HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 18, 15592-15599, May 3, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15592    most recent M111043200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shen, M.-R. Articles by Tang, M.-J. Search for Related Content PubMed PubMed Citation Articles by Shen, M.-R. Articles by Tang, M.-J. Social Bookmarking                      What's this?

A Novel Function of BCL-2 Overexpression in Regulatory Volume Decrease

ENHANCING SWELLING-ACTIVATED Ca²⁺ ENTRY AND Cl CHANNEL ACTIVITY^*

Meng-Ru Shen^§¶, Tzi-Peng Yang, and Ming-Jer Tang^**

From the  Department of Obstetrics and Gynecology and the  Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China and ^§ University Laboratory of Physiology, Parks Road, University of Oxford, Oxford OX1 3PT, United Kingdom

Received for publication, November 19, 2001, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 Ca²⁺ 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 Ca²⁺ entry that can be differentiated from the swelling-activated Ca²⁺ transient by kinetic analysis and sensitivity to Gd³⁺. 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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⁺, Cl, and a group of largely electroneutral organic solutes known as organic osmolytes. The predominant pathway for RVD is the opening of separate K⁺ and Cl channels (3). Much attention has been focused on the swelling-activated Cl 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 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 Ca²⁺ ([Ca²⁺]_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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 CO₂/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 µg/ml streptomycin (Sigma). For experiments, cells were seeded in a concentration of 2 × 10⁴ cells/cm² 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, MgCl₂ 1, CaCl₂ 2, glucose 10, HEPES 10, and mannitol 70, titrated to pH 7.4 with NaOH (300 ± 3 mosmol liter¹, n = 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 ± 3 mosmol liter¹). To measure the activity of the swelling-activated Cl channel, the KCl was replaced by CsCl in the media, and the pipette solutions contained (in mM) the following: CsCl 40, cesium aspartate 100, MgCl₂ 1, CaCl₂ 1.93, EGTA 5, ATP 2, GTP 0.5, HEPES 5. The pipette solutions were adjusted to pH 7.2 with CsOH. The free Ca²⁺ concentration in the pipette solution was buffered at 100 nM by EGTA, which was below the threshold for the activation of Ca²⁺-activated Cl 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 µg 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 ~30 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 ×400 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/V₀) was calculated from the cross-sectional surface area at the beginning (S₀) of the experiment and during (S) the experiments from the relation: V/V₀ = (S/S₀)^3/2 (12). Data were presented as the percentage of starting volume (V/V₀), 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 µg/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 current were obtained from either a ramp or a step protocol. The ramp protocol consisted of a step to 80 mV for 0.4 s and followed by a 1.3-s linear voltage ramp to +80 mV, after which the potential was stepped back to the holding potential of 20 mV. This voltage protocol was repeated every 15 s from a holding potential of 20 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 20 mV to test potentials from 80 to +80 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) relative to that of Cl (P_X/P_Cl) 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,

(Eq. 1)

where [Cl]_n and [Cl]_s are the Cl concentrations in the normal and substituted external solutions; [X]_s is the concentration of the substituting anion; F is the Faraday constant; R is the gas constant, and T is absolute temperature.

Fluorescence Measurements of [Ca²⁺]_i-- [Ca²⁺]_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 µM 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 ~10⁵ cells located in the excitation path. Excitation wavelength was alternated between 340 (I₃₄₀) and 380 nm (I₃₈₀), and fluorescence intensity was monitored at 510 nm. [Ca²⁺]_i was calculated from the I₃₄₀/I₃₈₀ ratio using Equation 2 proposed by Grynkiewicz et al. (19),

(Eq. 2)

