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Journal of Physiology (2001), 532.3, pp. 661-672
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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Hepatocyte neutral amino acid transport increases during the early period of liver regeneration (Walker & Whitfield, 1978; Wondergem & Potter, 1978). A corresponding hyperpolarization of the transmembrane electric potential (Vm) (Humphrey & Maeno, 1969; Wondergem & Harder, 1980; Wondergem, 1982; de Hemptine et al. 1985; Paloheimo et al. 1987) drives a volume regulatory Cl- efflux (Wang & Wondergem, 1992; Lidofsky & Roman, 1997). This compensates the hepatocyte swelling that results from an obligatory influx of water with amino acid uptake (Wang & Wondergem, 1993). Our aim has been to determine whether these transport events, which are attributable to metabolic demand on the liver remnant, also affect hepatocyte proliferation. Others have demonstrated that swelling-activated Cl- currents play an important role in mechanisms controlling proliferation of various cultured cells (Voets et al. 1995; Nilius et al. 1997a; Rouzaire-Dubois & Dubois, 1998).
Direct measurement of membrane Cl- conductances might well elucidate this matter in liver cells; however, such an approach is difficult. Isolated hepatocytes, which have been used extensively for metabolic studies in suspension or in primary culture, are non-proliferative yet show phenotypic change with time. Various liver cell lines can be useful in overcoming these limitations, but many are transformed or have phenotypes that differ markedly from hepatocytes. Recently, Fausto and colleagues (Wu et al. 1994) reported the establishment of the AML12 cell line, which derives from the liver of a mouse transgenically modified to include human transforming growth factor 
(TGF-). These cells are non-transformed and possess a large complement of differentiated hepatocyte traits (Wu et al. 1994).
This investigation utilized the AML12 cell line to show that hyperosmotic sucrose and various Cl- channel blockers, which respectively depolarize hepatocytes and inhibit swelling-activated membrane Cl- currents (ICl,swell), halt cell proliferation. Preliminary reports of these findings have appeared elsewhere (Wondergem et al. 1998b; Wondergem & Ecay, 1999).
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METHODS |
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Cell culture
The mouse hepatocyte AML12 cell line (Wu et al. 1994) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in 25 mm2 T-flasks with Dulbecco's modified Eagle's medium-Ham's F-12 medium (Sigma), which was supplemented with 10 % fetal bovine serum (HyClone), insulin-transferrin-selenium (ITS, Sigma), 0.1 µM dexamethasone, and gentamicin at 50 µg ml-1, and equilibrated with 95 % air-5 % CO2 in a water-jacketed tissue culture incubator. Medium was changed every other day, and cells were passaged every 5-7 days. The latter consisted of washing the cells twice with phosphate-buffered saline (PBS), incubating them for 5-7 min with porcine trypsin (1.2-2.5 mg ml-1 PBS), followed by pelleting of suspended cells (50 g, 7 min), aspirating the trypsin solution, diluting with fresh complete medium, and replating the cells.
Whole-cell voltage clamp technique
Cells were grown for 1-2 days on 4 mm 
4 mm sections of plastic coverslips, which were transferred to an acrylic chamber (Warner, New Haven, CT, USA) on the stage of an inverted microscope (Olympus IMT-2, Melville, NY, USA) equipped with Hoffman modulation contrast optics. Cells were superfused at room temperature with a standard external salt solution. The composition of this along with other solutions used throughout these experiments are given in Table 1. Cell heights were measured by Hoffman modulation contrast optics using the micrometer scale on the fine focus knob of the microscope, focusing first on the coverslip and then on the top of the cell. The microscope micrometer was calibrated in similar fashion using 2 µm and 6 µm latex beads (Sigma) placed onto the coverslip.
Borosilicate glass capillaries (1.2 mm o.d., 0.68 mm i.d., type EN-1, Garner Glass Co., Claremont, CA, USA) were cleaned in a sonicator and dried in a convection oven at 90 °C. Patch pipettes (3-8 M
in the bath solution) were fabricated from the cleaned glass with a Brown-Flaming horizontal micropipette puller (P-87, Sutter Instruments, San Rafael, CA, USA). These were coated to within 0.5 mm of the tip with polystyrene base coil dope (Polyweld 912, Amphenol, Wallingford, CT, USA), and the tips were heat polished prior to use. A micromanipulator (MO-202, Narishige, Tokyo) fixed to the microscope stage was used to position pipettes.
