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Originally published In Press as doi:10.1074/jbc.M411072200 on November 17, 2004

J. Biol. Chem., Vol. 280, Issue 4, 2653-2658, January 28, 2005
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Involvement of Chloride in Apoptotic Cell Death Induced by Activation of ATP-sensitive P2X7 Purinoceptor*

Mitsutoshi Tsukimoto, Hitoshi Harada{ddagger}, Akira Ikari, and Kuniaki Takagi

From the Department of Environmental Biochemistry and Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received for publication, September 27, 2004 , and in revised form, November 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ATP-gated P2X7 receptor is a plasma membrane receptor belonging to the family of P2X purinoceptors. Its activation leads to multiple downstream events including influx of ions, pore formation to allow the passage of larger molecular weight species, and cell death by apoptosis and/or necrosis. The cell death is thought to be correlated with the pore formation but does not directly result from the dilatation of pores. We have generated and characterized a clone of chicken DT40 lymphocytes stably transfected with the rat P2X7 receptor. In this study, we investigated the mechanism of P2X7 receptor-induced cell death using this clone. Treatment with P2X7 receptor agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP induced depolarization of membrane potential, pore formation, and cell shrinkage, an early hallmark of apoptosis in the buffer containing physiological concentrations of ions. Analysis by flow cytometry revealed that the activity of pore formation in shrunk cells was much higher than in non-shrunk cells. The activation of P2X7 receptor also caused the release of lactate dehydrogenase from cells. The P2X7 receptor-mediated cell shrinkage and lactate dehydrogenase release were blocked when media Cl– was replaced with gluconate. However, removal of extracellular Cl– did not affect plasma membrane depolarization and pore formation by treatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP. Therefore we concluded that pore formation plays a critical role in the P2X7 receptor-induced apoptotic cell death and that this is mediated by extracellular Cl– influx.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular ATP plays an important role in cell signaling, modulation of cell growth, differentiation, and induction of cell death by apoptosis and/or necrosis (1, 2). The actions of extra-cellular ATP are mediated through P2 purinoceptors, which are classified into two major subtypes: ionotropic P2X and metabotropic G protein-coupled P2Y receptors (3, 4). The P2X7 receptor, previously designated as P2Z receptor and now the seventh member of P2X receptors, utilizes extracellular ATP to increase cationic permeability with consecutive plasma membrane depolarization, and its intense or prolonged activation leads to the opening of a large non-selective pore allowing the passage of hydrophilic molecules of up to 900 Da in size. This receptor shares 35–40% homology with other members of the P2X family, all with the same predicted topology of two transmembrane regions, a cysteine-rich extracellular loop, and intracellular N and C termini. The C terminus is more than 100 amino acids longer than that of any other member of the family and is essential for the opening of the large pore (5). The activation of the P2X7 receptor has been reported to link to a number of other cellular events including cell fusion (6), membrane blebbing (7), and interleukin-1{beta} release (8). The P2X7 receptor was first cloned from rat brain (5) and subsequently from human monocytes (9) and mouse microglial cells (10). Expression of its mRNA, detected by Northern blot analysis, was found in bone marrow and in tissues containing abundant macrophages or monocytes (11). Thus the cellular events caused by the P2X7 receptor are thought to play a significant role in the regulation of immune and central nervous systems.