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 I₃₄₀/I₃₈₀ ratio at low [Ca²⁺]_i, respectively. F_max and R_max are the 380 nm fluorescence intensity and I₃₄₀/I₃₈₀ ratio at high [Ca²⁺]_i, and R is the I₃₄₀/I₃₈₀ ratio recorded during experiments. Calibration measurements of F_min and R_min were performed after incubating cells for 10 min in nominally Ca²⁺-free isotonic solution containing 3 mM EGTA. Cells were then superfused with isotonic solution containing 1 µM thapsigargin, 5 µM ionomycin, and 10 mM Ca²⁺ 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 ± S.E. Student's paired or unpaired t test was used for statistical analyses. Differences between values were considered significantly when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Enhanced Capability of RVD by BCL-2 Overexpression-- As shown in Fig. 1A, BCL-2 was differentially expressed in wild-type, 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 ± 0.08 of original cell size, n = 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 ± 0.05 (n = 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 ± 0.07 (n = 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 > B4 > C1 = wild-type MDCK cells, which is well correlated with BCL-2 levels in these clones.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Response of cell volume to hypotonic stress in MDCK cells with different BCL-2 expression. A, different BCL-2 levels in MDCK cells with stable BCL-2 transfection. Western blotting analysis demonstrates the expression of BCL-2 in homogenates of wild-type, plasmid control (C1), and BCL-2 transfected (B4 and B6) MDCK clones. B, time course of volume changes in C1, B4, B6, and wild-type MDCK cells following superfusion with hypotonic bath solution (230 mosmol liter1). **, p < 0.01, compared with the volume ratio with wild-type MDCK cells at 10 min, unpaired t test. The y axis (V/V0) depicts the cell volume at the indicated times divided by the cell volume at zero time. Each point represents mean ± S.E. (n = 60 cells). C, effect of BCL-2 antibodies on the time course of volume changes in response to hypotonicity for C1 and B6 cells. Antibodies were delivered by microinjection. Each point represents mean ± S.E. (n = 25 cells). **, p < 0.01, compared with the volume ratio of B6 cells without or with antibody treatment at 20 min. D, inducible BCL-2 expression by the lac operator/repressor system in MDCK cells. Time course induction of BCL-2 by 1 mM IPTG for the indicated time (upper panel). The levels of BCL-2 were determined by Western blotting. P, positive control, taken from the stable BCL-2 transfectants. Lower panel, results (n = 3) of densitometric analysis of the Western blotting. E, progressively enhancing capability of RVD by inducible BCL-2 expression. *, p < 0.05; **, p < 0.01, compared with the volume ratio with control groups (Day 0) at 10 min, unpaired t test. The y axis (V/V0) depicts the cell volume at the indicated time divided by the cell volume at zero time after superfusion with hypotonic bath solution (230 mosmol liter1). Each point represents mean ± S.E. (n = 60 cells).

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 Channel-- The swelling-activated Cl 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 channel.

Fig. 2, A and B, shows the representative recordings of swelling-activated Cl 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 ± 0.8 pA pF¹ at +80 mV and 5.5 ± 0.8 pA pF¹ at 80 mV and with a slope conductance of 0.084 ± 0.009 nS pF¹ (n = 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 +40 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 (E_Cl = 25 mV), indicating that the swelling-activated current is mainly carried by Cl (Fig. 2A). The sequence of anion permeability, calculated from the shifts in reversal potential, was I > Br > Cl (1.53 ± 0.20: 1.26 ± 0.10:1, n = 5).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Up-regulation of swelling-activated Cl channel by BCL-2 overexpression. Representative recordings of Cl current traces (step protocol) in isotonic (300 mosmol liter1) and hypotonic (230 mosmol liter1) solution for C1 cells (A) and B6 cells (B). Horizontal lines represent zero current levels. Current-voltage relationships were obtained from traces in isotonic and hypotonic solutions. Closed and open circles are hypotonic and isotonic currents, respectively. The step protocol for (A and B) consisted of a 1-s voltage step, applied every 15 s from a holding potential of 20 mV to test potentials from 80 to +80 mV with an increment of 20 mV. C, normalized swelling-activated Cl currents measured at +80 mV in wild-type MDCK cells and MDCK cells transfected with plasmid control (C1) and bcl-2 (B4 and B6) gene. Each column represents mean ± S.E. (n = 50). *, p < 0.05; **, p < 0.01 by unpaired t test, compared with wild-type MDCK cells.

