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A novel function of BCL-2 overexpression in regulatory volume decrease. Enhancing swelling-activated Ca(2+) entry and Cl(-) channel activity.

The cellular function of the oncogene bcl-2, a key regulator of apoptosis, is still debated. The goal of this study was to explore the relationship between BCL-2 overexpression and cell volume regulation by using two independent models, Madin-Darby canine kidney (MDCK) cells stably transfected with BCL-2 and MDCK clones with inducible BCL-2 expression by the lac operator/repressor. BCL-2 overexpression enhanced the capability of regulatory volume decrease (RVD), a cellular defensive process against hypotonic stress. In various clones of MDCK cells, hypotonic stress induced an outwardly rectified Cl(-) current that was significantly up-regulated by BCL-2 overexpression. Other fundamental characteristics of this channel were similar among different MDCK clones, such as sensitivity to Cl(-) channel inhibitor, anion permeability, and time-dependent inactivation at more positive potential. Most importantly, BCL-2 overexpression up-regulates the swelling-activated Ca(2+) transient that is a critical signaling for normal RVD and the activation of swelling-activated Cl(-) channel in MDCK cells. BCL-2 overexpression also enhances the capacitative Ca(2+) entry that can be differentiated from the swelling-activated Ca(2+) transient by kinetic analysis and sensitivity to Gd(3+). Moreover, neutralization of endogenous BCL-2 by antibody blocks the normal RVD response and the activation of swelling-activated Cl(-) channel in human cervical cancer HT-3 cells. These results provide a new insight into the novel function of BCL-2 overexpression in the regulation of cell volume and ion flux.

The cellular function of the oncogene bcl-2, a key regulator of apoptosis, is still debated. The goal of this study was to explore the relationship between BCL-2 overexpression and cell volume regulation by using two independent models, Madin-Darby canine kidney (MDCK) cells stably transfected with BCL-2 and MDCK clones with inducible BCL-2 expression by the lac operator/repressor. BCL-2 overexpression enhanced the capability of regulatory volume decrease (RVD), a cellular defensive process against hypotonic stress. In various clones of MDCK cells, hypotonic stress induced an outwardly rectified Cl ؊ current that was significantly upregulated by BCL-2 overexpression. Other fundamental characteristics of this channel were similar among different MDCK clones, such as sensitivity to Cl ؊ channel inhibitor, anion permeability, and time-dependent inactivation at more positive potential. Most importantly, BCL-2 overexpression up-regulates the swelling-activated Ca 2؉ transient that is a critical signaling for normal RVD and the activation of swelling-activated Cl ؊ channel in MDCK cells. BCL-2 overexpression also enhances the capacitative Ca 2؉ entry that can be differentiated from the swelling-activated Ca 2؉ transient by kinetic analysis and sensitivity to Gd 3؉ . Moreover, neutralization of endogenous BCL-2 by antibody blocks the normal RVD response and the activation of swellingactivated Cl ؊ channel in human cervical cancer HT-3 cells. These results provide a new insight into the novel function of BCL-2 overexpression in the regulation of cell volume and ion flux.
Mammalian cells have to avoid excessive changes of cell volume that jeopardize structural integrity and constancy of the intracellular milieu. Homeostasis of cell volume does not simply indicate a constant volume but rather serves as the integration of events in regulating cell function (1,2). Most mammalian cells defend themselves against hypotonic stress by losing solutes together with osmotically obligated water, a process termed regulatory volume decrease (RVD). 1 The principal solutes lost during RVD are K ϩ , Cl Ϫ , and a group of largely electroneutral organic solutes known as organic osmolytes. The predominant pathway for RVD is the opening of separate K ϩ and Cl Ϫ channels (3). Much attention has been focused on the swelling-activated Cl Ϫ channel, because it shows a broad sensitivity for different anions and organic osmolytes (3). In addition to volume regulation and osmolyte transport, the swelling-activated Cl Ϫ channel participates in several important physiological processes, such as metabolism, hormone release, cell proliferation, differentiation, and migration (1,4). In some cell types, osmotic swelling increases intracellular Ca 2ϩ ([Ca 2ϩ ] i ), which plays a critical role in the control of RVD (1,5).
