate is open and the ion binding sites are accessible from the cytosol. When the pump is in the E2 form, the outer gate is open and the ion binding sites are accessible from the extracellular space. To function as a pump, the inner and outer gates open and close in a sequential fashion but can never open together. However, when PTX binds to the NKA, the pump is locked into a conformation that allows simultaneous opening of both gates, giving rise to characteristic channel activity. Permeation studies have shown that the channels activated by PTX are not selective for either Na+ or K+ but, rather, are nonselective, exhibiting a slight but detectable permeability to Ca2+ (4). Although the ability of PTX channels to directly influence cytosolic free Ca2+ concentration ([Ca2+]i) has not been studied in detail, experiments in porcine coronary artery smooth muscle cells (18), human osteoblast-like Saos-2 cells (23), rabbit endothelial cells (1), and mouse spleen cells (29) with the use of fluorescent Ca2+ indicators suggest that PTX may increase [Ca2+]i indirectly via depolarization and activation of voltage-gated Ca2+ channels or via the Na+/Ca2+ exchanger. The ability of PTX to directly increase [Ca2+]i by stimulating Ca2+ influx via the NKA operating in channel mode has not been demonstrated in any cell type.

It is well established that PTX ultimately causes cell lysis, but the mechanism by which this occurs has not been investigated. Given the slight permeability of the PTX channel for Ca2+ and the high density of NKA pumps in the plasmalemma, a component of PTX-induced cell death may reflect elevated [Ca2+]i. In this regard, maitotoxin (MTX), another extremely potent marine toxin, initiates a well-characterized cascade of events that also culminates in cell lysis. First, MTX activates Ca2+-permeable nonselective cation channels and causes a concomitant elevation in [Ca2+]i. This is followed closely in time by the formation or activation of large endogenous pores that allow passage of low-molecular-mass molecules (<800 Da) across the plasma membrane (8, 9, 41, 45). These large pores have been termed cytolytic/oncotic pores, or COP, because their activation appears to be a prelude to oncotic cell death. 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. Our recent studies suggest that cell lysis is not associated with membrane rupture but, rather, may reflect the activation of a "death channel" (10). Lysis can be monitored at the single-cell level by measuring the release of transiently expressed green fluorescent protein (GFP; 27 kDa) or by measuring the uptake of propidium iodide (PI).

The purpose of the present study was to determine whether PTX 1) increases [Ca2+]i in vascular endothelial cells via the NKA pump and 2) initiates a cell death cascade similar or identical to that observed for MTX. Bovine aortic endothelial cells (BAECs) are useful for these studies because they lack voltage-gated Ca2+ channels and the Na+/Ca2+ exchanger (32). Thus changes in [Ca2+]i caused by PTX will presumably be related to Ca2+ influx via the NKA pump operating in channel mode. The results of these studies demonstrate that PTX causes a 1) ouabain-sensitive increase in [Ca2+]i that reflects Ca2+ influx from the extracellular space, 2) ouabain-sensitive biphasic increase in EB uptake, 3) glycine-sensitive release of cell-associated GFP, and 4) dramatic blebbing of the plasmalemma during the lysis phase. Although some details of the PTX-induced cell death cascade differ from those observed for MTX, the results are consistent with the hypothesis that Ca2+ entry via the NKA pump operating in channel mode is responsible for cell lysis. Thus, although the NKA pump is the target of PTX action, it is the rise in [Ca2+]i that initiates cellular damage and leads to rapid oncotic cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Solutions and reagents. Normal HEPES-buffered saline (HBS) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM D-glucose, 15 mM HEPES, and 0.1% bovine serum albumin, with pH adjusted to 7.4 at 37°C with NaOH. Ca2+-free HBS contained the same salts as HBS without added CaCl2. Fura-2 acetoxymethyl ester (fura-2 AM), sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI AM), EB, the propidium cation YO-PRO-1, and PI were obtained from Molecular Probes (Eugene, OR). MTX and PTX were obtained from Wako Bioproducts (Richmond, VA). MTX was stored as a stock solution in ethanol at –20°C. PTX was stored at –80°C as an aqueous stock solution in 1% bovine serum albumin. All other salts were of reagent grade.

Cell culture. BAECs, isolated from cow aorta (7), were cultured as previously described (33) at 37°C in a humidified air atmosphere with 5% CO2 by using Dulbecco's modified Eagle's medium (GIBCO) 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 contact-inhibited cobblestone appearance typical of endothelial cells. Human embryonic kidney (HEK)-293 cells were similarly grown in monolayer culture by using minimal essential medium supplemented with L-glutamine, 10% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin solution.