As depicted in Fig. 2B, B6 cells also presented a small isotonic background current of 10 ± 0.5 pA pF¹ at +80 mV and 8.0 ± 0.4 pA pF¹ at 80 mV, with a slope conductance of 0.10 ± 0.01 nS pF¹ (n = 50), which was not significantly different from those of C1 cells. Hypotonicity induced a remarkable outwardly rectified current (Fig. 2B) with the anion permeability of I > Br > Cl (1.58 ± 0.18:1.30 ± 0.15:1, n = 5). To compare the activities of swelling-activated Cl channel among different MDCK clones, we normalized the swelling-activated Cl 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 current was 56 ± 2.8 pA pF¹ (n = 50) at +80 mV which was similar to that of wild-type MDCK cells (Fig. 2C). For B4 and B6 cells, the normalized swelling-activated Cl current significantly increased to 80 ± 3.0 pA pF¹ (n = 50, p < 0.05, unpaired t test) and 105 ± 5.0 pA pF¹ (n = 50, p < 0.01), respectively.

In addition to altering the current amplitude, the activation rate of swelling-activated Cl 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 ± 0.03 (n = 50) and 0.28 ± 0.03 (n = 50) pA pF¹ s¹ at +80 mV, respectively. B4 and B6 cells expressed a faster current activation (B4 cells, 0.55 ± 0.05 pA pF¹ s¹, n = 50; B6 cells, 0.74 ± 0.02 pA pF¹ s¹, n = 50). These results clearly demonstrate that BCL-2 overexpression up-regulates the activation of swelling-activated Cl channel. Other fundamental characteristics of this channel were similar among these different MDCK clones, such as sensitivity to Cl channel inhibitor NPPB (Fig. 3A), time-dependent inactivation at more positive potential, and anion permeability.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Activation rate of the swelling-activated Cl channel is significantly increased by BCL-2 overexpression. A, time course of membrane currents activated at +80 mV in wild-type, plasmid control (C1), and BCL-2-transfected (B4 and B6) MDCK clones. Data points were obtained from the voltage ramp protocol that was applied every 15 s. Thick horizontal bar, application of hypotonic solution (HYPO; 230 mosmol liter1) or 100 µM NPPB. Horizontal dashed line, zero current level. B, summary of activation rate for swelling-activated Cl channel in various MDCK clones. Each column represents mean ± S.E. (n = 50) *, p < 0.05; **, p < 0.01; unpaired t test, compared with wild-type MDCK cells.

We also studied the activities of swelling-activated Cl channel in MDCK cells with inducible BCL-2 expression (Fig. 4). In the absence of IPTG (Day 0, control), the normalized swelling-activated Cl current was 50 ± 1.5 pA pF¹ s¹ at +80 mV (n = 50). In the presence of IPTG for 1 day, the normalized swelling-activated Cl current was 55 ± 2.7 pA pF¹ s¹ at +80 mV (n = 50), which was similar to that of the control group. However, after 2 days of IPTG induction, the normalized swelling-activated Cl current was significantly increased to 75 ± 2.3 pA pF¹ (n = 50) at +80 mV (p < 0.05, unpaired t test; Fig. 5B). After 3 days of IPTG induction, the normalized swelling-activated Cl current further increased to 85 ± 3.0 pA pF¹ (n = 50) at +80 mV (p < 0.05, unpaired t test; Fig. 5B). IPTG itself showed no effect on the activity of swelling-activated Cl current. These results confirm that the increasing activity of swelling-activated Cl channel is a specific effect of BCL-2 overexpression.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Progressively induced BCL-2 expression is accompanied by differential activation of swelling-activated Cl channel. A, representative recordings of swelling-activated Cl currents from ramp protocol in MDCK cells transfected with inducible BCL-2. Day 0 indicated MDCK cells cultured in the absence of 1 mM IPTG, which were used as the control. Trace 1 and 2, membrane currents recorded in the isotonic and hypotonic solutions, respectively. B, normalized swelling-activated Cl currents measured at +80 mV in MDCK cells in the absence or presence of IPTG. *, p < 0.05, by unpaired t test, compared with control group (Day 0). Each column represents mean ± S.E. (n = 50).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Extracellular Ca2+ ([Ca2+]o) is necessary for regulatory volume decrease and the activation of swelling-activated Cl channel. A, time course of volume changes in C1 and B6 cells following superfusion with hypotonic bath solution (230 mosmol liter1) containing 2 mM Ca2+ or 0 mM Ca2+ plus 1.5 mM EGTA. **, p < 0.01, compared with the volume ratio at 10 min, unpaired t test. The y axis (V/V0) depicts the cell volume at the indicated time divided by the cell volume at zero time. Each point represents mean ± S.E. (n = 60 cells). B, summary of swelling-activated Cl currents measured at +80 mV in the presence or absence of [Ca2+]o. The number of cells examined is indicated above each bar. *, p < 0.05; **, p < 0.01, unpaired t test, compared with groups in which membrane currents were measured in the presence of [Ca2+]o.