The gene of BCL-2 is located at chromosome 18q21 and encodes a 25-26-kDa protein (6). Overexpression of BCL-2 is known to convey resistance to apoptosis induced by many agents (7). Despite this fact, the function of BCL-2 on other cellular events is usually overlooked, and very little is known about the involvement of the BCL-2 family in the regulation of cell volume. Because volume constancy is one of the most critical events for cellular homeostasis and survival, it would be interesting to study the association of BCL-2 family with cell volume regulation.
The Madin-Darby canine kidney (MDCK) cell line is one of the best characterized preparations for the study of epithelial ion and water transport and its regulation. We have successfully developed two model systems to dissect the BCL-2 effects on phenotypic or morphological changes of MDCK cells (8 -10). One system is MDCK cells with differential expressions of stable BCL-2 transfectant, and the other system is MDCK cells with inducible expression of BCL-2 by lac operator/repressor. By using these two model systems, this study was aimed at exploring the relationship between BCL-2 and cell volume regulation. The results demonstrated a novel function of BCL-2 overexpression in RVD.

MATERIALS AND METHODS
Cell Culture-Wide-type MDCK cells and two human cervical cancer cell lines (HT-3 and SiHa) were obtained from the American Type Culture Collection (Manassas, VA). Another series of MDCK clones, successfully developed in our laboratory, were included in the study as follows: (i) stable transfection of bcl-2 gene (B4 and B6 cell lines) or empty vector (C1 cell) (9); (ii) inducible expression of BCL-2 by the lac operator/repressor system in MDCK cells (8). In this system, MDCK cells were cotransfected with the lac repressor gene and the human bcl-2 gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence (11). The induction of BCL-2 expression is dependent on the incubation time as well as the concentration of the lactose analog isopropyl-␤-D-thiogalactoside (IPTG). Cells were maintained at 37°C in a CO 2 /air (5:95%) atmosphere and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 80 IU/ml penicillin, and 80 g/ml streptomycin (Sigma). For experiments, cells were seeded in a concentration of 2 ϫ 10 4 cells/cm 2 and grown to 60 -80% confluence to obtain cultures in the logarithmic growth phase.
Chemicals and Solutions-5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was purchased from Research Biochemicals (Natick, MA). Other chemicals were obtained from Sigma. The osmolarity of solutions was measured using a vapor pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany). The isotonic medium contained (in mM) the following: NaCl 100, KCl 5, MgCl 2 1, CaCl 2 2, glucose 10, HEPES 10, and mannitol 70, titrated to pH 7.4 with NaOH (300 Ϯ 3 mosmol liter Ϫ1 , n ϭ 5). The components of the hypotonic medium are the same as those of the isotonic medium except mannitol was omitted, resulting in a 23% hypotonicity (230 Ϯ 3 mosmol liter Ϫ1 ). To measure the activity of the swelling-activated Cl Ϫ channel, the KCl was replaced by CsCl in the media, and the pipette solutions contained (in mM) the following: CsCl 40, cesium aspartate 100, MgCl 2 1, CaCl 2 1.93, EGTA 5, ATP 2, GTP 0.5, HEPES 5. The pipette solutions were adjusted to pH 7.2 with CsOH. The free Ca 2ϩ concentration in the pipette solution was buffered at 100 nM by EGTA, which was below the threshold for the activation of Ca 2ϩ -activated Cl Ϫ channel.
Western Blot Analysis-Expression of BCL-2 in wild-type MDCK and BCL-2 transfectants was determined by immunoblotting, as described previously (8,9). In brief, 50 g of protein extract from specific samples was resolved by 10% SDS-PAGE and was electrophoretically blotted onto nitrocellulose paper. The nitrocellulose paper was incubated with mouse anti-human BCL-2 monoclonal antibody (1:500 dilution, Transduction Laboratories), and immunocomplexes were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000 dilution); finally, the immunocomplexes were made visible by fluorography with an enhanced chemiluminescence detection kit (Amersham Biosciences). The results were analyzed by scanning densitometry and were expressed as arbitrary unit.
Measurements of Cell Volume-Cell volume was measured as described previously (12). Briefly, cells were harvested, transferred, and allowed to achieve cell attachment in Petri dish for ϳ30 min. A 2-ml bath, which was continuously superfused with isotonic solution or hypotonic solution, was then applied. Cells were viewed with magnification up to ϫ400 by an Olympus IX70 inverted microscope that was equipped with Hoffman modulation optics (Olympus, Tokyo, Japan). To monitor the change of cell size, the microscope was coupled to a video camera system, and the images were recorded in real time and stored on a video cassette recorder (National Inc., Tokyo, Japan). Images were then analyzed by the public domain NIH image program. The majority of cells observed were spheroid, and the relative volume change (V/V 0 ) was calculated from the cross-sectional surface area at the beginning (S 0 ) of the experiment and during (S) the experiments from the relation: V/V 0 ϭ (S/S 0 ) 3/2 (12). Data were presented as the percentage of starting volume (V/V 0 ), as a function of time. The validity of this approach to measure cell volume has been demonstrated in mouse thymocytes (12), renal A6 cells (13), and lymphocytes (14).