Measurement of apparent cytosolic free Ca2+ and Na+ concentration. [Ca2+]i and [Na+]i were measured using the fluorescent indicators fura-2 and SBFI as previously described (33). Experiments were performed with cells in the 12th to 20th passage and 1–3 days postconfluence. Briefly, cells were harvested and resuspended in HBS containing 20 µM fura-2 AM or SBFI AM. After 30 (fura-2) or 45 min (SBFI) of incubation at 37°C, the cell suspension was diluted ~10-fold with HBS, incubated for an additional 30 or 45 min, 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 using an SLM 8100 spectrophotofluorometer. Excitation wavelength alternated between 340 and 380 nm every second, and fluorescence intensity was monitored at an emission wavelength of 510 nm. Calibration of the fura-2 associated with the cells was accomplished using Triton lysis in the presence of a saturating concentration of Ca2+ followed by addition of EGTA (pH 8.5). [Ca2+]i was calculated using the equations of Grynkiewicz et al. (13) with a Kd value of 224 nM for Ca2+ binding to fura-2. The Kd of SBFI for Na+ is in the range of 3–12 mM, depending on the K+ concentration. To determine the maximum fluorescence ratio for SBFI, we increased the concentration of extracellular Na+ to 200 mM by adding a small aliquot of 1 M NaCl, and the cells were lysed with Triton. SBFI fluorescence as a function of time was normalized to the value obtained in the presence of 200 mM Na+. All measurements were performed at 37°C.

Measurement of vital dye uptake. An aliquot (2 ml) of dispersed cells suspended in HBS at 37°C was placed in a cuvette. After the addition of EB (final concentration 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 (extracellular) dye fluorescence and expressed as a percentage relative to the value obtained after complete permeabilization of the cells with 50 µM digitonin. Uptake of PI and YO-PRO-1 was determined as described for EB with excitation/emission wavelengths of 536/617 and 468/510 nm, respectively.

Transfection of BAECs 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 (10), using Lipofectamine 2000 (Invitrogen). Four to six hours after transfection, the cells were dispersed with trypsin/EDTA and reseeded onto 12-mm glass coverslips (24 coverslips per 35-mm dish).

Time-lapse video microscopy. BAECs in complete medium 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 on the stage of a Leica DMIRE2 inverted microscope. The cells were illuminated with light from a 175-W xenon lamp with the use of filter cubes appropriate for EB and PI (Leica N21) or GFP (Leica L5). Epifluorescence was recorded using a SPOT-RT camera (Diagnostic Instruments, Sterling Heights, MI), and images were acquired and analyzed using SimplePCI imaging software (Compix, Cranberry Township, PA). During each experiment, phase and dual-fluorescence images were sequentially collected at 30-s intervals with shutter controllers switching 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 (45). Phase images were digitally merged with the corresponding fluorescent images, and time-lapse videos were created using the 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 determined from a nearby control region lacking cells, over the entire region of interest. The background-subtracted GFP fluorescence was summed for all the pixels within the region of interest. This data was then normalized to the baseline established during the first 5 min to enable comparison between cells.

Statistical treatment of data. All experiments were performed at least three times. For cuvette-based experiments, fluorescence was collected at 1-s intervals, and the curves shown present the mean values from at least three independent experiments. Unless otherwise indicated, the symbols represent mean ± SE values that, for clarity, are only shown at selected time points. For single-cell measurements, fluorescence values were determined as described and are plotted for each cell in the field of view as 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 as supplemental material (the online version of this article contains supplemental data).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PTX on [Ca2+]i in BAECs. To examine the effect of PTX on [Ca2+]i, fura-2 loaded BAECs were suspended in a temperature-controlled cuvette and fluorescence was recorded as described in MATERIALS AND METHODS. As shown in Fig. 1A, addition of PTX produced a time- and concentration-dependent increase in [Ca2+]i. The dose-response relationship was unusual in that relatively modest increases in [Ca2+]i were observed between 3 and 60 nM, whereas 100 nM PTX produced a large and rapid change. Addition of PTX in the absence of extracellular Ca2+ had no effect (Fig. 1B), but subsequent readdition of Ca2+ produced a large increase in [Ca2+]i that was enhanced by elevating extracellular Ca2+ from 2 to 10 mM (Fig. 1C). These results demonstrate that PTX does not release Ca2+ from internal stores but, rather, stimulates Ca2+ influx from the extracellular space. PTX-induced Ca2+ influx was unaffected by nifedipine (10 µM) or diltiazem (10 µM), two antagonists of the voltage-gated Ca2+ channel (data not shown).