Normal RVD Requires Extracellular Ca²⁺ ([Ca²⁺]_o)-- In addition to Cl channels, Ca²⁺ signaling is deemed responsible for the normal RVD in some cell types (5). To ascertain the role of Ca²⁺ signaling in volume regulation of MDCK cells, we studied the RVD process in the absence of [Ca²⁺]_o. Compared with normal RVD process, removal of [Ca²⁺]_o almost abolished the RVD process of C1 and B6 cells (Fig. 5A). The activation of swelling-activated Cl channels also depends on [Ca²⁺]_o. In the presence of [Ca²⁺]_o, the activity of the swelling-activated Cl channel at +80 mV is 56 ± 2.8 pA pF¹ (n = 50) and 105 ± 5.0 pA pF¹ (n = 50) for C1 and B6 cells, respectively. On the other hand, more than 80% of activation of swelling-activated Cl channel was suppressed when [Ca²⁺]_o was removed (Fig. 5B). This indicates that Ca²⁺ entry plays a critical role in the volume regulation of these cell types.

BCL-2 Overexpression Enhances Hypotonicity-induced Ca²⁺ Entry-- We further studied Ca²⁺ signaling in response to hypotonicity. Superfusion of C1 cells with a hypotonic solution elicited a rise of [Ca²⁺]_i from the basal level of 100 ± 1 nM (n = 10) to a peak of 180 ± 6 nM with an activation rate of 1.27 ± 0.14 nM s¹ (Fig. 6, A and B). The initial rise of [Ca²⁺]_i was subsequently followed by a decay rate of 0.17 ± 0.08 nM s¹ to reach a plateau level (Fig. 6, A and B). BCL-2 overexpression did not change the steady-state [Ca²⁺]_i levels of MDCK cells (Fig. 6A). But hypotonic shock induced a steep rise of [Ca²⁺]_i transient in B6 cells with an activation rate of 6.1 ± 0.3 nM s¹ (n = 10), which is significantly faster than that of C1 cells (p < 0.01, unpaired t test). The initial steep rise of [Ca²⁺]_i transient was followed by a faster decay to return the original level (Fig. 6, A and B).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   BCL-2 overexpression is associated with enhanced swelling-activated [Ca2+]i transient. A, representative recordings of the changes of intracellular Ca2+ ([Ca2+]i) evoked by a hypotonic solution containing 2 mM extracellular Ca2+ ([Ca2+]o) in plasmid control (C1) and BCL-2 transfected (B6) MDCK clones. B, summary of the activation and decay rate of swelling-activated [Ca2+]i transient evoked by hypotonic solution containing 2 mM Ca2+. C, representative recordings from 10 different experiments to show the changes of [Ca2+]i evoked by hypotonic solution in the absence of [Ca2+]o plus 1.5 mM EGTA ([Ca2+]o = 0 mM). D, summary of the changes of [Ca2+]i evoked by hypotonic solution containing 2 or 0 mM Ca2+. [Ca2+]i is the initial rise of [Ca2+]i from the basal level to the peak in swelling-activated [Ca2+]i transient. Each column represents mean ± S.E. (n = 10). *, p < 0.05; **, p < 0.01; unpaired t test, compared with control groups (C1 cells). ISO, isotonic solution, 300 mosmol liter1; HYPO, hypotonic solution, 230 mosmol liter1.