The whole-cell and inside-out patch configurations were obtained using the standard patch clamp technique (Hamill et al. 1981). Membrane currents were measured with a patch clamp amplifier (Axopatch 1-D, Axon Instruments, Foster City, CA, USA) with the low-pass, Bessell filtering (-3 dB) set at 2 kHz. Whole-cell currents from the patch clamp amplifier were fed into a DMA-1 digital interface connected to a 486-SX computer equipped with Clampex software (Axon Instruments). Records were stored on an internal Jaz drive (Iomega, Roy, UT,USA). Ag-AgCl half-cells constituted the electrodes, and an agar bridge (4 % w/v in external solution) connected the reference electrode to the bath solution. The junction null potential was zeroed in the cell-attached mode prior to whole-cell access when recording with symmetrical transmembrane [Cl-]. Differences in junction potentials at the reference electrode due to changes in bath [Cl-] were measured directly, and current-voltage plots were corrected accordingly. Electrode series resistances were measured following whole-cell access, but they were not compensated prior to recordings.
Measurement of cell number and mitotic index
AML12 cells were plated in triplicate in 35 mm tissue culture dishes at 1 105 cells per dish in 2 ml of medium for a 6 day analysis of cell growth. Cells were harvested as described above and then counted on a haemocytometer by trypan blue (0.04 % w/v) exclusion to determine cell growth. After 2 days the cells entered log-phase growth (see Results), at which time the medium was changed to one containing one of the Cl- channel inhibitors: 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; Sigma), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; Research Biochemicals Internat., Natick, MA, USA), tamoxifen (Sigma), or mibefradil (a gift from P. Weber, Hoffmann-La Roche LTD, Basel). Hydrophobic inhibitors were dissolved fresh daily in dimethyl sulfoxide (DMSO) for a 103 stock solution, which was diluted with culture medium to obtain the desired final concentrations. DMSO was added in equal amounts (
0.2 %) to all control samples to exclude the effects of the solvent. Mibefradil was dissolved as a 10 mM stock solution in 0.9 % saline and diluted in medium to give the required concentrations.
AML12 cells in log phase growth on glass coverslips, which had been demarcated previously into quadrants, were photographed (one image per quadrant) with a digital camera (Kodak DC 120) attached to an inverted microscope (Olympus IMT) with phase contrast optics at 200 magnification. The flat cells were focused as dark phase objects and, thus, they were readily distinguished from the thicker round cells, which appeared as bright phase objects. Digitized images were stored on computer disk (Zipdisk, Iomega). Later they were imported into Adobe Photoshop (v. 4.0.1), and the images were overlaid with a grid to facilitate counting. All cells in the field were counted and designated as either flat or round. Cells were scored as mitotic if they were observed to be in either prophase, metaphase, anaphase or telophase. The mitotic index for flat and round cells was the percentage of cells in mitosis for each group.
Protein measurements
AML12 cells were plated in 96-well tissue culture plates at 1-2 104 cells per well. After 2 days the medium was aspirated and replaced with either control (DMSO) medium or medium plus increasing concentrations of the three Cl- channel inhibitors. Two days later the medium was aspirated and the plate was rinsed with 0.2 ml per well of ice-cold PBS (pH = 7.41). Each well then received 0.2 ml of 0.25 N NaOH, and the plate was shaken slowly for 4 h to solubilize cellular protein. The amount of protein in each well was determined by the BCA protein assay (Pierce, Rockford, IL, USA; Smith et al. 1985) using bovine serum albumin as the standard.
Measurement of adenosine triphosphate (ATP)
AML12 cells nearly confluent in 60 mm tissue culture dishes were treated for 48 h with either DMSO (control), NPPB (40 µM), DIDS (31 µM), tamoxifen (1.3 µM), or 2,4-dinitrophenol (500 µM for 1 h). Doses of Cl- channel blockers were selected based on the IC50 for growth inhibition (see Results). The medium was removed and replaced with 300 µl of 5 % perchloric acid-1 mM EDTA, and the cells were scraped from the dish. This was repeated; the scrapings were combined and centrifuged at 12 000 g for 30 s; the supernatants were decanted and saved for ATP/ADP measurements; and the acid insoluble pellet was dissolved in 300 µl of 0.25 N NaOH for protein determination (BCA; Pierce).