The mechanism of activation and repression of apoptosis has been a central focus of studies examining the role of programmed cell death in both normal and pathological conditions. The longer C terminus is required for cell death because no cell death occurs in HEK293 cells expressing a truncated receptor lacking the final 177 residues (5). P2X7 receptor-induced cell death has been thought to be correlated with the pore formation (7). However, little is known about the molecular mechanism. We have generated a clone of chicken DT40 B cell line stably transfected with the rat P2X7 receptor (DT40/P2X7 cells) and reported apoptotic cell death mediated via the P2X7 receptor (12). There are three reasons why we selected DT40 cells for the host cells. First, the P2X7 receptors were present on human B cells (15), and the polymorphisms were reported in B cell chronic lymphocytic leukemia (16, 17). However, DT40 cells appear to lack functional P2X7 purinoceptors. Second, they provide an excellent model system for the analysis of the B cell signaling (13). Third, stable transfections of this cell line are unique in that they demonstrate an unexpectedly high ratio of targeted to random integration into the homologous gene loci, rendering them highly genetically tractable (14). Apoptosis induced by the activation of P2X7 receptor in this clone was measured by exposure of phosphatidylserine on the outside of the cell membrane and DNA laddering. We also detected the activation of caspase-3 when the cloned cells were exposed to P2X7 receptor agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP).1 Caspase-3 activation plays a key role in apoptosis. However, the BzATP-induced cell death in this clone could not be prevented by the treatment with a general caspase blocker, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone. This suggested an unidentified system of cell death mediated by the P2X7 receptor. Although cell shrinkage or loss of cell volume has traditionally been viewed as a passive event during apoptosis, recent studies from several laboratories have shown that cell shrinkage or more specifically flux of ions associated with the change in cell size plays a critical role in the regulation of the cell death machinery (18). Cell death caused by the activation of P2X7 receptor in heterologous expression systems (including our system) and P2Z receptor in native cells have been documented (1, 2, 12). However, the cell shrinkage and its significance in the cell death mediated by P2X7 receptor have not been reported. In the present study, to understand the molecular mechanism by which P2X7 receptor-induced cell death occurs, we investigated the essential role of pore formation in the apoptotic cell death induced by the activation of P2X7 receptor in DT40/P2X7 cells and its ionic dependence and selectivity by measuring cell size, an early distinction between apoptosis and necrosis, with a flow cytometer.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), phenol red free Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F-12), fetal bovine serum, chicken serum, penicillin, streptomycin, amphotericin B, BzATP, and ethidium bromide were purchased from Sigma. Bis(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) was from Dojindo Laboratories (Kumamoto, Japan). The cytotoxicity detection kit was purchased from Roche Applied Science. All other chemicals used for the experiments were of the highest purity available.

Cell Culture—DT40 cells stably transfected with rat P2X7 receptors (DT40/P2X7) have been established and characterized as detailed previously (12). Cells used in this study were routinely maintained in DMEM/F-12 supplemented with 10% heat-inactivated fetal bovine serum, 1% heat-inactivated chicken serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B in an atmosphere of 5% CO2, 95% air at 37 °C.

Buffer Solution—Physiological salt buffer (Hanks' based salt solution (HBSS)) was made with the following composition: 136.9 mM NaCl, 5.5 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.25 mM CaCl2, 5.5 mM D-glucose, 4.2 mM NaHCO3, and 10 mM HEPES (pH 7.4). In some studies, Na+ or K+ was replaced with choline, and Cl– was replaced with gluconate. To remove Ca2+, Mg2+, , and , we made HBSS without CaCl2, MgSO4, Na2HPO4, KH2PO4, and NaHCO3, respectively. For high K+ HBSS, HBSS was initially made minus KCl and NaCl (solution A), and the experimental media were made up of solution A with NaCl and KCl to a total concentration of 142.4 mM.

Determination of Ethidium Bromide Uptake by Flow Cytometry— Cells were washed with and resuspended in the indicated buffer at 1 x 106 cells/ml and then incubated with BzATP and ethidium bromide (25 µM) at 37 °C. After incubation, the sample was analyzed using a flow cytometer (Beckman Coulter Epics XL and System II software version 3.0) with laser excitation at 488 nm and examined at 620 nm for ethidium fluorescence. The forward and side scatter signals from 50,000 particles were collected. With cellular debris and aggregates gated out by forward and side scatter, the fluorescence signals from ethidium were analyzed. Some data were converted to density plots using WIN-MDI software version 2.8. (J. Trotter, Scripps Institute, San Diego, CA).

Quantification of Lactate Dehydrogenase Release—Lactate dehydrogenase (LDH) release into cell culture supernatant was quantified by a cytotoxicity detection kit (Roche Applied Science), following the instructions. Cells (1 x 105 cells/well) were incubated in a 96-well plate at 37 °C for 9 h with BzATP in DMEM/F-12, HBSS, or Cl–-free HBSS. At the end of incubation, supernatants were collected, and the LDH content was measured. Lactate dehydrogenase release is expressed as the percentage of the total content determined by lysing an equal number of cells with 1% Triton X-100.