Figure 1
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  		Fig. 1. Effect of palytoxin (PTX) on cytosolic free Ca2+ concentration ([Ca2+]i) in bovine aortic endothelial cells (BAECs). A: fura-2-loaded BAECs were suspended in HEPES-buffered saline (HBS) containing 2 mM Ca2+. Several traces are shown superimposed. Various concentrations of PTX, as indicated at right of each trace, were added to each trace at the time indicated by the arrow. B: 3 traces are shown superimposed. Cells were suspended in Ca2+-free HBS. PTX (varying concentrations) was added at the indicated time, followed by addition of 2 mM Ca2+. C: same protocol as in B, except the concentration of Ca2+ added was raised to 10 mM. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
The unusual dose-response relationship suggested that the change in [Ca2+]i induced by PTX may reflect multiple mechanisms. To test this hypothesis, we compared the dose-response relationship of PTX for [Ca2+]i with that for [Na+]i. Preliminary experiments revealed that BAECs failed to load with SBFI AM, the Na+-sensitive dye. Therefore, these experiments were performed using HEK cells. As shown in Fig. 2A, PTX produced an increase in [Ca2+]i in HEK cells that was essentially identical to that observed in BAECs; relatively modest increases in [Ca2+]i were observed between 3 and 60 nM, but a large rapid increase was observed at 100 nM PTX. In sharp contrast, PTX produced a potent and graded increase in [Na+]i (Fig. 2B). The threshold concentration was ~0.3 nM, which after a delay of ~1 min produced a slow increase in [Na+]i. At 10 nM PTX, the response was rapid, and within 1 min [Na+]i was fully equilibrated with extracellular Na+, i.e., 140 mM. Thus, at a concentration of 10 nM, PTX rapidly increased [Na+]i but had only a small effect on [Ca2+]i. For comparison, we examined the effect of MTX on both [Ca2+]i and [Na+]i. MTX is known to activate Ca2+-permeable, nonselective cation channels and to increase [Ca2+]i with an EC50 of ~0.3 nM (8, 34). As shown in Fig. 2, C and D, MTX produced an increase in both [Ca2+]i and [Na+]i in HEK cells over the same concentration range. Together, these results suggest that PTX may affect [Ca2+]i through multiple mechanisms.


Figure 2
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  		Fig. 2. Comparison of PTX- and maitotoxin (MTX)-induced changes in [Ca2+]i and [Na+]i in human embryonic kidney (HEK) cells. HEK cells were loaded with either fura-2 (A and C) or sodium-binding benzofuran isophthalate (SBFI; B and D) to measure [Ca2+]i or [Na+]i, respectively. Several traces are shown superimposed in each panel. At the times indicated by the arrows, PTX (A and B) or MTX (C and D) was added at the concentration indicated at right of each trace. SBFI fluorescence was normalized to the value obtained in the presence of 200 mM Na+. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
Effect of ouabain on PTX-induced responses. Previous studies have shown that PTX binds with high affinity to the NKA pump and that the effects of PTX can be blocked by cardiac glycosides. Therefore, if the effect of PTX on [Ca2+]i reflects interaction with the NKA pump, the response should be completely inhibited by ouabain. To test this hypothesis, fura-2-loaded BAECs were pretreated with ouabain for ~2 min before challenge with PTX (Fig. 3). Ouabain produced a dose-dependent blockade of the PTX-induced increase in [Ca2+]i. A concentration of 6 µM ouabain was sufficient to completely block the response to 100 nM PTX. To determine whether blockade of the NKA could reverse the increase in [Ca2+]i, we added ouabain at various times after PTX (Fig. 4). Preliminary studies showed that much higher concentrations of ouabain were required to displace PTX once bound to the pump. Therefore, these experiments were initially performed using 10 nM PTX. To observe a change in [Ca2+]i at this concentration of toxin, we increased Ca2+ in the extracellular buffer to 10 mM. As shown in Fig. 4, A and B, addition of PTX under this condition caused a rapid but small increase in [Ca2+]i over the first minute that was followed by a slowly rising phase that reached near saturation of the fura-2 after 25 min. A similar biphasic increase in [Ca2+]i was seen after 100 nM PTX in normal extracellular Ca2+ (Fig. 4C). Addition of ouabain at 100, 300, or 500 s after 10 nM PTX immediately stopped further increase and caused a slow return of [Ca2+]i toward basal levels (Fig. 4, A and B). Importantly, the increase in [Ca2+]i produced by PTX was almost fully reversed by subsequent addition of ouabain, even after challenge with higher concentrations of PTX (100 nM; Fig. 4C). Thus the change in [Ca2+]i must be related to PTX interaction with the NKA. The reason for the small, rapid increase in [Ca2+]i shown in Fig. 4C immediately after addition of ouabain is unknown but may reflect an increase in Ca2+ permeability of the PTX channels when bound with ouabain.