ATP and ADP concentrations were measured by high performance liquid chromatography (HPLC) according to Hill et al. (1987). An aliquot of supernatant (400 µl) was mixed with 1.5 M KHCO3 (160 µl). The KClO4 was pelleted by centrifugation at 12 000 g for 30 s, and 10 µl samples of the supernatants were injected into an HPLC radial compression column (Waters Nova-Pak C18; 5 100 mm; 4 µm; Waters Corp., Milford, MA, USA). The HPLC system included a Waters M-45 solvent delivery system, a 717-plus autosampler, a Lambda Max Model-480 LC spectrophotometer, and a 746-Data Module recording integrator. Adenine nucleotides were eluted at 0.5 ml min-1 using a mobile phase of 0.1 M sodium phosphate, pH 6, plus 4 % (v/v) methanol; they were detected at 259 nm and quantified by interpolation using external ATP-ADP (Sigma) combined standards ranging from 0.03 to 0.5 mM. The linear correlation coefficients for standard plots of peak area vs. concentration for both ATP and ADP were r2 > 0.999. The average elution times for the ATP and ADP peaks were 3.2 ± 0.01 and 3.6 ± 0.01 min (n = 5), respectively.
Data analysis
Cell growth rates in control or inhibitor-treated cells were compared by plotting log10 cell number versus days in culture. Slopes (regression coefficients, b) of linear functions fitted to these plots were determined by regression analysis using the method of least squares. The significance of b was determined first by rejecting the null hypothesis (H0) that b = 0 at P < 0.05. Growth rates of cells in experimental treatment groups differed from those of the control if the numerical values of the experimental slopes were outside the 95 % confidence interval of the control slope. Dose effects of Cl- channel blockers on cell protein accumulation were determined by fitting protein data versus log10 drug concentration to a sigmoidal function by means of an iterative, non-linear curve fit (SlideWrite; Advanced Graphics Software, Inc., Encinitas, CA, USA), which employed a Levenberg-Marquardt algorithm. Inhibitor concentrations producing half-maximal growth inhibition (IC50) were computed from these fitted curves. Differences among means were determined by ANOVA combined with the Student-Newman-Keuls or the least-significant-difference multiple comparison tests at P < 0.05.
The permeability ratios PX/PCl, where X- was the substituted anion, were computed according to the modified Goldman-Hodgkin-Katz (GHK) equation:
where 
Erev is the difference in reversal potentials of the I-V plots for Cl- and X- following the anion substitution, [Cl-]n and [Cl-]s are the Cl- concentrations in the normal and substituted external hypotonic solutions, [X-]s the concentration of the substituted anion, F is Faraday's constant, R the gas constant, and T the absolute temperature.
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RESULTS |
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AML12 cell membrane chloride currents
AML12 cells adhered to the culture substratum within hours of passage. They appeared initially as single cells that were loosely attached. As the cells proliferated over 48 h, they formed islands of flattened cells with a mean height of 3.3 ± 0.2 µm (n = 30). This was significantly less (P < 0.001) than the mean height of 10.0 ± 0.4 µm (n = 30) for round cells, which were interspersed among the flat cells. Cells in log-phase growth (> 48 h) were scored under phase contrast microscopy as either flat or round. Of 1513 cells counted, 1352 (89.4 %) were flat, and 161 (10.6 %) were round. Cytokinesis was readily evident among the round cell population, and 74/161 (46 %) were mitotic. In contrast, 35/1352 (2.6 %) of the flat cells were mitotic. Round cells were not observed in dishes of confluent cells, which comprised a sheet of very flat, tightly juxtaposed cells.
AML12 cells readily formed gigohm seals with patch pipettes, and seal resistance was 2.4 ± 0.2 G (mean ± S.E.M.; n = 31). Whole-cell access of round cells was obtained by mild suction, and average values for pipette access resistance, whole-cell resistance and cell capacitance were, respectively, 8.6 ± 1.3 M, 491 ± 95 M and 25.6 ± 1.3 pF (means ± S.E.M.; n = 31).