Determination of Cell Size by Flow Cytometry—Cell size and changes in the light scattering properties of the cells were determined by flow cytometry. Cells were washed with and resuspended in the indicated buffer at 1 x 106 cells/ml and then incubated with BzATP at 37 °C. After incubation, 100,000 cells for each sample were examined with a flow cytometer by exciting the cells with a 488-nm argon laser and determining their distribution on a forward scatter versus side scatter dot plot. Light scattered in the forward direction is proportional to cell size, whereas light scattered at a 90° angle (side scatter) is proportional to cell density (19). Therefore, as a cell shrinks a decrease in the amount of forward scattered light is observed. A gate based on the properties of the control cells was set on each forward scatter versus side scatter dot plot to separate the normal and shrunk populations of cells and remained constant throughout the analysis. Cell shrinkage is expressed as the percentage of shrunk cells in total cells determined by statistical analysis of the dot plots using Beckman Coulter System II software version 3.0. Some data were converted to density plots using WINMDI software version 2.8 for presentation.

Measurement of Plasma Membrane Potential—Alternations in the plasma membrane potential were measured using a fluorescent potential-sensitive anionic dye, DiBAC4(3) (20). Cells were washed twice and resuspended at a density of 1 x 106 cells/ml. The cells were incubated with 1 µM DiBAC4(3) for 10 min at 37 °C before the addition of stimulants. Fluorescence was monitored at excitation/emission wavelengths of 493/516 nm with a fluorescence spectrophotometer (F-2000, Hitachi). For analysis with a flow cytometer, the sample was analyzed with laser excitation at 488 nm and examined at 520 nm for DiBAC4 (3) fluorescence.

Statistical Analysis—Values are given as mean ± S.E. Comparison between two values was performed by unpaired Student's t test. For multiple comparisons among different groups of data, significant differences were determined by the Bonferroni method. Significance was defined at p < 0.001 using the Instat version 3.0 statistical package (GraphPad Software).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Apoptotic Cell Death During P2X7 Receptor Activation—The activation of P2X7 receptor initially leads to the opening of ionic channels, which in turn results in the secondary opening of membrane pores that allow the passage of larger molecular species (5). Fig. 1 shows the BzATP-induced ethidium bromide uptake and LDH release after 10 min and 9 h incubation of DT40 cells expressing rat P2X7 receptors (DT40/P2X7 cells), respectively, in the normal physiological solution (DMEM/F-12). Treatment with BzATP induced ethidium uptake and LDH release in a similar dose-dependent manner. These results confirm our report that apoptosis is induced by the activation of P2X7 receptor in DT40/P2X7 cells (12). Recently, cell volume loss or cell shrinkage has been demonstrated to be an early detectable event in the apoptotic program (18). We therefore investigated the effect of activation of P2X7 receptor on the change in cell size of DT40/P2X7 cells by flow cytometry. When cells were incubated in DMEM/F-12 and exposed to BzATP (100 µM for 90 min), cell shrinkage was detected (Fig. 2A). This cell shrinkage was completely prevented in DMEM/F-12 supplemented with 5 mM MgCl2 (Fig. 2A) that is known to block the activation of P2X7 receptor. The BzATP-induced cell shrinkage was time- and dose-dependent (Fig. 2, B and C). Moreover, the simultaneous analysis of pore formation and cell shrinkage by flow cytometry revealed that the ethidium uptake was much higher in the shrunk cells than in the non-shrunk cells (Fig. 3).



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  		FIG. 1.
BzATP-induced ethidium uptake and LDH release. A, DT40/P2X7 cells were incubated for 10 min with 25 µM ethidium bromide and BzATP in DMEM/F-12, and ethidium bromide uptake was measured with a flow cytometer. B, DT40/P2X7 cells (1 x 105 cells/well) were incubated in a 96-well plate for 9 h with BzATP in DMEM/F-12. At the end of incubation, supernatants were collected and LDH content was measured. The release is expressed as the percentage of total content determined by lysing an equal number of cells with 1% Triton X-100.