Figure 3
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  		Fig. 3. Effect of ouabain pretreatment on PTX-induced changes in[Ca2+]i. Six traces are shown superimposed. Ouabain, at the concentrations indicated at right of each trace, was added to fura-2-loaded BAECs at time 0. PTX (100 nM) was added at the time indicated by the arrow (100 s). Ob, ouabain. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 

Figure 4
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  		Fig. 4. Effect of high ouabain level on [Ca2+]i after PTX. Several traces are shown superimposed in each panel. A and B: fura-2-loaded BAECs were suspended in HBS containing 10 mM Ca2+. PTX (10 nM) was added at the time indicated by the arrow (100 s). Ouabain (0.5 mM) was added (+Ob) either before PTX (time 0) or at various times after PTX as indicated. –Ob, no ouabain added. Note longer time course in B. C: same protocol as in A with extracellular [Ca2+] reduced to 2 mM and PTX increased to 100 nM. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
It is known that ouabain does not block the PTX-induced channels but, rather, causes a slow displacement of PTX from the NKA (4). Thus the ability of ouabain to immediately stop further increases in [Ca2+]i seemed paradoxical. The result suggests, however, that the slowly rising phase of [Ca2+]i observed before ouabain reflects binding of PTX and a progressive increase in the number of active channels, whereas the decline in [Ca2+]i after addition of ouabain reflects PTX dissociation and channel closure. Because ouabain binds with low affinity to the PTX-modified form of the pump and with high affinity for the unmodified form (4), we reasoned that addition of a low concentration of ouabain sometime after PTX should selectively block further increase in [Ca2+]i without displacing PTX, i.e., without initiating a reduction in [Ca2+]i. As shown in Fig. 5A, the addition of 100 nM PTX caused a biphasic influx of Ca2+ as shown above. Addition of 30 µM ouabain ~2 min after PTX immediately blocked further influx but did not reverse the rise in [Ca2+]i. An identical result was obtained when the extracellular Ca2+ was increased to 10 mM (Fig. 5B). These results provide strong support for the hypothesis that the second phase of [Ca2+]i increase reflects the rate of PTX binding to unoccupied pump units.


Figure 5
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  		Fig. 5. Effect of low ouabain level on [Ca2+]i after PTX. Two traces are shown superimposed in each panel. A: fura-2-loaded BAECs were suspended in HBS containing normal Ca2+ (2 mM). PTX (100 nM) was added at the time indicated by the arrow (100 s). Ouabain (30 µM) was added 50 s after PTX as indicated. B: same protocol as in A with extracellular Ca2+ concentration increased to 10 mM. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
Effect of PTX on vital dye uptake. Previous experiments have shown that subsequent to the increase in [Ca2+]i, MTX causes the uptake of vital dyes such as EB via a pathway referred to as the cytolytic/oncotic pore, or COP (8, 34, 35). Furthermore, COP is tightly linked to channel activity, because blockade of MTX-induced Ca2+ influx rapidly terminates EB uptake (9). To determine whether PTX also activates COP, we monitored EB fluorescence as a function of time after challenge with PTX. As shown in Fig. 6A, PTX caused a time- and concentration-dependent increase in EB uptake. At 3 nM PTX, EB uptake was delayed in time ~3 min but subsequently increased at a rate that was significantly greater than basal EB uptake determined in the absence of PTX. Increasing the concentration of PTX from 3 to 100 nM shortened the delay and increased the rate of EB uptake. Interestingly, the PTX-induced uptake of EB was monophasic, suggesting that cell lysis does not occur over the time course of these experiments (~20 min; see Real-time evaluation of PTX-induced cell death). As was seen in the fura-2 experiments, the effect of PTX on EB uptake could be blocked by prior addition of ouabain (Fig. 6B) or by addition of ouabain after PTX (Fig. 6C). Thus the uptake of EB is clearly related to PTX interaction with the NKA. However, addition of ouabain after PTX did not immediately stop further EB uptake, which continued for 3–4 min before returning to basal levels (Fig. 6C). Thus attenuation of EB uptake by ouabain may reflect dissociation of PTX or may be related to the decrease in [Ca2+]i.