Whole-cell recordings of membrane current, which were taken immediately after accessing the cytoplasm and dialysing most (~80 %) round dividing cells, yielded linear current-voltage relationships in response to voltage steps from -100 to 80 mV (Fig. 1A, Bath). The patch pipettes for this and subsequent measurements of hypotonic stress-activated current contained internal solution plus 1-2 mM ATP. We noted in some instances that outward current increased with time (> 20 min) after achieving the whole-cell recording mode, and this current was inhibited either by addition of 100 µM DIDS or by substituting external Cl- with equimolar gluconate (S. H. Monen & R. Wondergem, unpublished observations). Hence, we postulated that cell swelling following dialysis of the cytoplasm activated an anionic current in AML12 cells, as had been reported for other epithelial cells (Worrell et al. 1989; Solc & Wine, 1991). To test this, we measured whole-cell currents before and after subjecting AML12 cells to hyposmotic stress by reducing the external NaCl concentration by 40 mM (from 280 to 221 mosmol kg-1 with change in ionic strength, 
). This activated an outwardly rectifying current, (Fig. 1A, Hypotonic) which took 10 min to reach maximal levels (results not shown). The outward current often inactivated rapidly at intracellular potentials 
60 mV (Fig. 1A, Hypotonic). The extent of inactivation varied considerably among cells, and even within the same cell during repeated measurements (not shown). Hypotonic stress activation of the membrane current reversed after restoring the control osmolality (Fig. 1A, Washout). Current-voltage (I-V) relationships obtained from data points taken at the end of the voltage pulses showed similar results with repeated measurements from different cells (Fig. 1B). The shift in I-V reversal potential from 0 mV (Bath) to 8 mV (Hypo ()) was consistent with that predicted by the 7 mV change in the transmembrane Nernst equilibrium potential for Cl- with the hypotonic solution (Table 1). This indicated that hypotonic stress of AML12 cells activated an anionic current.
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Figure 1. Effect of hypotonic stress on whole-cell Cl- current in round dividing AML12 cells A, whole-cell currents recorded during voltage pulses ranging from -100 to 80 mV in consecutive, 20 mV increments. Arrows indicate current at a holding potential of -50 mV. Bath, hypotonic, and washout traces recorded in the same cell. Hypotonic stress trace recorded 10 min after reducing external NaCl by 45 mM. Time and current scale bars apply to all traces. B, summary current-voltage (I-V) plots in which currents were measured from single points toward the end of the voltage steps. Hypo (
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Hypotonic stress was also administered without altering the external ionic strength. Cells were equilibrated with external solution in which 80 mM sucrose was substituted for 40 mM NaCl (280 mosmol kg-1, Table 1). Hypotonic stress achieved by eliminating the sucrose (221 mosmol kg-1 with constant ionic strength, 
) activated inward anionic current identical to that effected by hypotonicity with decreased ionic strength (Fig. 1B). The apparent larger outward current from this hypotonicity resulted from less current inactivation under these conditions since currents were measured at the end of the voltage pulses (Fig. 1B).
Various Cl- channel blockers also inhibited hypotonic stress-activated anionic current. Results from repeated measurements in different cells (n = 3) are shown in Fig. 2 as I-V plots of currents acquired at the end of the voltage pulses. Addition of DIDS (100 µM) inhibited completely the membrane outward current induced by hypotonic stress (Fig. 2A). This inhibition reversed when the DIDS was washed out (not shown). NPPB (50 µM) and tamoxifen (10 µM) also inhibited hypotonic stress-activated outward current along with inward current (Fig. 2B and C). NPPB inhibition reversed completely and immediately after the drug washout, whereas tamoxifen inhibition reversed partially only after 1 h of the drug washout (not shown). Since the I-V plots generated following the addition of the latter two agents were linear throughout the voltage range measured, we plotted the cell specific conductance (nS pF-1 at Vm = 40 mV) versus NPPB doses, and versus tamoxifen doses (plots not shown). The IC50 for NPPB inhibition of the cell Cl- conductance was 2.3 µM, and the IC50 for tamoxifen inhibition of the cell Cl- conductance was 0.7 µM. To exclude the possibility that secondary effects of these hydrophobic agents accounted for their inhibition of ICl,swell, mibefradil, which is a hydrophilic Ca2+ channel antagonist that has been shown also to block ICl,swell (Nilius et al. 1997b), was added to the hypotonic solution. Mibefradil at 10 µM also inhibited ICl,swell (Fig. 2D), and the IC50 was 6.3 µM.
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Figure 2. Effects of Cl- channel inhibitors and anion substitution on hypotonic stress-activation of membrane Cl- current Current-voltage (I-V) plots for whole-cell currents recorded in AML12 cells during voltage pulses ranging from -100 to 80 mV in consecutive 20 mV increments. Holding potential = -50 mV. Currents were obtained from single points taken at the end of the voltage steps. A, effect of DIDS (100 µM) in the hypotonic solution. B, effect of NPPB (50 µM) in the hypotonic solution. C, effect of tamoxifen (10 µM) in the hypotonic solution. D, effect of mibefradil (10 µM) in the hypotonic solution. (Means ± S.E.M.; n = 3 cells).
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We determined the anionic selectivity of the swelling-activated outward current by replacinging 95 mM NaCl (external hypotonic, Table 1) with Na[X], where X- represents the substituted anions SCN-, I-, Br-, or gluconate. Membrane current traces in response to voltage ramps from -100 to 100 mV (applied over 3 s) were recorded from the same cell for each anion substitution. The relative anion conductances, measured by slope conductance at the reversal potentials, were SCN- I- Br- Cl- > gluconate (Table 2). The permeability sequence obtained from reversal potentials was SCN- > I- > Br- > Cl- > gluconate, and the corresponding relative permeabilities from the GHK equation are presented in Table 2.
Some of the round dividing cells (~20 %) had outwardly rectifying membrane currents immediately on achieving the whole-cell configuration (Fig. 3A). These currents at positive voltages showed time-dependent activation, and they either did not inactivate or showed only modest inactivation. Subsequent application of hypotonic stress (280-221 mosmol kg-1) to these cells induced no further increase in outward current (not shown). However, hyperosmotic stress (280-330 mosmol kg-1) induced by superfusing the cells with external solution containing 50 mM sucrose markedly reduced this current (Fig. 3A). In contrast, I-V plots from whole-cell currents recorded in flat, tightly juxtaposed AML12 cells were linear throughout, with no outward rectification (not shown). Moreover, hyposmotic stress applied to the flat AML12 cells did not further activate the membrane Cl- current, as occurred with the round dividing cells, at least not within 10-12 min following the onset of hypotonic stress. The slope conductance (nS pF-1) for flat cells following 12 min of hypotonic solution (221 mosmol kg-1) was 0.60 ± 0.16 nS pF-1 (n = 6), which did not differ (P = 0.35) from that of 0.54 ± 0.11 nS pF-1 (n = 6) for control solution (280 mosmol kg-1). In spite of the smaller capacitance of the flat cells, 11.5 ± 4.4 pF (n = 6), their specific conductances did not differ from those of dividing cells of 0.44 ± 0.09 nS pF-1 (n = 7).
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Figure 3. Whole-cell Cl- current activated spontaneously in dividing AML12 cells A, whole-cell currents obtained immediately on whole-cell access of a round dividing cell and of the same cell after superfusing with hypertonic medium created by adding 50 mM sucrose to external solution containing 135 mM NaCl. Whole-cell currents recorded during voltage pulses ranging from -100 to 80 mV in consecutive, 20 mV increments. Arrows indicate current at a holding potential of -50 mV. Time and current scale bars apply to all traces. B, summary current-voltage (I-V) plots for whole-cell currents obtained from round dividing cells before and after hypertonic solution. Symbols and bars represent means ± S.E.M. (n = 3 cells).
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Growth measurements
AML12 cells entered the log-phase of cell proliferation 2 days after they were plated with medium containing 10 % FBS. The cells grew significantly more slowly in medium lacking serum. The rate of cell proliferation in medium containing serum was reduced significantly by adding increasing concentrations of each of the Cl- channel inhibitors used to inhibit hypotonic stress-activated Cl- current in AML12 cells: NPPB (50 and 100 µM, Fig. 4A); DIDS (100 and 250 µM, Fig. 4B); tamoxifen (0.5 and 5 µM, Fig. 4C); and mibefradil (5 and 10 µM, Fig. 4D). The growth rate of AML12 cells treated with 50 µM NPPB had accelerated 2 days after washing out the NPPB (Fig. 5A), and the same effect was achieved in cells treated with of 10 and 12.5 µM mibefradil (Fig. 5B). In contrast, neither the DIDS- nor the tamoxifen-induced growth inhibition reversed after the drug washout.
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Figure 4. Effect of Cl- inhibitors on growth rates of AML12 cells in monolayer tissue culture A, effect of NPPB; B, effect of DIDS; C, effect of tamoxifen; D, effect of mibefradil. Drugs were added on day 2. Values are means ± S.E.M. (n = 3 tissue culture dishes). * Growth rate differs from that of control, P < 0.05.
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Figure 5. Effect of washout of Cl- inhibitors on growth rates of AML12 cells in monolayer tissue culture A, cells treated with NPPB. Control, cells grown in 10 % FBS; NPPB, cells grown in 10 % FBS plus 50 µM NPPB added on day 2; NPPB Wash, cells grown in 10 % FBS plus 50 µM NPPB added on day 2 and washed out on day 4. Values are means ± S.E.M. (n = 3 tissue culture dishes). * Differs from Control and NPPB, P < 0.02; ** differs from Control, P < 0.001. B, cells treated with mibefradil. Control, cells grown in 10 % FBS; Mibefradil, cells grown in either 10 µM or 12.5 µM mibefradil added on day 2; Mibefradil Wash, cells grown in 10 % FBS plus either 10 or 12.5 µM mibefradil added on day 2 and washed out on day 4. * Differs from Control and Mibefradil, P < 0.02; ** differs from Control, P < 0.001. C, cells treated with sucrose. Control, cells grown in 10 % FBS; 50 mM sucrose, sucrose added on day 4 to growth medium plus 10 % FBS; 100 mM sucrose, sucrose added on day 4 to growth medium plus 10 % FBS. * Regression coefficient (b) for number of cells vs. time following added sucrose was not different from zero. H0: b = 0, P > 0.05.
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We measured the effect of increasing doses of each of the hydrophobic Cl- channel inhibitors on protein accumulation in AML12 cells after 2 days to determine the half-maximal inhibitory doses (IC50). Cells were plated in 96-well plates and allowed to attach and grow for 48 h. At this time cells in some wells were harvested for later protein measurement (time zero) and either DMSO (vehicle control) or Cl- channel inhibitors were added to the remaining wells for an additional 48 h. Growth rates in the presence of increasing concentrations of Cl- channel inhibitors were compared with control growth, and the IC50 values for NPPB, DIDS, tamoxifen and mibefradil were 40 µM, 31 µM, 1.3 µM and 7 µM, respectively. Only in the case of DIDS, at 250 µM, did the amount of protein accumulated after 2 days fall below that of cells seeded at time zero, P < 0.05.
To exclude the possibility that secondary effects of the Cl- channel blockers accounted for their inhibition of hepatocyte growth, we measured cell proliferation in the presence of hyperosmotic conditions that were created by adding sucrose to the medium. We had shown previously that hyperosmotic stress depolarizes the Vm of mouse hepatocytes (Howard & Wondergem, 1987; Wang & Wondergem, 1991), and increases their intracellular chloride activities (Wang & Wondergem, 1992). Rouzaire-Dubois & Dubois (1998) have shown that sustained hyperosmolarity in neuroblastoma cells effects a paradoxical increase in cell volume. Thus, our rationale was that hyperosmotic sucrose would depolarize AML12 cell Vm, increase intracellular Cl- activity, and thereby swell the cells. Sucrose, added to the complete medium on day 4 at either 50 or 100 mM stopped proliferation of AML12 cells in log-phase growth (Fig. 5C). The regression coefficients (b) for growth curves (days 4-6) for sucrose treated cells were not different from zero (P > 0.2 and 0.5 for H0: b = 0). In contrast, the untreated cells grew normally.
ATP and ADP measurements
It was necessary to determine whether the hydrophobic, membrane-permeable drugs used in this study affected AML12 cell ATP content, since ATP is required for volume-activated Cl- channels (Jackson et al. 1994) for phosphorylation-dependent cell signalling, or for the metabolic requirements of cell division. The ATP/ADP amounts in AML12 cells following 48 h treatments with either NPPB, DIDS, or tamoxifen at the IC50 for growth inhibition are shown in Table 3. Neither NPPB nor tamoxifen treatments of AML12 cells for 48 h significantly reduced cellular ATP content. However, 48 h treatments with DIDS or 1 h treatment with 2,4-dinitrophenol (2,4-DNP at 500 µM) significantly reduced cellular ATP amounts along with corresponding increases in the quantity of ADP.
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DISCUSSION |
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The Cl- channel blocking agents used in these experiments inhibit cell proliferation and growth at doses comparable to those that inhibit swelling-activated Cl- currents in AML12 cells. Taking the data together, we conclude that membrane Cl- currents in mouse hepatocytes play a role in mechanisms controlling cell proliferation. Thus, membrane hyperpolarization (Humphrey & Maeno, 1969; Wondergem & Harder, 1980; Wondergem, 1982; de Hemptine et al. 1985; Paloheimo et al. 1987) and volume regulatory Cl- effluxes (Wang & Wondergem, 1992), which heretofore have been attributed to compensatory metabolic demand on the liver remnant following partial hepatectomy, also play a role in compensatory hepatic growth.
Hypotonic stress to round AML12 cells, but not to flattened cells, elicits an outwardly rectifying Cl- current. Moreover, a similar Cl- current is evident immediately on accessing the cytoplasm in ~20 % of the round cells. Since the mitotic index of the round cells was considerably greater than that of the flat cells, we conclude that these AML12 cells, like many cultured cells, loosen their attachment to the substratum and round up when they divide. Rounding may in part reflect osmotic swelling from substrate accumulation. However, it is likely that activation of volume-activated Cl- current ensues during certain phases of the cell cycle and cell rounding. We conjecture that kinetic changes in the equilibria of cytoskeletal elements, e.g. the dynamic instability of microtubules and 'treadmilling' of actin filaments, yield increases in cytoplasmic concentrations of either monomeric tubulin or actin at critical instances during shape change. These could effect an increase in intracellular osmolality and alter osmotic balance, which if uncompensated would result in swelling. In either case, activation of the Cl- current at these times constitutes part of a physiological volume regulatory response. If the Cl- current is blocked, the AML12 cells do not proliferate. Shen et al. (2000) accordingly reported that blocking ICl,swell in human cervical cancer cells arrests proliferation in the G0/G1 stage of the cell cycle.
Volume-activated Cl- currents (ICl,swell) have been identified with the family of voltage-sensitive Cl- channels, i.e. ClC-2 (Gründer et al. 1992) and ClC-3 (Duan et al. 1997). Their molecular identification is still uncertain (Clapham, 1998; Strange, 1998), and we have yet to characterize the channels that account for the volume-activated Cl- current in AML12 cells. Nevertheless, based on its outward rectification, rapid inactivation at Vm > 60 mV, anion selectivity and sensitivity to Cl- channel blockers, the hypotonic stress-activated anionic current in AML12 cells is consistent with the ICl,swell, which has been reported for numerous cells (Nilius et al. 1996; Strange et al. 1996; Okada, 1997) and has been linked to ClC-3 (Duan et al. 1997). Similar swelling-activated Cl- currents have been found in isolated hepatocytes (Meng & Weinman, 1996) and the HTC hepatoma cell line (Bodily et al. 1997).
We note that the spontaneous outwardly rectifying current in round dividing cells differs from that induced by hypotonic stress in its time-dependent activation and the absence or modest level of inactivation at Vm > 60 mV. We do not know if this reflects different Cl- channels or different functional states of the same Cl- channel. Calcium-activated membrane Cl- currents rectify outwardly without inactivation at positive Vm (Meyer & Korbmacher, 1996; Pedersen et al. 1998). However, the free [Ca2+] in the pipette-filling solution was computed to be 18 nM (Chang et al. 1988), which is below the threshold for Ca2+ activation of Cl- channels in other cells (Nilius et al. 1997b; Pedersen et al. 1998). Inactivation is a sine qua non in identifying ICl,swell. Nonetheless, the degree of inactivation varies among cells (Nilius et al. 1996), within batches of the same cells (Strange et al. 1996), and with extracellular pH and ionic concentrations (Voets et al. 1997a). Since intracellular ionic strength modulates the volume sensor for activation of ICl,swell (Cannon et al. 1998; Nilius et al. 1998; Voets et al. 1999), it is possible that the inactivation kinetics of ICl,swell varies with ionic strength differences across the plasma membrane. The latter are substantial for swelling effected by a decrease in external NaCl concentration; however, swelling effected by a rise in intracellular organic osmolyte concentration provides only a small ionic strength difference across the plasma membrane. We have shown that anionic current inactivation in AML12 cells diminishes with hypotonicity induced without a change in ionic strength across the plasma membrane. Thus, the differences in inactivation of anionic current in hypotonically stressed dividing cells compared with those showing spontaneous activation may reflect the response of ICl,swell to ionic strength differences across the plasma membrane. However, we cannot exclude the involvement of additional Cl- channels in accounting for the various time-dependent responses of Cl- currents at positive voltages.
