Involvement of chloride in apoptotic cell death induced by activation of ATP-sensitive P2X7 purinoceptor.

The ATP-gated P2X(7) 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 P2X(7) receptor. In this study, we investigated the mechanism of P2X(7) receptor-induced cell death using this clone. Treatment with P2X(7) 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 P2X(7) receptor also caused the release of lactate dehydrogenase from cells. The P2X(7) 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 P2X(7) receptor-induced apoptotic cell death and that this is mediated by extracellular Cl(-) influx.

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 extracellular 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 P2X 7 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 fam-ily, 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 P2X 7 receptor has been reported to link to a number of other cellular events including cell fusion (6), membrane blebbing (7), and interleukin-1␤ release (8). The P2X 7 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 P2X 7 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). P2X 7 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 P2X 7 receptor (DT40/ P2X7 cells) and reported apoptotic cell death mediated via the P2X 7 receptor (12). There are three reasons why we selected DT40 cells for the host cells. First, the P2X 7 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 P2X 7 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 P2X 7 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 P2X 7 receptor agonist, 2Ј-3Ј-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 * 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.
‡ 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@u-shizuoka-ken.ac.jp. by the P2X 7 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 P2X 7 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 P2X 7 receptor have not been reported. In the present study, to understand the molecular mechanism by which P2X 7 receptor-induced cell death occurs, we investigated the essential role of pore formation in the apoptotic cell death induced by the activation of P2X 7 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.
Determination of Ethidium Bromide Uptake by Flow Cytometry-Cells were washed with and resuspended in the indicated buffer at 1 ϫ 10 6 cells/ml and then incubated with BzATP and ethidium bromide (25 M) 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 ϫ 10 5 cells/well) were incubated in a 96-well plate at 37°C for 9 h with BzATP in DMEM/F-12, HBSS, or Cl Ϫ -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 ϫ 10 6 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 o 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 potentialsensitive anionic dye, DiBAC 4 (3) (20). Cells were washed twice and resuspended at a density of 1 ϫ 10 6 cells/ml. The cells were incubated with 1 M DiBAC 4 (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 DiBAC 4 (3) fluorescence.
Statistical Analysis-Values are given as mean Ϯ 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 Ͻ 0.001 using the Instat version 3.0 statistical package (GraphPad Software).

RESULTS
Apoptotic Cell Death During P2X 7 Receptor Activation-The activation of P2X 7 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 P2X 7 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 P2X 7 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 P2X 7 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 M for 90 min), cell shrinkage was detected ( Fig. 2A). This cell shrinkage was completely prevented in DMEM/F-12 supplemented with 5 mM MgCl 2 ( Fig. 2A) that is known to block the activation of P2X 7 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).
Ionic Effects on P2X 7 Receptor-mediated Cell Shrinkage-Previous studies demonstrated that P2X 7 receptor function is affected by a wide range of ions (7,(21)(22)(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 P2X 7 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 ϩ , K ϩ , Ca 2ϩ , Mg 2ϩ , Cl Ϫ , PO 4 3Ϫ and HCO 3 Ϫ (HBSS). Replacing Na ϩ with choline, removing PO 4 3Ϫ or HCO 3 Ϫ in HBSS did not affect the BzATP-induced cell shrinkage (Fig.  4, B-D). On the other hand, removing Ca 2ϩ or Mg 2ϩ or replacing K ϩ with choline stimulated the BzATP-induced cell shrinkage (Fig. 4, E-G). Interestingly, when Cl Ϫ in HBSS was re-placed with gluconate, the BzATP-induced cell shrinkage was suppressed (Fig. 4H).
To further study the role of extracellular Cl Ϫ on the P2X 7 receptor-mediated cell death, we investigated the effect of removal of extracellular Cl Ϫ on the P2X 7 -mediated events. Removal of extracellular Cl Ϫ 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 Ϫ could not block the BzATPinduced pore formation but did block cell shrinkage and LDH release (Fig. 5, B-D). The activation of P2X 7 receptor also triggers plasma membrane depolarization caused by transmembrane cation influx (25). Therefore we monitored the change in membrane potential using a fluorescent potentialsensitive anionic dye, DiBAC 4 (3). As shown in Fig. 6, the treatment with BzATP evoked fast membrane depolarization, and it was not suppressed by removal of extracellular Cl Ϫ .