Microinjection of Cells-Antibodies (0.5 g/ml) of BCL-2 were delivered into the cytoplasm by microinjection as described previously (15,16). The antibodies were generated from human BCL-2 (amino acid 49 -179 as immunogen, purchased from Transduction Laboratories). For preparing the micropipettes for microinjection, the GD-1 glass capillaries (Narishige Scientific Instrument Laboratories, Tokyo, Japan) were heated and pulled by gravity using a two-step, vertical micropipette puller (PC-10; Narishige). To ascertain the procedure of microinjection caused no damaging effect, the RVD responses of 30 MDCK cells (B6) injected with pipette solution containing denatured BCL-2 antibodies were analyzed. The preliminary results showed the RVD responses were not affected by the procedure of microinjection.
Electrophysiological Measurements-The whole-cell mode of the patch clamp technique was used to measure membrane currents as described previously (17). Cells were bathed at room temperature (22-25°C) and continuously superfused with isotonic or hypotonic solution. When the patch pipettes were connected to the input stage of an Axopatch-200A amplifier (Axon Instruments, Burlingame, CA), their DC resistance varied between 3 and 5 megohms. The current-voltage relationship and time course of swelling-activated Cl Ϫ current were obtained from either a ramp or a step protocol. The ramp protocol consisted of a step to Ϫ80 mV for 0.4 s and followed by a 1.3-s linear voltage ramp to ϩ80 mV, after which the potential was stepped back to the holding potential of Ϫ20 mV. This voltage protocol was repeated every 15 s from a holding potential of Ϫ20 mV. Currents were sampled at 2-ms intervals (1024 points per record). The step protocol consisted of a 1-s voltage step, applied every 15 s from a holding potential of Ϫ20 mV to test potentials from Ϫ80 to ϩ80 mV with an increment of 20 mV. Currents were sampled at 1-ms intervals. Current densities were determined by normalizing the whole-cell current to the membrane capacitance. The anion selectivity of the swelling-activated currents was examined by the ramp protocol. At maximal current activation, the normal hypotonic solution was replaced by hypotonic solutions containing NaI or NaBr. The permeability of various anions (X Ϫ ) relative to that of Cl Ϫ (P X Ϫ /P Cl Ϫ ) was determined from the shift of the reversal potential in anion substitution experiments. In this case, an agar bridge was used to minimize junction potential, and permeability ratios were calculated from a modified Goldman-Hodgkin-Katz Equation 1, where ] i was measured with the fura-2 fluorescence ratio method on an a fluorimeter (F-2000, spectrophotometer, Hitachi, Tokyo, Japan) as described previously (18). In brief, cells attached on coverslips were loaded with 2 M fura-2/acetoxymethyl ester (fura-2/AM) in Dulbecco's modified Eagle's culture medium at room temperature for 40 min and then at 37°C for 20 min. After loading, cells were washed three times with phosphate-buffered saline. After washing, the coverslip was mounted in a custom-made holder and placed in a 5-ml quartz cuvette. Fluorescence emission was collected from a group of ϳ10 5 cells located in the excitation path. Excitation wavelength was alternated between 340 (I 340 ) and 380 nm (I 380 ), and fluorescence intensity was monitored at 510 nm. [Ca 2ϩ ] i was calculated from the I 340 /I 380 ratio using Equation 2 proposed by Grynkiewicz et al. (19), where K d is the dissociation constant for fura-2 in the cytosol (250 nM), F min and R min are the 380 nm fluorescence intensity and I 340 /I 380 ratio at low [Ca 2ϩ ] i , respectively. F max and R max are the 380 nm fluorescence intensity and I 340 /I 380 ratio at high [Ca 2ϩ ] i , and R is the I 340 /I 380 ratio recorded during experiments. Calibration measurements of F min and R min were performed after incubating cells for 10 min in nominally Ca 2ϩ -free isotonic solution containing 3 mM EGTA. Cells were then superfused with isotonic solution containing 1 M thapsigargin, 5 M ionomycin, and 10 mM Ca 2ϩ to evaluate F max and R max . Data Recording and Analysis-Data from electrophysiological experiments were digitized and analyzed using pCLAMP software (version 6.0.3, Axon Co., Foster City, CA). All