Figure 6
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  		Fig. 6. Effect of PTX and ouabain on ethidium bromide (EB) uptake. Several traces are shown superimposed in each panel. A: EB uptake was measured after addition of PTX at the concentration indicated at right of each trace. B: EB uptake in response to 100 nM PTX was measured in the absence and presence of ouabain at the concentration indicated at right of each trace. Ouabain was added before PTX at time 0. C: ouabain was added either before or after PTX as indicated. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
Previous studies also have shown that MTX-induced COP is permeable to larger vital dyes such as YO-PRO-1 (molecular mass 345 kDa), but much less permeable to PI (molecular mass 475 kDa). An example of MTX-induced YO-PRO-1 uptake is shown Fig. 7. As previously shown (8), YO-PRO-1 uptake in response to MTX was biphasic; the first phase reflects COP activity, whereas the second phase reflect cell lysis. Much to our surprise, YO-PRO-1 was essentially impermeable via the PTX-induced COP pathway. This result suggests that although PTX can cause a ouabain-sensitive formation of large pores that allow EB influx, the PTX-induced COP pathway has a smaller molecular weight cutoff compared with that activated by MTX.


Figure 7
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  		Fig. 7. Comparison of MTX and PTX on YO-PRO-1 uptake. Two traces are shown superimposed. YO-PRO-1 uptake was measured in BAECs in response to addition of either 0.3 nM MTX or 100 nM PTX as indicated. Curves represent mean values of 3 independent experiments; symbols represent means ± SE at selected time points.

 
Real-time evaluation of PTX-induced cell death. As shown in Fig. 6, the uptake of EB in response to PTX was monophasic, suggesting the cells remain viable for at least 20 min after PTX. To determine the actual time course of cell death, and to evaluate changes in cell morphology, we examined the effect of PTX in greater detail at the single-cell level. We previously developed a single-cell assay for the real-time evaluation of cell lysis that was based on release of GFP (molecular mass 27 kDa) (10, 45). For these experiments, BAECs were transfected with a GFP-plasmid construct and seeded onto glass coverslips. The cells were incubated in bath solution containing EB. Changes in cell morphology, loss of GFP, and the uptake of EB were monitored in real-time with the use of simultaneous phase and fluorescence video microscopy. An example of one such experiment is shown as a montage in Fig. 8, and the time-lapse video of this experiment is available as Supplemental Video 1. The time 0 images show a single, isolated GFP-expressing BAEC with typical cell morphology. The cell exhibits green fluorescence with no detectable EB fluorescence (red). PTX (100 nM) was added at 5 min. Between 5 and 40 min, GFP fluorescence remains essentially constant, but a small, monophasic increase in cell-associated EB fluorescence is observed. This initial phase of EB uptake is indicative of COP as shown in the cuvette experiments. Between 40 and 43 min, GFP fluorescence is rapidly lost from the cell. This release of GFP is associated with a rapid uptake of EB as indicated by the intense staining of the cell nucleus. This rapid phase of EB uptake corresponds in time with GFP release and therefore reflects cell lysis. Note that during GFP release, distinct membrane blebs develop on the surface of the cell. However, the blebs do not rupture or burst but, rather, continue to dilate and grow in size during and after cell lysis. This is most evident in the time-lapse video, which also demonstrates that the blebbing profile is a general phenomenon observed in all cells, even those not expressing GFP. These experiments were repeated on a number of coverslips, and the loss of GFP and the uptake of EB were quantified as a function of time after addition of PTX. For some coverslips, the loss of GFP and the uptake of PI were simultaneously monitored. PI is not permeable via COP (45), and therefore uptake of PI represents a second index of cell lysis. Individual cell responses are shown in Fig. 9 as different colored lines. Although there is perfect correlation between the time of GFP loss and the rapid phase of EB or PI uptake into the cell, there is considerable variation in the time to cell lysis. The most sensitive cell dies at ~20 min, whereas the most resistant cells die at ~90 min; only 3 of 214 cells examined were still alive at 90 min.