We cannot state with certainty that Cl- currents in flattened cells do not respond to hypotonic stress, only that they do not activate within the same time span as do the round dividing cells. We note that the magnitude of the specific anionic conductance of the flat cells compared well with that of hyposmotically stressed dividing cells. However, the former did not rectify outwardly nor inactivate with voltage. This could have resulted from expression of a different membrane Cl- channel in flat AML12 cells. Voets et al. (1997b) reported on the downregulation of volume-activated Cl- currents during muscle cell differentiation. In spite of their proliferative capability, AML12 cells retain many differentiated hepatocyte phenotypes, such as connexins and LDH enzyme isoforms (Wu et al. 1994). Dumenco et al. (1995) also reported that serum-free medium enhanced the differentiated function of albumin secretion and slowed growth of AML12 cells. It is possible that the appearance of another Cl- current in the flat AML12 cells is consistent with downregulation of ICl,swell with cell differentiation, as seen in muscle cells.
ICl,swell currents are either active spontaneously in some of the round mitotic AML12 cells, or they are elicited in these cells by hypotonic stress. Inhibiting these currents with Cl- channel blockers or with hyperosmotic sucrose suppresses cell proliferation. Each treatment swells cultured neuroblastoma cells (Rouzaire-Dubois & Dubois, 1998) either by blocking volume regulatory ionic currents or by sustaining volume regulatory increase in the case of prolonged hyperosmolarity. Rouzaire-Dubois & Dubois (1998) also reported that swelling of neuroblastoma cells by either means lowers their rate of proliferation. Thus, uncompensated cell swelling inhibits cell proliferation. NPPB, tamoxifen and mibefradil accordingly suppress swelling-activated Cl- currents and proliferation in bovine endothelial cells (Voets et al. 1995; Nilius et al. 1997a). NPPB and DIDS also inhibit small conductance Cl- channels and phytohaemagglutinin induced proliferation in human T lymphocytes (Phipps et al. 1996). Bubien et al. (1990), using human lymphocytes, were the first to report that membrane Cl- permeability increases with the G1/S phase of the cell cycle.
NPPB inhibited Cl- current and cell growth without lowering cellular ATP levels. Moreover, this inhibition reversed with washout of the drug. Thus, we exclude metabolic inhibition in accounting for the inhibitory effects of NPPB on AML12 cells. This is of particular importance in relating ICl,swell currents to mechanisms regulating cell proliferation, since ICl,swell is an ATP-dependent Cl- current (Jackson et al. 1994). The specificity of tamoxifen treatment, however, is less unequivocal. Anti-oestrogen effects of tamoxifen are well established, and they account in part for tamoxifen-induced inhibition of rat liver regeneration (Francavilla et al. 1989). Tamoxifen also blocks voltage-dependent K+ channels and cell proliferation in neuroblastoma cells (Rouzaire-Dubois & Dubois, 1990), and it inhibits acidification of cells independently of the oestrogen receptor (Altan et al. 1999). The results obtained using DIDS are also less certain. DIDS block of anionic current in AML12 cells was voltage dependent, occurring only at positive voltages, above the physiological Vm. DIDS also lowered the cellular ATP content, and at a high dose it decreased the total protein in the proliferative assay below that of the control at time zero. Both results indicate metabolic toxicity, which may result in cell death and loss. In addition, inhibition of growth either by DIDS or by tamoxifen did not reverse with washout of the drug. Ballatori and colleagues (Ballatori et al. 1995; Ballatori & Wang, 1997) reported that NPPB markedly decreases cellular ATP levels in skate hepatocytes and HepG2 cells, whereas DIDS has no effect. We cannot explain this disparity between our findings and theirs, except to point out that we used different cells and longer exposure to the inhibitors.
The IC50 values for inhibition of ICl,swell by tamoxifen and mibefradil compare well with values for inhibition of cell proliferation. This suggests that each agent effects both actions through a common mechanism. In contrast, the IC50 for NPPB-induced inhibition of cell proliferation was an order of magnitude greater than that of ICl,swell. The former is most probably a high estimate, because serum was present in the growth medium (although it was not present in the electrophysiological studies). Serum proteins readily bind > 90 % of NPPB (Fryklund et al. 1993), which reduces drug availability and thus increases the concentration required for the drug to be effective. Serum proteins similarly bind glibenclamide, which increases the concentration required for effective inhibition of growth of human bladder tumour cells compared with that needed to block K+ channels in serum-free conditions (Monen et al. 1998; Wondergem et al. 1998a).
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[Abstract] |
Acknowledgements
This work was supported by a TN-SBR scholarship to S.H.M., a grant from the American Heart Association (TN97N74) to T.W.E. and a grant from the Cancer Research Group of ETSU to R.W. We thank M. Miyamoto for evaluating the manuscript.
Corresponding author
R. Wondergem: Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70,576, Johnson City, TN 37614-0576, USA.
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