The swelling-activated [Ca²⁺]_i transient may result from Ca²⁺ influx from extracellular space or release from the internal store. To dissect the responsible source, we examined the swelling-activated [Ca²⁺]_i transient in the absence of [Ca²⁺]_o. Under this condition, the isotonic basal of [Ca²⁺]_i was 53 ± 2.0 nM (n = 10) and 58 ± 1.6 nM (n = 10) for C1 and B6 cells (C1 cells versus B6 cells, p > 0.05), respectively, and hypotonicity induced a progressive increase of [Ca²⁺]_i to reach a plateau of 70 ± 1.5 and 76 ± 2.0 nM (Fig. 6C). These results suggest the initial steep rise of [Ca²⁺]_i results mainly from Ca²⁺ entry from extracellular space. To study whether Ca²⁺ entry or internal release is enhanced by BCL-2 overexpression, we analyzed the initial rise of [Ca²⁺]_i ([Ca²⁺]_i) in swollen C1 and B6 cells. In the presence of [Ca²⁺]_o, the [Ca²⁺]_i is 81 ± 3 (n = 10) and 200 ± 6 (n = 10) for C1 and B6 cells, respectively (p < 0.01, unpaired t test, Fig. 6D). However, in the absence of [Ca²⁺]_o, there was no significant difference in [Ca²⁺]_i (Fig. 6D). Taken together, BCL-2 overexpression up-regulates the hypotonicity-induced Ca²⁺ entry and has a better buffering capability for swelling-activated [Ca²⁺]_i transient.

Hypotonicity-induced Ca²⁺ influx in a wide range of cell types has been reported to be blocked effectively by trivalent metal cations (12). Gadolinium (Gd³⁺) inhibited the swelling-activated [Ca²⁺]_i transient of B6 cells in a dose-dependent manner. The swelling-activated [Ca²⁺]_i transient in C1 cells is also sensitive to Gd³⁺ (data not shown).

It has been reported that BCL-2 overexpression results in an up-regulation of capacitative Ca²⁺ entry (CCE) in human promyeloid leukemia cell line and human B-cell lymphoma cell line (20). CCE is the specific gating of Ca²⁺ entry across the plasma membrane in response to depletion of intracellular stores during Ca²⁺ signaling and can be triggered by thapsigargin (TG), an irreversible inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (21). A prompt question arises: is swelling-activated [Ca²⁺]_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. [Ca²⁺]_i was measured in fura-2/AM-loaded cells in the absence of [Ca²⁺]_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, Ca²⁺ is released immediately from intracellular stores, resulting in an elevation of [Ca²⁺]_i. However, in these experiments, cells have been incubated in Ca²⁺-free media plus 1.5 mM EGTA for 30 min before [Ca²⁺]_i measurement, which is long enough to deplete intracellular Ca²⁺ stores; therefore, no immediate Ca²⁺ release was detected. Only after [Ca²⁺]_o is replenished 500 s later does [Ca²⁺]_i rise as the ion crosses the plasma membrane. B6 cells had significantly higher levels of [Ca²⁺]_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).