Involvement of Cl Ϫ Influx in High K ϩ -induced Cell Shrinkage-To further study the involvement of extracellular Cl Ϫ in the P2X 7 receptor-mediated cell death, we examined the effect of extracellular K ϩ on cell size. The cells were incubated in buffers with differing concentrations of K ϩ , where concentrations of Na ϩ were varied reciprocally to maintain osmolarity and a univalent cation concentration of 142.4 mM. Increasing the extracellular K ϩ concentration up to 70 mM had no effect on the BzATP-induced cell shrinkage (data not shown). Interestingly, high extracellular K ϩ concentrations (over 70 mM) induced cell shrinkage of our clone and even normal DT40 cells. Treatment with high extracellular K ϩ (100 mM) for 90 min induced cell shrinkage and membrane depolarization but not ethidium uptake (Fig. 7A) in normal DT40 cells. The high K ϩ -induced cell shrinkage was concentration-dependent and was completely inhibited by removal of extracellular Cl Ϫ (Fig.  7B). Moreover, treatment with an anion channel/exchanger inhibitor, DIDS, blocked the cell shrinkage induced by high extracellular K ϩ (100 mM) (Fig. 7C). DISCUSSION Activation of P2X 7 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 P2X 7 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 in-  clude 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 P2X 7 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 P2X 7 receptor occurred by apoptosis rather than by necrosis under our experimental conditions. The P2X 7 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 P2X 7 receptorspecific longer C terminus is required for induction of cell death. Other P2X receptors such as P2X 2 or P2X 4 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 DiBAC 4 (3) fluorescence and the uptake of ethidium bromide, providing evidence for activation of the cation channel and for the pore formation, respectively. The uptake 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 P2X 7 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 uptake 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 P2X 7 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 P2X 7 receptor induced cell shrinkage in the minimum and essential ion solution (HBSS), suggesting the involvement of ions in the P2X 7 receptor-mediated cell shrinkage. Removing Ca 2ϩ or Mg 2ϩ 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 Ca 2ϩ or Mg 2ϩ (3); therefore the removal of Ca 2ϩ or Mg 2ϩ enhanced BzATP-induced cell shrinkage by the reduction of an activation threshold of the receptor. Replacing K ϩ with choline stimulated the BzATP-induced cell shrinkage. Unfortunately, we have little information on the effect of extracellular K ϩ on the BzATP-induced events. Because efflux of intracellular K ϩ is one of the important features of apoptosis (31), removal of extracellular K ϩ may have enhanced cell shrinkage via the acceleration of intracellular K ϩ efflux. Previous reports indi- cated that elevated extracellular K ϩ concentration inhibits death receptor-mediated and chemical-mediated apoptosis in Jurkat T cells (32)(33)(34). However, increasing extracellular K ϩ concentration (5-70 mM) had no effect on the BzATP-induced cell shrinkage (data not shown), and high K ϩ concentration (over 70 mM) induced cell shrinkage by itself. Therefore the attempt to demonstrate the involvement of intracellular K ϩ efflux was unsuccessful. We know there is room for further investigation on the role of extracellular K ϩ , but we were concerned with the inhibitory effect by removal of extracellular Cl Ϫ on the P2X 7 -mediated cell death in this paper.
The BzATP-induced cell shrinkage was blocked when media Cl Ϫ was replaced with gluconate. Removal of extracellular Cl Ϫ blocked the BzATP-induced LDH release, but not the pore formation and the membrane depolarization. These observations indicated that removal of extracellular Cl Ϫ did not prevent BzATP from binding to P2X 7 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 Ϫ influx via the pore formed in the P2X 7 receptor-mediated apoptotic cell death. Moreover, increasing extracellular K ϩ induced membrane depolarization and cell shrinkage of DT40 cells. This high K ϩ -induced cell shrinkage was completely inhibited by removal of extracellular Cl Ϫ . Because transmembrane K ϩ and/or Na ϩ gradients, specifically membrane potential, should be the major energy source to keep the intracellular Cl Ϫ concentration low, we speculate that the high K ϩ -induced cell shrinkage was triggered by influx of extracellular Cl Ϫ into DT40 cells. The pretreatment with an anion channel/transporter inhibitor, DIDS, also inhibited the high K ϩ -induced cell 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 Ϫ -free HBSS containing various concentrations of K ϩ , and the cell shrinkage was measured by flow cytometry. C, DT40 cells were incubated for 90 min with 50 or 100 M DIDS in HBSS (5 mM K ϩ ) or HBSS containing 100 mM K ϩ , and the percentage of shrunk cells in total cells was determined from four independent experiments. * (p Ͻ 0.001) indicates statistically significant differences compared with data in HBSS containing 100 mM K ϩ .
shrinkage. Thus these observations strongly support the presence of an induction system of cell shrinkage triggered by extracellular Cl Ϫ influx in DT40 cells. Buisman et al. (35) have already reported that the ATP-induced non-selective pore was permeable to not only Na ϩ and K ϩ but also Cl Ϫ in macrophagelike cell line J774.2; however we have no definite information on the mobilization of intracellular Cl Ϫ of DT40 cells. The attempt to measure Cl Ϫ influx by using Cl Ϫ -sensitive fluorescent probe MQAE was unsuccessful because we were not able to distinguish the Cl Ϫ -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 Ϫ concentration in lymphocytes is in the range of 50 -85 mM (36,37), and the extracellular Cl Ϫ concentration is likely to be from 120 to 135 mM. Given the gradient of Cl Ϫ concentration across the membrane, opening a non-selective pore should result in Cl Ϫ moving into the cell. In fact, we detected significant inhibition of cell shrinkage by removal of extracellular Cl Ϫ at a concentration of 90 mM. This finding corresponded well with the above reports about intracellular Cl Ϫ concentration in lymphocytes. Therefore we propose extracellular Cl Ϫ entry through the non-selective pore by the activation of P2X 7 receptor.
We demonstrate in this paper the critical role of extracellular Cl Ϫ influx in P2X 7 receptor-mediated cell death. Previous studies also suggested that activation of P2X 7 receptor induces a complete collapse of ionic gradients that switches the cytosol from a high K ϩ -low Na ϩ -low Cl Ϫ ionic milieu to a low K ϩ -high-Na ϩ -high Cl Ϫ environment (35,38), but the main stress has fallen on the coordinated increase in intracellular Na ϩ and decrease in intracellular K ϩ in the signaling cascade of P2X 7 receptor. The reason why little attention has been given to increase in intracellular Cl Ϫ was that intracellular K ϩ efflux was essential for the induction of cell shrinkage and apoptosis by other apoptosis inducers, such as staurosporine, tumor necrosis factor-␣, or Fas ligand (32)(33)(34). Recent studies revealed that the early phase of apoptotic cell shrinkage, termed apoptotic volume decrease, is induced by Cl Ϫ efflux coupled to K ϩ 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 ϩ is critical because the elevated extracellular K ϩ concentration inhibits apoptotic events. Nevertheless, we were not able to detect the suppression by the elevated extracellular K ϩ 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 Ϫ influx into cells by the activation of P2X 7 receptor should cause intracellular Cl Ϫ reflux. Therefore we speculate that the P2X 7 receptor can also utilize a mechanism by which Cl Ϫ efflux induces cell shrinkage. Future studies will elucidate the mechanism of the apoptotic cell death mediated by increase in intracellular Cl Ϫ .
To our knowledge, Wang et al. (39) have only reported the presence of a Cl Ϫ /HCO 3 Ϫ -dependent mechanism in the P2X 7 receptor-induced ␥-aminobutyric acid release from astrocytes, but their system is different from our system in its sensitivity for HCO 3 Ϫ . The role of extracellular Cl Ϫ in the signal transduction pathway of apoptotic cell death by the activation of P2X 7 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 Ϫ influx in P2X 7 receptor-mediated apoptotic cell death.