Figure 8
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  		Fig. 8. Simultaneous measurement of PTX-induced green fluorescent protein (GFP) loss and EB uptake in single BAECs. BAECs, transfected with GFP, were grown on glass coverslips, mounted on 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 60 min as described in MATERIALS AND METHODS. PTX (100 nM) was added to the bath at time 5 min. A: each row of the montage shows 4 images from a selected cell (phase, GFP, EB, and merged phase/dual fluorescence) taken at the indicated time points. B: GFP (blue) and EB (red) fluorescence from each image was quantified as described in MATERIALS AND METHODS and is shown as a function of time. Time-lapse video of this experiment (Video 1) is available as part of the supplemental data online.

 

Figure 9
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  		Fig. 9. Composite single-cell fluorescence data. Experiments were performed and the fluorescence quantified as described for Fig. 8. Each line represents a single cell from several independent experiments. In all panels, PTX (100 nM) was added at time 5 min. The total number of cells evaluated for GFP loss and EB or PI uptake was 72, 120, and 94, respectively. Note that the loss of GFP fluorescence was simultaneously monitored in parallel experiments with either EB or PI uptake.

 
Previous studies on MTX showed that glycine blocks cell lysis without affecting either the increase in [Ca2+]i or the uptake of EB via COP. To determine whether glycine exhibits a similar cytoprotective effect against PTX-induced cell death, we monitored GFP release and EB uptake at the single-cell level in the presence of 5 mM glycine. As shown in Fig. 10, A and B, none of the cells examined (n = 52) showed the rapid loss of GFP in the presence of glycine, and only 12 cells exhibited the rapid phase of EB uptake, i.e., 113 of 125 cells examined were still alive at the 90-min time point. Note that the initial phase of EB uptake was unaffected by the presence of glycine. Thus glycine is specific for cell lysis and does not affect PTX-induced COP activity.


Figure 10
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  		Fig. 10. A and B: effect of the cytoprotective amino acid glycine on PTX-induced responses. The loss of cell-associated GFP and the uptake of EB were simultaneously recorded as described for Figs. 8 and 9, in the presence of 5 mM glycine. In 6 independent experiments, the total number of cells evaluated for GFP loss and EB was 43 and 108, respectively. C and D: effect of Ca2+ removal on PTX-induced responses. The loss of cell-associated GFP and the uptake of EB were simultaneously recorded as described for Figs. 8 and 9, in zero-Ca2+ HBS. In 7 independent experiments, the total number of cells evaluated for GFP loss and EB was 52 and 125, respectively. Time-lapse video of this experiment (Video 2) is available as part of the supplemental data online.

 
We wondered whether either the activation of COP or cell lysis caused by PTX was dependent on Ca2+. In the absence of Ca2+, cell lysis following addition of 100 nM PTX was greatly delayed; only 7 of 52 cells exhibited a rapid loss of GFP between 90 and 120 min (Fig. 10C). Likewise, only 7 of 125 cells examined showed a rapid second phase of EB uptake (Fig. 10D). Interestingly, although PTX-induced EB uptake was not blocked by Ca2+ removal, the first phase was delayed, consistent with a role for Ca2+ in the time course of COP activation or formation as previously described for MTX (45). We also noted that in some cells, both the GFP and EB fluorescence traces showed periods of oscillations. Close inspection of the videos showed that these oscillations were an artifact due to a unique blebbing profile called zeosis. Zeosis, which is derived from a Greek word meaning "to boil over," is characterized by rapid cytokinesis with continuous bleb extrusion and retraction (5). Zeosis has been associated with apoptosis (9, 21, 22, 37). As shown in supplemental video 2 (the online version of this article contains supplemental data), some cells round up and blebs are rapidly extruded and retracted on the surface of the cell, giving rise to rapid movement artifacts that cause the apparent oscillation profiles shown in the fluorescence traces. Together, the results suggest that in the presence of Ca2+, the cells rapidly die by necrotic/oncotic mechanisms, but in the absence of Ca2+, the cells are resistant to lysis.

The cuvette experiments demonstrate that the PTX-induced change in [Ca2+]i recovers to near normal levels when ouabain is added at various times after PTX (Fig. 4). This result was interesting in that the Na+ and K+ gradients are not expected to return to normal under this condition because the NKA pump is poisoned by ouabain. Thus, despite complete blockade of the NKA, the cell apparently has sufficient energy to remove Ca2+ from the cytosol, most likely via extrusion of Ca2+ across the plasmalemma by the PMCA pump. If a continuous elevation of Ca2+ is necessary for oncotic cell death, the cells should be protected from lysis by addition of ouabain after PTX. However, if the change in Na+ and K+ is sufficient to cause oncotic cell death, addition of ouabain after PTX should have no effect on the time to cell lysis. To test these possibilities, we examined the loss of GFP and the uptake of EB in response to PTX (100 nM) following addition of ouabain (1 mM) 5 min after PTX (Fig. 11). There were two striking results from this experiment. First, addition of ouabain substantially blocked EB uptake (compare EB uptake in Fig. 10, D vs. B). Second, only 2 of 47 cells examined exhibited a rapid loss of GFP and rapid uptake of EB indicative of cell lysis. As shown in Fig. 2, 100 nM PTX is sufficient to completely equilibrate intracellular Na+ with the extracellular space in <1 min. Thus it is remarkable that these cells do not lyse over the time course of this experiment (i.e., 90 min). These results demonstrate that a sustained increase in Ca2+ plays a key role in the lytic event and also may be necessary for continued activation of COP.


Figure 11
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  		Fig. 11. A and B: effect of ouabain addition after PTX. The loss of cell-associated GFP and the uptake of EB were simultaneously recorded as described for Figs. 8 and 9. PTX (100 nM) was added at time 5 min, and ouabain (1 mM) was added at time 10 min as indicated by the arrows. In 3 independent experiments, the total number of cells evaluated for GFP loss and EB was 33 and 47, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is well-established that PTX binds with high affinity to the NKA pump and converts the pump into a nonselective cation channel with low conductance to Na+ and K+. The PTX channel also exhibits a slight permeability to Ca2+ (4). The results of the present study show that PTX causes a concentration- and time-dependent change in both [Na+]i and [Ca2+]i. At a concentration necessary to produce a maximal change in [Na+]i, only a small increase in [Ca2+]i was seen, suggesting that there may be multiple mechanisms by which PTX causes an increase in [Ca2+]i. In fact, the PTX-induced increase in [Ca2+]i was biphasic, further suggesting multiple mechanisms. The [Ca2+]i response to PTX, however, was completely blocked by pretreatment of the cells with ouabain. Furthermore, the rise in [Ca2+]i caused by PTX could be reversed by subsequent addition of ouabain, even in the continued presence of PTX. The ability of ouabain to block and reverse the actions of PTX strongly suggests that the rise in [Ca2+]i is related to the action of PTX on the NKA pump. A clue to the possible explanation for these results came from the observation that addition of ouabain after PTX immediately stopped further increases in [Ca2+]i. Although this might reflect a blockade of the PTX channels by ouabain, previous studies have shown that ouabain has no acute effect on channel currents (4). Thus the binding of ouabain should have no immediate effect on Ca2+ influx. The only possible explanation is that the rising phase of [Ca2+]i reflects a slow time-dependent binding of PTX to the NKA and conversion to channel mode. In this scenario, addition of ouabain after PTX will prevent further binding of PTX to additional pump units and thus terminate a further rise in [Ca2+]i. The subsequent decline in [Ca2+]i would then reflect the actual unbinding of PTX (i.e., displacement by ouabain) from the NKA and the return of [Ca2+]i to basal levels, presumably via the action of SERCA and/or PMCA pumps. Because unbound NKA pumps have a higher affinity for ouabain than PTX-bound pumps (4), the prediction would be that addition of low concentrations of ouabain after PTX should only block further Ca2+ influx without initiating a return of [Ca2+]i to resting levels. Indeed, this was the exact profile observed. Thus it is clear that both phases of the [Ca2+]i response reflect interaction of PTX with the NKA pump. But why does PTX have a slow apparent on-rate during the second phase of the [Ca2+]i increase? One possibility is that two forms of the NKA exist in the plasmalemma that differ in affinity for PTX. The high affinity form would be rapidly activated by PTX, giving rise to the dramatic increase in [Na+]i and a small but detectable increase in [Ca2+]i, i.e., the first phase of [Ca2+]i increase. The slowly rising phase of [Ca2+]i would reflect a time-dependent conversion of the low-affinity form of the pump into the high-affinity form. It is equally possible that the time-dependent binding of PTX reflects two pools of the NKA (with equal affinity for PTX), one that is readily accessible and one that exists in a limited-access membrane compartment that gives rise to the slow on-rate. For example, a large molecule like PTX (molecular mass ~3,000 kDa) may have limited access to NKA pumps in caveolae, a membrane structure that is abundant in endothelial cells (12). Although we cannot distinguish between these two possibilities, it is clear that the rise in [Ca2+]i during both phases reflects interaction of PTX with the NKA and thus reflects Ca2+ influx via the channel mode of the modified pump units.