values in the present study were reported as mean Ϯ S.E. Student's paired or unpaired t test was used for statistical analyses. Differences between values were considered significantly when p Ͻ 0.05. Fig. 1A, BCL-2 was differentially expressed in wildtype, plasmid control (C1), and BCL-2-transfected (B4 and B6) MDCK clones. The typical volume changes induced by hypotonic stress in wild-type MDCK and C1 cells could be divided into three phases as follows: 1) an initial and rapid osmotic swelling, reaching a peak cell volume (1.27 Ϯ 0.08 of original cell size, n ϭ 60; Fig. 1B) at 2.8 min; 2) a rapid shrinkage in the following 2 min; and 3) a more gradual decrease of cell volume that finally reached a plateau that was 13% above the original cell size at 7-10 min (Fig. 1B). Hypotonic stress rapidly triggered B4 cells to reach a peak volume of 1.19 Ϯ 0.05 (n ϭ 60) of initial cell volume at about 2 min. A rapid decrease of cell volume subsequently appeared in the following time course, and cell volume returned to the original size at about 7-10 min (Fig. 1B). Moreover, osmotic swelling B6 cells reached a peak volume of 1.14 Ϯ 0.07 (n ϭ 60) of initial cell volume at 1.5 min and then returned to the original cell size in about 5 min (Fig.  1B). These results indicate the sequence of RVD capability is B6 Ͼ B4 Ͼ C1 ϭ wild-type MDCK cells, which is well correlated with BCL-2 levels in these clones.

Enhanced Capability of RVD by BCL-2 Overexpression-As shown in
To investigate whether the advantage of B6 cells in RVD is a specific effect, BCL-2 antibody was delivered into cells by microinjection. Compared with control group, the treatment of BCL-2 antibody increased initial osmotic swelling, attenuated the shrinkage phase, and inhibited the gradual decrease of volume regulation in B6 cells (Fig. 1C). In addition, B6 cells treated with BCL-2 antibody showed a similar responsive curve with osmotic swelling C1 cells or wild-type MDCK cells (Fig. 1B). However, BCL-2 antibody has no effect on the volume regulation of C1 cells (Fig. 1C). This indicates that the advantage of B6 cells in volume regulation results from BCL-2 overexpression.
We also studied the RVD response in a clone of MDCK cells that differentially expressed BCL-2 induced by the lactose analog IPTG. Compared with the control group (Day 0), the RVD process was not significantly changed after 1-day induction of 1 mM IPTG, in spite of the fact that a certain amount of BCL-2 was expressed (Fig. 1, D and E). The progress of capability for RVD became significant after 2 and 3 days of IPTG induction, suggesting that BCL-2 overexpression enhances the RVD capability in a dose-dependent manner.
To ensure the changing ability of volume regulation did not result from the drug effect of IPTG, we also investigated the effect of IPTG on RVD process. IPTG (1 mM) did not affect the RVD response of wild-type and other clones of MDCK cells (data not shown).
BCL-2 Overexpression Up-regulates the Swelling-activated Cl Ϫ Channel-The swelling-activated Cl Ϫ channel plays a critical role in RVD (3). We subsequently investigated whether the changing RVD capability of MDCK cells is because of up-regulating the swelling-activated Cl Ϫ channel. Fig. 2, A and B, shows the representative recordings of swelling-activated Cl Ϫ currents, obtained from MDCK cells with plasmid control (C1 cell) and stable BCL-2 transfectant (B6 cell). C1 cells had a small isotonic background current, averaging 8.0 Ϯ 0.8 pA pF Ϫ1 at ϩ80 mV and Ϫ5.5 Ϯ 0.8 pA pF Ϫ1 at Ϫ80 mV and with a slope conductance of 0.084 Ϯ 0.009 nS pF Ϫ1 (n ϭ 50). Application of a hypotonic solution induced cell swelling, which was accompanied by an activation of large outwardly rectifying currents. At potentials more positive than ϩ40 mV, the currents showed time-dependent inactivation, which became more pronounced at higher membrane potentials. The swelling-activated current was reversed at a potential close to the theoretical equilibrium potential for Cl Ϫ (E Cl ϭ Ϫ25 mV), indicating that the swelling-activated current is mainly carried by Cl Ϫ (Fig. 2A). The sequence of anion permeability, calculated from the shifts in reversal potential, was I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ (1.53 Ϯ 0.20: 1.26 Ϯ 0.10:1, n ϭ 5).