View larger version (34K): [in this window] [in a new window]   Fig. 7.   BCL-2 overexpression enhances CCE which can be differentiated from swelling-activated [Ca2+]i transient. A and B, representative recordings from 10 different experiments to show [Ca2+]i induced by 2 µM TG in extracellular Ca2+ ([Ca2+]o)-free media followed by replenishment of [Ca2+]o in the isotonic (A) or hypotonic (B) solution. In these experiments, cells were incubated in Ca2+-free media plus 1.5 mM EGTA for 30 min before [Ca2+]i measurement. C, comparison of the activation rate of [Ca2+]i transient in replenishment of [Ca2+]o in the isotonic or hypotonic solution. Each column represents mean ± S.E. (n = 10). *, p < 0.05, unpaired t test. ISO, isotonic solution, 300 mosmol liter1; HYPO, hypotonic solution, 230 mosmol liter1. D, dose-response curves of Gd3+ on capacitative Ca2+ entry (open square) and swelling-activated [Ca2+]i transient (closed circle) in B6 cells. The IC50 for capacitative Ca2+ entry and swelling-activated [Ca2+]i transient is 6 and 65 µM, respectively. Each point represents mean ± S.E. (n = 10). E, swelling-activated [Ca2+]i transient can be generated after 10 µM Gd3+ blocks Ca2+ influx through capacitative Ca2+ entry, a representative recording from 5 different experiments. Arrowheads indicate that B6 cells were treated with 2 µM TG, 10 µM Gd3+, and hypotonic solution (HYPO, 230 mosmol liter1).

However, swelling-activated [Ca²⁺]_i transient could be distinguished from the CCE. In TG-treated B6 cells, [Ca²⁺]_i rose to a peak at ~150 s with an activation rate of 1.5 ± 0.1 nM s¹ (n = 10), after [Ca²⁺]_o is replenished in isotonic solution. In contrast, [Ca²⁺]_i rose rapidly to a peak at ~80 s with an activation rate of 2.0 ± 0.1 nM s¹ (n = 10), after [Ca²⁺]_o is replenished in hypotonic solution. In TG-treated C1 cells, swelling-activated [Ca²⁺]_i transient could also be distinguished from the CCE (Fig. 7, A-C). In addition, in B6 cells, Gd³⁺ blocked Ca²⁺ entry during CCE activation more potently than Ca²⁺ influx during swelling-activated [Ca²⁺]_i transient with an IC₅₀ of 6 and 65 µM, respectively (Fig. 7D). Moreover, the swelling-activated [Ca²⁺]_i transient could be elicited after 10 µM Gd³⁺ completely inhibited the CCE activation (Fig. 7E). These results indicate that swelling-activated [Ca²⁺]_i transient and CCE represent separate pathways for Ca²⁺ 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 Ca²⁺ entry for the normal RVD response and the activation of swelling-activated Cl 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 swelling-activated Cl channel in HT-3 cells (Fig. 8D, n = 10). In contrast, the activation of swelling-activated Cl channel was not affected by BCL-2 antibody in SiHa cells (Fig. 8E, n = 10).

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Neutralization of endogenous BCL-2 by antibody blocks the normal RVD response. A, Western blotting analysis demonstrates that human cervical cancer HT-3 cells express the endogenous BCL-2, but cervical cancer SiHa cells do not. P, positive control for BCL-2 expression, taken from the stable BCL-2 transfectants (B6 cells). B and C, effects of BCL-2 antibody (0.5 µg/ml) on the time course of volume changes in response to hypotonicity for HT-3 and SiHa cells. Antibody was delivered by microinjection. The y axis (V/V0) depicts the cell volume at the indicated time divided by the cell volume at zero time. Each point represents mean ± S.E. (n = 20 cells). **, p < 0.01, compared with the volume ratio between groups without or with antibody treatment at 20 min. D and E, time courses of membrane currents activated at +80 mV or 80 mV for HT-3 and SiHa cells with or without intracellular dialysis of BCL-2 antibody. Data points were obtained from the voltage ramp protocol that was applied every 15 s. Horizontal bars indicate the application of hypotonic solution (HYPO, 230 mosmol liter1). Each point represents mean ± S.E. (n = 10).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 Ca²⁺ influx pathways mediated by swelling or store depletion. There are three lines of evidence consistent with the notion that these two Ca²⁺ influxes represent separate pathways for Ca²⁺ entry in MDCK cells. (i) In thapsigargin-treated B6 cells, [Ca²⁺]_i rose steadily to a peak at ~150 s with an activation rate of 1.5 ± 0.1 nM s¹, after [Ca²⁺]_o is replenished in isotonic solution. This [Ca²⁺]_i profile is the CCE pathway stimulated by store depletion with thapsigargin. In contrast, [Ca²⁺]_i rose rapidly to a peak at ~80 s with an activation rate of 2.0 ± 0.1 nM s¹, after [Ca²⁺]_o was replenished in hypotonic solution. This [Ca²⁺]_i profile under hypotonic solution resulted from the simultaneous activation of CCE and swelling-activated [Ca²⁺]_i transient. (ii) Gd³⁺ blocked Ca²⁺ entry during CCE activation more strongly than the swelling-activated [Ca²⁺]_i transient, with the IC₅₀ of 6 and 65 µM, respectively. (iii) The swelling-activated [Ca²⁺]_i transient could be elicited after 10 µM Gd³⁺ completely inhibited CCE activation. Swelling-activated Ca²⁺ entry is a critical signal for normal volume regulation of MDCK cells. BCL-2 overexpression results in enhanced swelling-activated Ca²⁺ entry and has a better buffering capacity for this [Ca²⁺]_i transient. Although the molecular identity is not available, the stretch-activated cation channels are presumably the route for Ca²⁺ entry in the hypotonic condition for most cell types (5, 12).