 



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  		FIG. 2.
Flow cytometric analysis of BzATP-induced cell shrinkage. A, DT40/P2X7 cells were incubated for 90 min with vehicle (control) or 100 µM BzATP in DMEM/F-12 or DMEM/F-12 supplemented with 5 mM MgCl2. B, DT40/P2X7 cells were incubated with vehicle (control) or 100 µM BzATP for the indicated time. C, DT40/P2X7 cells were incubated with the indicated concentration of BzATP for 90 min. At the end of incubation, the sample was analyzed with a flow cytometer as described in "Experimental Procedures." Gates were set up based on the control cells to determine the percentage of cells that had a decrease in forward scatter light (cell size) compared with the entire population of cells. The gates and the percentages of shrunk cells in total cells are indicated on each panel in A. The percentage of shrunk cells in total cells was determined from four independent experiments ± S.E. (B and C).

 



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  		FIG. 3.
Time-dependent ethidium uptake on BzATP-induced cell shrinkage. DT40/P2X7 cells were incubated with 25 µM ethidium bromide and 100 µM BzATP in DMEM/F-12 for the indicated time. At the end of incubation, the sample was analyzed with a flow cytometer as described in "Experimental Procedures." Data were converted to forward scatter versus ethidium bromide fluorescence density plots.

 
Ionic Effects on P2X7 Receptor-mediated Cell Shrinkage— Previous studies demonstrated that P2X7 receptor function is affected by a wide range of ions (7, 21–23) and that ionic homeostasis plays an important role in apoptosis including the induction of cell shrinkage (18, 24). To examine the contribution of ions to P2X7 receptor-mediated apoptotic cell death, we investigated the effects of ions on the BzATP-induced cell shrinkage of DT40/P2X7 cells. As shown in Fig. 4A, BzATP also induced cell shrinkage in the minimum and essential ion solution including Na+, K+, Ca2+, Mg2+, Cl–, and (HBSS). Replacing Na+ with choline, removing or in HBSS did not affect the BzATP-induced cell shrinkage (Fig. 4, B–D). On the other hand, removing Ca2+ or Mg2+ or replacing K+ with choline stimulated the BzATP-induced cell shrinkage (Fig. 4, E–G). Interestingly, when Cl– in HBSS was replaced with gluconate, the BzATP-induced cell shrinkage was suppressed (Fig. 4H).



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  		FIG. 4.
Ionic effects on BzATP-induced cell shrinkage. DT40/P2X7 cells were incubated with vehicle (control) or 100 µM BzATP for 90 min in normal HBSS (A), replacing Na+ with choline (B), removing (C), removing HCO –, (D) removing Ca2+ (E), removing Mg2+ (F), replacing K+ with choline (G) or replacing Cl– with gluconate (H). The change in cell size was analyzed on forward-scatter versus side scatter density plots by flow cytometry as described under "Experimental Procedures."

 
To further study the role of extracellular Cl– on the P2X7 receptor-mediated cell death, we investigated the effect of removal of extracellular Cl– on the P2X7-mediated events. Removal of extracellular Cl– inhibited the BzATP-induced cell shrinkage in a concentration-dependent fashion and was significant at less than 90 mM of extracellular concentration (Fig. 5A). Removal of extracellular Cl– could not block the BzATP-induced pore formation but did block cell shrinkage and LDH release (Fig. 5, B–D). The activation of P2X7 receptor also triggers plasma membrane depolarization caused by transmembrane cation influx (25). Therefore we monitored the change in membrane potential using a fluorescent potential-sensitive anionic dye, DiBAC4(3). As shown in Fig. 6, the treatment with BzATP evoked fast membrane depolarization, and it was not suppressed by removal of extracellular Cl–.