As depicted in Fig. 2B, B6 cells also presented a small isotonic background current of 10 Ϯ 0.5 pA pF Ϫ1 at ϩ80 mV and Ϫ8.0 Ϯ 0.4 pA pF Ϫ1 at Ϫ80 mV, with a slope conductance of 0.10 Ϯ 0.01 nS pF Ϫ1 (n ϭ 50), which was not significantly different from those of C1 cells. Hypotonicity induced a remark- To compare the activities of swelling-activated Cl Ϫ channel among different MDCK clones, we normalized the swellingactivated Cl Ϫ current, which was defined as the differences of current densities between isotonic and hypotonic solutions and was expressed as per unit membrane capacitance. For C1 cells, the normalized swelling-activated Cl Ϫ current was 56 Ϯ 2.8 pA pF Ϫ1 (n ϭ 50) at ϩ80 mV which was similar to that of wild-type MDCK cells (Fig. 2C). For B4 and B6 cells, the normalized swelling-activated Cl Ϫ current significantly increased to 80 Ϯ 3.0 pA pF Ϫ1 (n ϭ 50, p Ͻ 0.05, unpaired t test) and 105 Ϯ 5.0 pA pF Ϫ1 (n ϭ 50, p Ͻ 0.01), respectively.
In addition to altering the current amplitude, the activation rate of swelling-activated Cl Ϫ channel was also significantly increased by BCL-2 overexpression (Fig. 3). In wide-type MDCK and C1 cells, exposure to hypotonicity induced an outward rectifying current with an activation rate of 0.30 Ϯ 0.03 (n ϭ 50) and 0.28 Ϯ 0.03 (n ϭ 50) pA pF Ϫ1 s Ϫ1 at ϩ80 mV, respectively. B4 and B6 cells expressed a faster current activation (B4 cells, 0.55 Ϯ 0.05 pA pF Ϫ1 s Ϫ1 , n ϭ 50; B6 cells, 0.74 Ϯ 0.02 pA pF Ϫ1 s Ϫ1 , n ϭ 50). These results clearly demonstrate that BCL-2 overexpression up-regulates the activation of swelling-activated Cl Ϫ channel. Other fundamental characteristics of this channel were similar among these different MDCK clones, such as sensitivity to Cl Ϫ channel inhibitor NPPB (Fig. 3A), time-dependent inactivation at more positive potential, and anion permeability.
We also studied the activities of swelling-activated Cl Ϫ channel in MDCK cells with inducible BCL-2 expression (Fig. 4). In the absence of IPTG (Day 0, control), the normalized swellingactivated Cl Ϫ current was 50 Ϯ 1.5 pA pF Ϫ1 s Ϫ1 at ϩ80 mV (n ϭ 50). In the presence of IPTG for 1 day, the normalized swelling-activated Cl Ϫ current was 55 Ϯ 2.7 pA pF Ϫ1 s Ϫ1 at ϩ80 mV (n ϭ 50), which was similar to that of the control group. However, after 2 days of IPTG induction, the normalized swelling-activated Cl Ϫ current was significantly increased to 75 Ϯ 2.3 pA pF Ϫ1 (n ϭ 50) at ϩ80 mV (p Ͻ 0.05, unpaired t test; Fig. 5B). After 3 days of IPTG induction, the normalized swelling-activated Cl Ϫ current further increased to 85 Ϯ 3.0 pA pF Ϫ1 (n ϭ 50) at ϩ80 mV (p Ͻ 0.05, unpaired t test; Fig. 5B). IPTG itself showed no effect on the activity of swelling-activated Cl Ϫ current. These results confirm that the increasing activity of swelling-activated Cl Ϫ channel is a specific effect of BCL-2 overexpression.
Normal RVD Requires Extracellular Ca 2ϩ ([Ca 2ϩ ] o )-In addition to Cl Ϫ channels, Ca 2ϩ signaling is deemed responsible for the normal RVD in some cell types (5). To ascertain the role of Ca 2ϩ signaling in volume regulation of MDCK cells, we  (Fig. 5A). The activation of swelling-activated Cl Ϫ channels also depends on [Ca 2ϩ ] o . In the presence of [Ca 2ϩ ] o , the activity of the swelling-activated Cl Ϫ channel at ϩ80 mV is 56 Ϯ 2.8 pA pF Ϫ1 (n ϭ 50) and 105 Ϯ 5.0 pA pF Ϫ1 (n ϭ 50) for C1 and B6 cells, respectively. On the other hand, more than 80% of activation of swelling-activated Cl Ϫ channel was suppressed when [Ca 2ϩ ] o was removed (Fig. 5B). This indicates that Ca 2ϩ entry plays a critical role in the volume regulation of these cell types.
BCL-2 Overexpression Enhances Hypotonicity-induced Ca 2ϩ Entry-We further studied Ca 2ϩ signaling in response to hypotonicity. Superfusion of C1 cells with a hypotonic solution elicited a rise of [Ca 2ϩ ] i from the basal level of 100 Ϯ 1 nM (n ϭ 10) to a peak of 180 Ϯ 6 nM with an activation rate of 1.27 Ϯ 0.14 nM s Ϫ1 (Fig. 6, A and B). The initial rise of [Ca 2ϩ ] i was subsequently followed by a decay rate of 0.17 Ϯ 0.08 nM s Ϫ1 to reach a plateau level (Fig. 6, A and B). BCL-2 overexpression did not change the steady-state [Ca 2ϩ ] i levels of MDCK cells (Fig. 6A). But hypotonic shock induced a steep rise of [Ca 2ϩ ] i transient in B6 cells with an activation rate of 6.1 Ϯ 0.3 nM s Ϫ1 (n ϭ 10), which is significantly faster than that of C1 cells (p Ͻ 0.01, unpaired t test). The initial steep rise of [Ca 2ϩ ] i transient was followed by a faster decay to return the original level (Fig.  6, A and B).  (Fig. 6C). These results suggest the initial steep rise of [Ca 2ϩ ] i results mainly from Ca 2ϩ entry from extracellular space. To study whether Ca 2ϩ entry or internal release is enhanced by BCL-2 overexpression, we analyzed the initial rise of [Ca 2ϩ ] i (⌬[Ca 2ϩ ] i ) in swollen C1 and B6 cells. In the presence of [Ca 2ϩ ] o , the ⌬[Ca 2ϩ ] i is 81 Ϯ 3 (n ϭ 10) and 200 Ϯ 6 (n ϭ 10) for C1 and B6 cells, respectively (p Ͻ 0.01, unpaired t test, Fig. 6D). However, in the absence of [Ca 2ϩ ] o , there was no significant difference in ⌬[Ca 2ϩ ] i (Fig. 6D). Taken together, BCL-2 overexpression up-regulates the hypotonicityinduced Ca 2ϩ entry and has a better buffering capability for swelling-activated [Ca 2ϩ ] i transient.
Hypotonicity-induced Ca 2ϩ influx in a wide range of cell types has been reported to be blocked effectively by trivalent metal cations (12). Gadolinium (Gd 3ϩ ) inhibited the swellingactivated [Ca 2ϩ ] i transient of B6 cells in a dose-dependent manner. The swelling-activated [Ca 2ϩ ] i transient in C1 cells is also sensitive to Gd 3ϩ (data not shown).
It has been reported that BCL-2 overexpression results in an up-regulation of capacitative Ca 2ϩ entry (CCE) in human promyeloid leukemia cell line and human B-cell lymphoma cell line (20). CCE is the specific gating of Ca 2ϩ entry across the plasma membrane in response to depletion of intracellular stores during Ca 2ϩ signaling and can be triggered by thapsigargin (TG), an irreversible inhibitor of the endoplasmic reticulum Ca 2ϩ -ATPase (21). A prompt question arises: is swellingactivated [Ca 2ϩ ] i transient different from CCE in BCL-2 overexpressed cells?
Therefore, C1 and B6 cells were analyzed to determine the level of CCE after stimulation with thapsigargin. [Ca 2ϩ ] i was measured in fura-2/AM-loaded cells in the absence of [Ca 2ϩ ] o (Fig. 7A). TG, a well established inducer of CCE, was added at 100 s to trigger the opening of the plasma membrane calcium release-activated calcium channels. Generally, after the addition of TG, Ca 2ϩ is released immediately from intracellular stores, resulting in an elevation of [Ca 2ϩ ] i . However, in these experiments, cells have been incubated in Ca 2ϩ -free media plus 1.5 mM EGTA for 30 min before [Ca 2ϩ ] i measurement, which is long enough to deplete intracellular Ca 2ϩ stores; therefore, no immediate Ca 2ϩ release was detected. Only after [Ca 2ϩ ] o is replenished 500 s later does [Ca 2ϩ ] i rise as the ion crosses the plasma membrane. B6 cells had significantly higher levels of [Ca 2ϩ ] i than C1 cells, indicating that CCE is up-regulated in BCL-2-overexpressing cells (Fig. 7A). This result is consistent with the findings in human promyeloid leukemia cells and human B-cell lymphoma cells (20).
However, swelling-activated [Ca 2ϩ ] i transient could be distinguished from the CCE. In TG-treated B6 cells, [Ca 2ϩ ] i rose to a peak at ϳ150 s with an activation rate of 1.5 Ϯ 0.1 nM s Ϫ1 (n ϭ 10), after [Ca 2ϩ ] o is replenished in isotonic solution. In contrast, [Ca 2ϩ ] i rose rapidly to a peak at ϳ80 s with an activation rate of 2.0 Ϯ 0.1 nM s Ϫ1 (n ϭ 10), after [Ca 2ϩ ] o is replenished in hypotonic solution. In TG-treated C1 cells, swelling-activated [Ca 2ϩ ] i transient could also be distinguished from the CCE (Fig. 7, A-C). In addition, in B6 cells, Gd 3ϩ blocked Ca 2ϩ entry during CCE activation more potently than Ca 2ϩ influx during swelling-activated [Ca 2ϩ ] i transient with an IC 50 of 6 and 65 M, respectively (Fig. 7D). Moreover, the swelling-activated [Ca 2ϩ ] i transient could be elicited after 10 M Gd 3ϩ completely inhibited the CCE activation (Fig. 7E). These results indicate that swelling-activated [Ca 2ϩ ] i transient and CCE represent separate pathways for Ca 2ϩ entry in MDCK cells.
BCL-2 Antibody Affects the RVD Response of Cells Expressing Endogenous BCL-2-We further investigated the volume regulation of cells with endogenous BCL-2 expression. As shown in Fig. 8A, human cervical cancer HT-3 cells expressed the endogenous BCL-2, whereas cervical cancer SiHa cells did not. These two cell lines need hypotonicity-induced Ca 2ϩ entry for the normal RVD response and the activation of swelling- activated Cl Ϫ channel (15,22). Delivered by microinjection, BCL-2 antibody showed a significantly inhibitory effect on RVD response of HT-3 cells but had no effect on the volume regulation of SiHa cells (Fig. 8, B and C). Moreover, in patch clamp recordings, intracellular dialysis of BCL-2 antibody significantly blocked the activation rate and amplitude of swellingactivated Cl Ϫ channel in HT-3 cells (Fig. 8D, n ϭ 10). In contrast, the activation of swelling-activated Cl Ϫ channel was not affected by BCL-2 antibody in SiHa cells (Fig. 8E, n ϭ 10). DISCUSSION Here we show the novel function of BCL-2 overexpression in the regulation of cell volume and ion flux. In this study, various clones of MDCK cells were initially seeded in a similar cell density and were used for experiments in the logarithmic growth phase. Moreover, neutralization of endogenous BCL-2 by antibody blocks the normal RVD response in human cervical cancer cells, supporting that BCL-2 can play an important role in volume regulation. Therefore, the presented observation is a specific effect of BCL-2 on cellular function, rather than a bias from different cell proliferation rate or different culture condition or overexpression of a foreign protein. To our knowledge, this is the first study to demonstrate a direct correlation between BCL-2 function and cell volume regulation.
BCL-2 overexpression can up-regulate Ca 2ϩ influx pathways mediated by swelling or store depletion. There are three lines of evidence consistent with the notion that these two Ca 2ϩ influxes represent separate pathways for Ca 2ϩ entry in MDCK cells. (i) In thapsigargin-treated B6 cells, [Ca 2ϩ ] i rose steadily to a peak at ϳ150 s with an activation rate of 1. Although the molecular identity is not available, the stretch-activated cation channels are presumably the route for Ca 2ϩ entry in the hypotonic condition for most cell types (5,12).
In addition to up-regulation of swelling-activated [Ca 2ϩ ] i transient, BCL-2 overexpression in MDCK cells enhances the capacitative Ca 2ϩ entry which is thought to be essential for maintaining [Ca 2ϩ ] i homeostasis and may therefore be an important regulator of apoptosis during both the induction and execution phase, because both phases contain Ca 2ϩ -dependent components (20). In human promyeloid leukemia cells and B-cell lymphoma cells, BCL-2 overexpression results in upregulation of capacitative Ca 2ϩ entry and resistance to apoptosis induced by the inhibitor of capacitative Ca 2ϩ entry (20). However, the swelling-activated [Ca 2ϩ ] i transient apparently does not share the same pathway with capacitative Ca 2ϩ entry and has an unknown role in the antiapoptotic effect of BCL-2.
Osmotic swelling of MDCK cells led to a transient hyperpolarization followed by a sustained depolarization of cell mem- brane (23,24). Further studies confirmed that this was because of a transient activation of K ϩ channel and a sustained activation of Cl Ϫ channel (25,26), indicating that the swelling-activated Cl Ϫ channel plays a critical role in the control of RVD in MDCK cells. Activation of swelling-activated Cl Ϫ channel requires the Ca 2ϩ entry in MDCK cells. The amplitude and activation rate of swelling-activated Cl Ϫ channel was also upregulated by BCL-2 overexpression, thereby identifying a functional link between BCL-2 function and Cl Ϫ channel activation. It is likely that there exists a cause-effect relationship between the enhancing effects exerted by BCL-2 overexpression on Ca 2ϩ entry and activation of swelling-activated Cl Ϫ channel. However, a direct interaction of BCL-2 with swelling-activated Cl Ϫ channel cannot be completely ruled out by the current evidence.
An obvious question that arises is "what is the functional significance or benefit from the enhancing capability of volume regulation by BCL-2 overexpression?" The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Even at constant extracellular osmolarity, volume constancy of animal cells is constantly challenged by transport of osmotically active materials across cell membrane and formation or disappearance of cellular osmolarity by metabolism. Cell volume also undergoes a significant change during cell cycle progression, which perturbs cell vol-ume homeostasis and should be counterbalanced by volume regulation. The close linkage of cell volume homeostasis, cell growth, and metabolism implies that volume-regulatory transport pathways definitely play an important role in the normal physiological function or pathological conditions. Osmotically swollen cells restore their volume, exhibiting RVD by releasing intracellular K ϩ , Cl Ϫ , organic solutes, and obligated water (3). In many cell types, the volume regulatory effluxes of Cl Ϫ and some organic osmolytes are known to be induced by swellinginduced activation of the Cl Ϫ channel that is characterized by the moderate outward rectification, cytosolic ATP dependence, and intermediate unitary conductance (10 -100 pS) (3). In addition to volume regulation, the activations of the swellingactivated Cl Ϫ channel have been reported to participate in several important physiological processes, such as metabolism, hormone release, cell proliferation, differentiation, migration, and potential transport pathway for metabolic compounds (e.g. amino acids) that are required for cell growth (1,4). We found previously (17) that the differential expressions of swellingactivated Cl Ϫ channel associated with the cell cycle progression. The malignant transformation of human cervical epithelial cells is accompanied by the significant up-regulation of swelling-activated Cl Ϫ channel (27,28). The swelling-activated Cl Ϫ channel has also been suggested to be involved in pHregulatory steps, and its inhibition may induce cell alkalinization and arrest cell proliferation (1). Accordingly, increasing capability of RVD by up-regulation of swelling-activated Cl Ϫ channels may give cells an advantage on growth and metabolism and a better ability to handle stress.
Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. Little information is available on the role of ion channels in apoptosis. Lang and coworkers (14,29) have proposed a model of lymphocytes for the relationship among ion channels, cell volume, and apoptotic cell death. Stimulation of the CD95 receptors leads to a rapid activation of outwardly rectified Cl Ϫ channel (ORCC), which shares some similar characteristics with volume-sensitive Cl Ϫ channel. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces the activation of ORCC in cell-attached patch clamp experiments. The activation of this type of Cl Ϫ channel is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor or by genetic deficiency of p56 lck (14). These results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes. Another study demonstrated that apoptotic volume decrease was an early prerequisite to apoptotic cell death, and this apoptotic volume decrease process could be prevented by blocking the volumeregulatory Cl Ϫ or K ϩ channel (30). However, the role of cell volume-regulatory mechanisms in programmed cell death is still ill-defined, and the functional importance remains a matter of speculation.