In addition to up-regulation of swelling-activated [Ca²⁺]_i transient, BCL-2 overexpression in MDCK cells enhances the capacitative Ca²⁺ entry which is thought to be essential for maintaining [Ca²⁺]_i homeostasis and may therefore be an important regulator of apoptosis during both the induction and execution phase, because both phases contain Ca²⁺-dependent components (20). In human promyeloid leukemia cells and B-cell lymphoma cells, BCL-2 overexpression results in up-regulation of capacitative Ca²⁺ entry and resistance to apoptosis induced by the inhibitor of capacitative Ca²⁺ entry (20). However, the swelling-activated [Ca²⁺]_i transient apparently does not share the same pathway with capacitative Ca²⁺ 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 membrane (23, 24). Further studies confirmed that this was because of a transient activation of K⁺ channel and a sustained activation of Cl channel (25, 26), indicating that the swelling-activated Cl channel plays a critical role in the control of RVD in MDCK cells. Activation of swelling-activated Cl channel requires the Ca²⁺ entry in MDCK cells. The amplitude and activation rate of swelling-activated Cl channel was also up-regulated by BCL-2 overexpression, thereby identifying a functional link between BCL-2 function and Cl channel activation. It is likely that there exists a cause-effect relationship between the enhancing effects exerted by BCL-2 overexpression on Ca²⁺ entry and activation of swelling-activated Cl channel. However, a direct interaction of BCL-2 with swelling-activated Cl 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 volume 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⁺, Cl, organic solutes, and obligated water (3). In many cell types, the volume regulatory effluxes of Cl and some organic osmolytes are known to be induced by swelling-induced activation of the Cl 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 swelling-activated Cl 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 swelling-activated Cl 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 channel (27, 28). The swelling-activated Cl channel has also been suggested to be involved in pH-regulatory 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 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 co-workers (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 channel (ORCC), which shares some similar characteristics with volume-sensitive Cl 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 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 volume-regulatory Cl or K⁺ 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.

	FOOTNOTES

^* This work was supported in part by National Science Council of Taiwan Grants NSC 89-2320-B-006-135 and NSC 90-2321-B006-004 (to M. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a Swire Scholarship from John Swire & Sons Ltd.

^** To whom correspondence should be addressed: Dept. of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China. Fax: 886-6-2362780; E-mail: mjtang1@mail. ncku.edu.tw.

Published, JBC Papers in Press, February 22, 2002, DOI 10.1074/jbc.M111043200

	ABBREVIATIONS

The abbreviations used are: RVD, regulatory volume decrease; IPTG, isopropyl--D-thiogalactoside; MDCK, Madin-Darby canine kidney; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; [Ca²⁺]_i, intracellular Ca²⁺; [Ca²⁺]_o, extracellular Ca²⁺; CCE, capacitative Ca²⁺ entry; TG, thapsigargin; ORCC, outwardly rectified Cl channel.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES