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
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ABSTRACT |
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necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2
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, 35 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 23 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.
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MATERIALS AND METHODS |
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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 23 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 9095% 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 (69 coverslips/35-mm dish).
Time-lapse videomicroscopy. BAECs in complete DMEM were sparsely seeded on circular glass coverslips and used within 13 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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RESULTS |
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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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DISCUSSION |
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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 0.10.5 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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 14) is available online at http://ajpcell.physiology.org/cgi/content/full/00473.2003/DC1.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Cai H and Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840844, 2000.
3. Daly JW, Lueders J, Padgett WL, Shin Y, and Gusovsky F. Maitotoxin-elicited calcium influx in cultured cells effect: effect of calcium-channel blockers. Biochem Pharmacol 50: 11871197, 1995.[CrossRef][ISI][Medline]
4. Elliott SJ, Meszaros JG, and Schilling WP. Effect of oxidant stress on calcium signaling in vascular endothelial cells. Free Radic Biol Med 13: 635650, 1992.[CrossRef][ISI][Medline]
5. Estacion M and Schilling WP. Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells. BMC Physiol 1: 2, 2001.
6. Estacion M and Schilling WP. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis. BMC Physiol 2: 2, 2002.
7. Estacion M, Weinberg JS, Sinkins WG, and Schilling WP. Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine. Am J Physiol Cell Physiol 284: C1006C1020, 2003. First published December 11, 2002; 10.1152/ajpcell.00258.2002.
8. Gill DL and Chueh SH. An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line. J Biol Chem 260: 92899297, 1985.
9. Gu C and Cooper DM. Ca2+, Sr2+ and Ba2+ identify distinct regulatory sites on adenylyl cyclase (AC) types VI and VIII and consolidate the apposition of capacitative cation entry channels and Ca2+-sensitive ACs. J Biol Chem 275: 69806986, 2000.
10. Gusovsky F and Daly JW. Maitotoxin: a unique pharmacological tool for research on calcium-dependent mechanisms. Biochem Pharmacol 39: 16331639, 1990.[CrossRef][ISI][Medline]
11. Lum H and Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280: C719C741, 2001.
12. Mills JC, Stone NL, Erhardt J and Pittman RN. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 140: 627636, 1998.
13. Nuttle LC and Dubyak GR. Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes. J Biol Chem 269: 1398813996, 1994.
14. Orrenius S, McConkey DJ, Bellomo G, and Nicotera P. Role of Ca2+ in toxic cell killing. TIPS 10: 281285, 1989.[Medline]
15. Pearson JD. Endothelial cell biology. Radiology 179: 914, 1991.[Abstract]
16. Schiffrin EL. A critical review of the role of endothelial factors in the pathogenesis of hypertension. J Cardiovasc Pharmacol 38, Suppl 2: S3S6, 2001.
17. Schilling WP and Elliott SJ. Ca2+ signaling mechanisms of vascular endothelial cells and their role in oxidant-induced endothelial cell dysfunction. Am J Physiol Heart Circ Physiol 262: H1617H1630, 1992.[Abstract]
18. Schilling WP, Rajan L, and Strobl-Jager E. Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells: saturability, selectivity and kinetics. J Biol Chem 264: 1283812848, 1989.
19. Schilling WP, Sinkins WG, and Estacion M. Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts. Am J Physiol Cell Physiol 277: C755C765, 1999.
20. Schilling WP, Wasylyna T, Dubyak GR, Humphreys BD, and Sinkins WG. Maitotoxin and P2Z/P2X7 purinergic receptor stimulation activates a common cytolytic pore. Am J Physiol Cell Physiol 277: C766C776, 1999.
21. Shine KI, Douglas AM, and Ricchiuti NV. Calcium, strontium, and barium movements during ischemia and reperfusion in rabbit ventricle. Circ Res 43: 712720, 1978.[ISI][Medline]
22. Smart ML, Panchal RG, Bowser DN, Williams DA, and Petrou S. Pore formation is not associated with macroscopic redistribution of P2X7 receptors. Am J Physiol Cell Physiol 283: C77C84, 2002. First published February 13, 2002; 10.1152/ajpcell.00456.2001.
23. Soergel DG, Yasumoto T, Daly JW, and Gusovsky F. Maitotoxin effects are blocked by SK&F 96365, an inhibitor of receptor-mediated calcium entry. Mol Pharmacol 41: 487493, 1992.[Abstract]
24. Steiger GJ, Brady AJ, and Tan ST. Intrinsic regulatory properties of contractility in the myocardium. Circ Res 42: 339350, 1978.[ISI][Medline]
25. Tatham PER and Lindau M. ATP-induced pore formation in the plasma-membrane of rat peritoneal mast cells. J Gen Physiol 95: 459476, 1990.[Abstract]
26. Torgerson RR and McNiven MA. The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing. J Cell Sci 111: 29112922, 1998.
27. Trump BF, Berezesky IK, Chang SH, and Phelps JM. The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol 25: 8288, 1997.[ISI][Medline]
28. Vanderkooi JM, Martonosi A. Sarcoplasmic reticulum. XIII. Changes in the fluorescence of 8-anilino-1-naphthalene sulfonate during Ca2+ transport. Arch Biochem Biophys 144: 99106, 1971.[ISI]
29. Yu SP, Canzoniero LM, and Choi DW. Ion homeostasis and apoptosis. Curr Opin Cell Biol 13: 405411, 2001.[CrossRef][ISI][Medline]