After the rise in [Ca2+]i, PTX caused an increase in EB uptake into the cell. The rate of EB uptake was dependent on PTX concentration and was blocked by prior treatment with ouabain. In addition, the uptake of EB was blocked by addition of ouabain after PTX. These results suggest that EB uptake is related to the interaction of PTX with the NKA. The mechanism of EB uptake following either PTX or MTX remains essentially unknown. A similar phenomenon was first reported after activation of P2X7 purinergic receptors (6, 11, 26, 44). P2X receptors form Ca2+-permeable cation channels activated by extracellular ATP. After stimulation by ATP or the ATP analog 3'-O-(4-benzoyl)benzoyl-ATP, it was suggested that P2X7 receptors dilate or aggregate to form a larger pore structure that would allow uptake of vital dyes such as EB and YO-PRO-1 (19, 42). However, it was subsequently shown that the dye-permeable pore activated or formed after stimulation of P2X7 was indistinguishable from that observed following MTX (35), suggesting that COP is a unique molecular entity activated downstream of Ca2+ entry via either P2X7- or MTX-activated cation channels. In fact, MTX-induced EB uptake was observed in a variety of different cell types, including those that do not normally express P2X7 receptors. One of the striking observations in the present study was that COP activated by PTX does not allow entry of YO-PRO-1. This result suggests that the COP activated by PTX may in fact be different from that activated by MTX or P2X7 receptor. Thus COP may in fact be unique to each specific agonist. Irrespective of the identity or mechanism of activation, it is clear the PTX activates COP and that this precedes cell lysis.

After the formation or activation of COP, the final phase of the cell death cascade is the actual lytic event. PTX-induced cell lysis, as indicated by the rapid phase of EB or PI uptake or the rapid loss of GFP from the cell, was greatly delayed by removal of Ca2+ or by the presence of the cytoprotective amino acid glycine. In addition, cell lysis was associated with dramatic membrane blebbing. Similar results have been noted for the lytic phase associated with both MTX and P2X7 receptor stimulation (10, 35, 40, 41). Thus it would appear that the cellular mechanisms responsible for cell lysis are the same for each of these toxic insults. As previously noted for both MTX and P2X7 receptor stimulation (8, 10, 40, 41), the membrane blebs do not rupture or burst during lysis but, rather, continue to dilate and grow in size. Undoubtedly, water movement is required for bleb dilation, but how is it possible for water to move into the blebs at a time when large macromolecules like GFP are leaving the cell? One possibility is that the actual osmotically responsive element within the cell is the endoplasmic reticulum (ER) and that swelling and blebbing reflect water movement into the ER. Swelling of the ER would then stretch the plasmalemma, allowing release of GFP without obvious bursting or rupturing of the surface membrane blebs. Our previous experiments using GFP-concatomers with molecular masses ranging from 27 to 162 kDa indicate that the lytic pore lets even the largest of these molecules leave the cell during lysis (10). This might suggest that swelling of the ER simply causes tears in the outer cell membrane. However, lysis is greatly attenuated (present study) or completely blocked (10) by glycine or L-alanine, in a stereospecific manner. Thus cell lysis does not appear to be a random, nonspecific event such as membrane rupture. Although the ER-swelling hypothesis seems plausible, it remains speculative and awaits further investigation and the identification of the channel responsible for the water movement in these cells.

In summary, by converting the NKA pump into a channel, PTX causes a small but significant increase in [Ca2+]i in vascular endothelial cells, which in turn leads to the activation of large dye-permeable pores. In the presence of a continuous sustained increase in [Ca2+]i, the cells ultimately lyse, releasing large macromolecules. These results demonstrate that PTX causes rapid oncotic cell death via a Ca2+ overload mechanism. This cascade of events has now been observed in a variety of cell types and in response to PTX, MTX, and purinergic receptor stimulation, suggesting a common cellular mechanism initiated by the rise in [Ca2+]i.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-65323.


   ACKNOWLEDGMENTS
 
We thank Milana A. B. Applegate 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 Dr., 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.


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