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  		FIG. 5.
Effects of removal of extracellular Cl– on BzATP-induced ethidium uptake, cell shrinkage, and LDH release. A, DT40/P2X7 cells were incubated for 90 min with vehicle (control) or 100 µM BzATP in HBSS containing various concentrations of Cl–, and the cell shrinkage was measured by flow cytometry. B, DT40/P2X7 cells were incubated for 10 min with 25 µM ethidium bromide and vehicle (control) or 100 µM BzATP in HBSS or Cl–-free HBSS. The ethidium uptake was measured with a flow cytometer. C, DT40/P2X7 cells were incubated for 90 min with vehicle (control) or 100 µM BzATP in HBSS or Cl–-free HBSS, and the percentage of shrunk cells in total cells was determined from four independent experiments. D, DT40/P2X7 cells were incubated for 9 h with vehicle (control) or 100 µM BzATP in HBSS or Cl–-free HBSS, and the LDH release from cells was measured. * (p < 0.001) indicates statistically significant differences compared with data obtained for the treatment with BzATP in HBSS.

 



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  		FIG. 6.
Effect of removal of extracellular Cl– on BzATP-induced depolarization of plasma membrane potential. DT40/P2X7 cells were incubated for 10 min with 1 µM DiBAC4(3) in HBSS or Cl–-free HBSS and stimulated by 100 µM BzATP. Fluorescence was monitored at excitation/emission wavelengths of 493/516 nm with a fluorescence spectrophotometer.

 
Involvement of Cl– Influx in High K+-induced Cell Shrinkage—To further study the involvement of extracellular Cl– in the P2X7 receptor-mediated cell death, we examined the effect of extracellular K+ on cell size. The cells were incubated in buffers with differing concentrations of K+, where concentrations of Na+ were varied reciprocally to maintain osmolarity and a univalent cation concentration of 142.4 mM. Increasing the extracellular K+ concentration up to 70 mM had no effect on the BzATP-induced cell shrinkage (data not shown). Interestingly, high extracellular K+ concentrations (over 70 mM) induced cell shrinkage of our clone and even normal DT40 cells. Treatment with high extracellular K+ (100 mM) for 90 min induced cell shrinkage and membrane depolarization but not ethidium uptake (Fig. 7A) in normal DT40 cells. The high K+-induced cell shrinkage was concentration-dependent and was completely inhibited by removal of extracellular Cl– (Fig. 7B). Moreover, treatment with an anion channel/exchanger inhibitor, DIDS, blocked the cell shrinkage induced by high extracellular K+ (100 mM) (Fig. 7C).



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  		FIG. 7.
High K+-induced cell shrinkage and its inhibition by removal of extracellular Cl–. A, DT40 cells were incubated for 90 min with 1 µM DiBAC4(3) or 25 µM ethidium bromide in HBSS (5 mM K+) or HBSS containing 100 mM K+. The change in cell size, plasma membrane potential, and ethidium uptake were examined with a flow cytometer. B, DT40 cells were incubated for 90 min in HBSS or Cl–-free HBSS containing various concentrations of K+, and the cell shrinkage was measured by flow cytometry. C, DT40 cells were incubated for 90 min with 50 or 100 µM DIDS in HBSS (5 mM K+) or HBSS containing 100 mM K+, and the percentage of shrunk cells in total cells was determined from four independent experiments. * (p < 0.001) indicates statistically significant differences compared with data in HBSS containing 100 mM K+.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of P2X7 receptor has been reported to induce cell death by necrosis and/or apoptosis in a variety of cells (1, 2). We have also reported apoptotic cell death mediated via activation of P2X7 receptor by measuring exposure of phosphatidylserine on the outside of cell membrane and DNA laddering in DT40/P2X7 cells (12). The morphological changes by apoptosis include cell shrinkage, plasma and nuclear membrane blebbing, organelle relocalization and compaction, chromatin condensation, and the production of membrane-enclosed particles that contain intracellular material known as "apoptotic bodies" (26–28). Cell shrinkage is a major hallmark of apoptosis and starts before cell fragmentation. On the other hand, the necrotic alterations are characterized by swelling and the random spillage of cellular contents into the extracellular milieu (29). In this report, not cell swelling but cell shrinkage was detectable in advance of the P2X7 receptor-mediated cell death of DT40/P2X7 cells in the buffer containing physiological concentrations of ions. Consequently, it was confirmed that the cell death by activation of P2X7 receptor occurred by apoptosis rather than by necrosis under our experimental conditions.

The P2X7 receptor exhibits an interesting and seemingly unique property; as well as behaving as a rapidly activating non-selective cation channel, intense or prolonged activation leads to the opening of a large non-selective pore allowing the passage of molecules of up to 900 Da in size. The P2X7 receptor-specific longer C terminus is required for induction of cell death. Other P2X receptors such as P2X2 or P2X4 receptors also mediate rapid membrane depolarization but never induce cell death (3, 30). Also in our system the addition of BzATP induced rapid depolarization of the plasma membrane detected from the increase in DiBAC4(3) fluorescence and the uptake of ethidium bromide, providing evidence for activation of the cation channel and for the pore formation, respectively. The up-take of membrane-impermeable nucleic acid-binding molecules such as ethidium and propidium has been a standard tool for measuring cell permeabilization, and the cells are presumed to be dead by necrosis and late phase apoptosis. However, it was confirmed that the early dye uptake by the activation of P2X7 receptor results selectively from the pore formation and not directly from the cell lysis (7). The analysis by flow cytometry revealed that the ethidium uptake in shrunk cells was much higher than that in the non-shrunk cells. Although we are not able to say for certain whether the difference of ethidium up-take between the shrunk cells and the non-shrunk cells was dependent on the expression of receptor protein or the activity of pore, these observations suggest that pore formation should participate in the P2X7 receptor-induced cell shrinkage in DT40/P2X7 cells.

Because membrane pores can cause movement of ions across the plasma membrane, next we investigated the ionic effects on BzATP-induced cell shrinkage of DT40/P2X7 cells. The activation of P2X7 receptor induced cell shrinkage in the minimum and essential ion solution (HBSS), suggesting the involvement of ions in the P2X7 receptor-mediated cell shrinkage. Removing Ca2+ or Mg2+ stimulated the BzATP-induced cell shrinkage. It is widely known that the effect of BzATP (or ATP) is greatly potentiated by reducing the concentration of extracellular Ca2+ or Mg2+ (3); therefore the removal of Ca2+ or Mg2+ enhanced BzATP-induced cell shrinkage by the reduction of an activation threshold of the receptor. Replacing K+ with choline stimulated the BzATP-induced cell shrinkage. Unfortunately, we have little information on the effect of extracellular K+ on the BzATP-induced events. Because efflux of intracellular K+ is one of the important features of apoptosis (31), removal of extracellular K+ may have enhanced cell shrinkage via the acceleration of intracellular K+ efflux. Previous reports indicated that elevated extracellular K+ concentration inhibits death receptor-mediated and chemical-mediated apoptosis in Jurkat T cells (32–34). However, increasing extracellular K+ concentration (5–70 mM) had no effect on the BzATP-induced cell shrinkage (data not shown), and high K+ concentration (over 70 mM) induced cell shrinkage by itself. Therefore the attempt to demonstrate the involvement of intracellular K+ efflux was unsuccessful. We know there is room for further investigation on the role of extracellular K+, but we were concerned with the inhibitory effect by removal of extracellular Cl– on the P2X7-mediated cell death in this paper.

The BzATP-induced cell shrinkage was blocked when media Cl– was replaced with gluconate. Removal of extracellular Cl– blocked the BzATP-induced LDH release, but not the pore formation and the membrane depolarization. These observations indicated that removal of extracellular Cl– did not prevent BzATP from binding to P2X7 receptor but affected the process of apoptotic cell death. Taking the involvement of pore formation in cell shrinkage into consideration, it seems reasonable to suppose that the involvement of extracellular Cl– influx via the pore formed in the P2X7 receptor-mediated apoptotic cell death. Moreover, increasing extracellular K+ induced membrane depolarization and cell shrinkage of DT40 cells. This high K+-induced cell shrinkage was completely inhibited by removal of extracellular Cl–. Because transmembrane K+ and/or Na+ gradients, specifically membrane potential, should be the major energy source to keep the intracellular Cl– concentration low, we speculate that the high K+-induced cell shrinkage was triggered by influx of extracellular Cl– into DT40 cells. The pretreatment with an anion channel/transporter inhibitor, DIDS, also inhibited the high K+-induced cell shrinkage. Thus these observations strongly support the presence of an induction system of cell shrinkage triggered by extracellular Cl– influx in DT40 cells. Buisman et al. (35) have already reported that the ATP-induced non-selective pore was permeable to not only Na+ and K+ but also Cl– in macrophage-like cell line J774.2; however we have no definite information on the mobilization of intracellular Cl– of DT40 cells. The attempt to measure Cl– influx by using Cl–-sensitive fluorescent probe MQAE was unsuccessful because we were not able to distinguish the Cl–-dependent fluorescence change from the effect of leakage of MQAE (molecular weight 326) through the membrane pore formed in response to BzATP. The reported value of intracellular Cl– concentration in lymphocytes is in the range of 50–85 mM (36, 37), and the extracellular Cl– concentration is likely to be from 120 to 135 mM. Given the gradient of Cl– concentration across the membrane, opening a non-selective pore should result in Cl– moving into the cell. In fact, we detected significant inhibition of cell shrinkage by removal of extracellular Cl– at a concentration of 90 mM. This finding corresponded well with the above reports about intracellular Cl– concentration in lymphocytes. Therefore we propose extracellular Cl– entry through the non-selective pore by the activation of P2X7 receptor.

We demonstrate in this paper the critical role of extracellular Cl– influx in P2X7 receptor-mediated cell death. Previous studies also suggested that activation of P2X7 receptor induces a complete collapse of ionic gradients that switches the cytosol from a high K+-low Na+-low Cl– ionic milieu to a low K+-high-Na+-high Cl– environment (35, 38), but the main stress has fallen on the coordinated increase in intracellular Na+ and decrease in intracellular K+ in the signaling cascade of P2X7 receptor. The reason why little attention has been given to increase in intracellular Cl– was that intracellular K+ efflux was essential for the induction of cell shrinkage and apoptosis by other apoptosis inducers, such as staurosporine, tumor necrosis factor-{alpha}, or Fas ligand (32–34). Recent studies revealed that the early phase of apoptotic cell shrinkage, termed apoptotic volume decrease, is induced by Cl– efflux coupled to K+ efflux, and one of the lead players would be the volume-sensitive outwardly rectifying chloride channel (25). In these systems, the efflux of intracellular K+ is critical because the elevated extracellular K+ concentration inhibits apoptotic events. Nevertheless, we were not able to detect the suppression by the elevated extracellular K+ concentration of the BzATP-induced cell shrinkage in our system. These observations suggested that differences in the pathway of apoptotic cell death might exist depending on species specificity, cell type specificity, or the model system being examined. However, the extracellular Cl– influx into cells by the activation of P2X7 receptor should cause intracellular Cl– reflux. Therefore we speculate that the P2X7 receptor can also utilize a mechanism by which Cl– efflux induces cell shrinkage. Future studies will elucidate the mechanism of the apoptotic cell death mediated by increase in intracellular Cl–.

To our knowledge, Wang et al. (39) have only reported the presence of a -dependent mechanism in the P2X7 receptor-induced {gamma}-aminobutyric acid release from astrocytes, but their system is different from our system in its sensitivity for . The role of extracellular Cl– in the signal transduction pathway of apoptotic cell death by the activation of P2X7 receptor has not been addressed before. This is the first study to demonstrate the essential role of pore formation and the selective involvement of subsequent extracellular Cl– influx in P2X7 receptor-mediated apoptotic cell death.


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

{ddagger} To whom correspondence should be addressed: School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. Tel.: 81-054-264-5670; Fax: 81-054-264-5672; E-mail: harada{at}u-shizuoka-ken.ac.jp.

1 The abbreviations used are: BzATP, 2'-3'-O-(4-benzoylbenzoyl)-ATP; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; DMEM/F-12, Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham; DiBAC4(3), bis(1,3-dibutylbarbituric acid)-trimethine oxonol; HBSS, Hanks' based salt solution; LDH, lactate dehydrogenase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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