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【摘要】 Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K +, Cl -, and H 2 O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na + reabsorption via elevation of epithelial Na + channel ( -ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl - concentration ([Cl - ] i ) stimulated the Na + reabsorption. These suggest that RVD would reveal its stimulatory action on the Na + reabsorption by reducing [Cl - ] i. However, the reduction of [Cl - ] i during RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl - ] i change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N -(6-methoxyquinolyl) acetoethyl ester. The [Cl - ] i in A6 cells in an isotonic medium was 43.6 ± 3.1 mM. After hypotonic shock (268 to 134 mosmol/kgH 2 O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl - ] i from 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl - channel blocker) or quinine (K + channel blocker), we did not detect the reduction of [Cl - ] i. Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl - ] i and that RVD shows its action via reduction of [Cl - ] i acting as an intracellular signal regulating cellular physiological functions.
【关键词】 A epithelial cell cell volume N (methoxyquinolyl) acetoethyl ester highresolution flow cytometer Cell Lab Quanta
THE RENAL EPITHELIA ARE AMONG the tissues routinely exposed to variable extracellular osmolalities under physiological conditions that require cells to maintain appropriate cell volume and intracellular osmolality. Hypotonicity causes a biphasic change in cell volume; i.e., initial cell swelling followed by regulatory volume decrease (RVD) returning its cell volume toward the original one. The mechanism generating RVD is well understood; i.e., KCl release occurs through volume-sensitive K + and Cl - channels. Although this KCl efflux may be thought to affect the intracellular Cl - concentration ([Cl - ] i ), no evidence is available on [Cl - ] i during RVD. The chloride ion is characterized as a major physiological anion in living tissues and is critically involved in the regulation of cell volume, intracellular pH, acid-base balance, and fluid secretion ( 41 ). In particular, the change in [Cl - ] i has been reported to modify gene expression, activity of some proteins, and activity of ion channels; e.g., the intracellular Cl - regulates the cyclooxygenase (COX)-2 gene expression in macula densa cells ( 8, 44 ), the activity of the epithelial Na + channel (ENaC) ( 25 ), the activity of Na + -K + -2Cl - cotransporter (NKCC) in dog tracheal cells ( 19, 20 ) and in human trabecular meshwork cells ( 35 ). Moreover, our recent study ( 34 ) suggests that the intracellular Cl - could act as a signal regulating Na + reabsorption through changes in -subunit of the ENaC ( -ENaC) mRNA expression in renal epithelial A6 cells via activation of a pathway dependent on protein tyrosine kinases. This result indicates that a change of [Cl - ] i is one of the important intracellular signals for regulation of cellular function. Despite the importance of the Cl - for many cellular functions, no accurate information on [Cl - ] i during RVD is available. This means that we have no idea whether a change in [Cl - ] i occurs during RVD. This lack of information is primarily due to technical difficulties in the measurement of [Cl - ] i during RVD. The [Cl - ] i has been tried to be measured by using conventional techniques such as nuclear magnetic resonance ( 6 ), microelectrode ( 1, 23 ), X-ray probe electron microanalysis ( 18, 36 ), or 36 Cl - tracer ( 14 ). However, these methods are limited to measure [Cl - ] i, because of the technical expertise required to make reliable measurements and the imperfect selectivity and sensitivity to Cl -. There are other approaches for measurements of [Cl - ] i using quinoline-based halide-sensitive fluorescent dyes sensitive to Cl - such as 6-methoxy-1-(3-sulfopropyl) quinolinium (SPQ), N -(6-methoxyquinolyl) acetoethyl ester (MQAE), and 6-methoxy- N -ethylquinolinium (MEQ). These dyes have a high selectivity to Cl - and have been used for measurements of [Cl - ] i in a variety of preparation such as neurons, glia, fibroblast, and different types of epithelial and endothelial cells ( 4, 5, 13, 37, 43 ). However, there are difficulties and problems in the use of these indicators for measurement of [Cl - ] i during RVD. This complication is a change of intracellular dye concentration during RVD, since fluorescent intensities of these indicators depend not only on [Cl - ] i, but also on the intracellular concentration of dye. Namely, these Cl - indicators lack the Cl - -dependent change in spectra shape precluding ratio-metric measurements. These problems described above indicate that although the [Cl - ] i would be correctly measured under a cell-volume-unchanged condition, the [Cl - ] i cannot be accurately determined under a cell-volume-changeable condition such as RVD.
In this study, we developed a new method for measuring [Cl - ] i by using a unique flow cytometer, Cell Lab Quanta (Beckman Coulter, Fullerton, CA), to overcome the problem of [Cl - ] i measurement during RVD by using a fluorescent Cl - indicator, MQAE. This flow cytometer enables us to simultaneously measure a cellular fluorescent intensity and exact cell volume by Coulter principle ( 9 ). Therefore, we can correct the fluorescent intensity of MQAE by using the data of cellular volume during RVD, getting the exact [Cl - ] i without any effects of changes in MQAE concentration caused by cell volume changes. In the present study, we demonstrated that the [Cl - ] i was drastically decreased during RVD. This remarkable reduction of [Cl - ] i was blocked by inhibition of RVD with NPPB (Cl - channel blocker) or quinine (K + channel blocker). These results suggest that a change in the extracellular osmolality is converted into the change in [Cl - ] i, that the change of [Cl - ] i may be one of the primary hypotonic signals, and that a physiological significance of RVD is reduction of [Cl - ] i.
MATERIALS AND METHODS
Materials. NCTC-109 medium and MQAE were purchased from Invitrogen (Carlsbad, CA). Cl - -substituted NCTC-109 with NO 3 salts was obtained from Cell Science and Technology Institute (Sendai, Japan). Nigericin, tributyltin chloride, and carbonyl cyanide 3-chloropenylhydrazone (CCCP) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Quinine, valinomycin, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) were purchased from Sigma (St. Louis, MO).
Cell preparation. Renal epithelial A6 cells derived from Xenopus laevis were purchased from American Type Culture Collection (Manassas, VA). A6 cells ( passages 76-84 ) were grown on plastic culture flasks in culture medium modified for amphibian cells, which contained 75% (vol/vol) NCTC-109 medium, 15% (vol/vol) distilled water, and 10% fetal bovine serum (osmolality 265 mosmol/kgH 2 O) ( 15, 39 ). The flasks were kept in a humidified incubator at 27°C with 1.0% CO 2 in air. For [Cl - ] i measurements, cells were seeded on 25-cm 2 plastic culture flask at a density of 5.0 x 10 4 cells/flask and were cultured for 14 days. For measurement of water content in A6 cells, cells were seeded on 75-cm 2 flasks at density of 1.5 x 10 5 cells/flask and cultured for 14 days.
Solutions. The 120 mM NaCl isotonic medium (NaCl buffer) contained 120 mM NaCl, 3.5 mM KCl, 1.0 mM CaCl 2, 1.0 mM MgCl 2, 5.0 mM glucose, and 10 mM HEPES. The 120 mM NaNO 3 solution (NaNO 3 buffer) contained 120 mM NaNO 3, 3.5 mM KNO 3, 1.0 mM Ca(NO 3 ) 2, 1.0 mM Mg(NO 3 ) 2, 5.0 mM glucose, and 10 mM HEPES. For calibration of intracellular MQAE fluorescent intensity against [Cl - ] i, the 0 mM Cl -, 10 mM NaNO 3, 105 mM KNO 3, 1.0 mM Ca(NO 3 ) 2, 1.0 mM Mg(NO 3 ) 2, 8.5 mM CsNO 3, 5.0 mM glucose, and 10 mM HEPES, the 100 mM Cl - [10 mM NaNO 3, 100 mM KCl, 5.0 mM KNO 3, 1.0 mM Ca(NO 3 ) 2, 1.0 mM Mg(NO 3 ) 2, 8.5 mM CsNO 3, 5.0 mM glucose, and 10 mM HEPES], and the KSCN [120 mM KSCN, 3.5 mM KCl, 1.0 mM Ca(NO 3 ) 2, 1.0 mM Mg(NO 3 ) 2, 5.0 mM glucose, and 10 mM HEPES] buffers were prepared. CsNO 3 was used for adjusting osmolality in each buffer without affecting other ionic compositions for calibration of Cl -. The osmolality of buffers was 268 mosmol/kgH 2 O. All solutions used in the present study were adjusted to pH 7.40.
Measurement of water volume. To detect the exact concentration of intracellular MQAE, we measured the intracellular water (MQAE accessible) content of A6 cells. Cells were seeded on 75-cm 2 plastic culture flasks at a density of 1.5 x 10 5 cells/flask and were cultured for 14 days. Cells were washed with PBS and then detached from the flask by using Trypsin-EDTA. A6 cells were transferred to 10-ml glass centrifuge tubes and then centrifuged at 800 rpm for 5 min. After the centrifugation, the supernatant was discarded and tubes were weighed to determine the wet cell volume. To measure the dry weight, A6 cells were subsequently dried to reach the constant weight over a period of 24 h in a 90°C oven.
Measurements of cell volume and fluorescence. Cell volume and fluorescence measurements were performed by using a high-resolution flow cytometer, Cell Lab Quanta (Beckman Coulter). This flow cytometer is designed to simultaneously measure the fluorescent intensity and the electronic volume (EV) ( 26 ). The EV measured with the Coulter Principle ( 9 ) is much more reliable and accurate than the volume detected by forward angle light scattering (FALS) analyzes by the conventional flow cytometer. The MQAE was excited by using an Hg lamp with 360-nm wavelength. The fluorescent intensity of MQAE was measured by using 460-nm band-pass filter. All data on EV and fluorescent intensity were analyzed by Quanta control software. EV and MQAE fluorescence of more than 10,000 cells were collected.
MQAE loading and intracellular calibration of intracellular Cl - concentration. A6 cells were incubated with MQAE (10 mM) in a Cl - -substituted NCTC-109 medium for 12 h at 27°C in a CO 2 incubator (1% CO 2 ). Calibration of the fluorescence in terms of [Cl - ] i was accomplished with 5 µM nigericin (K + /H + ionophore), 5 µM valinomycin (K + ionophore), 5 µM CCCP (protonophore), and 10 µM tributyltin chloride (Cl - /OH - ionophore) in high-K + calibration buffers (pH 7.4) containing various Cl - concentrations ( 22, 24, 41 ) to keep the [Cl - ] i at the level identical to the extracellular Cl - concentration with constant [K + ] i and pH i. Calibration buffers were made by appropriately mixing 0 mM Cl - and 100 mM Cl - buffers. In the high-K + buffer with these ionophores, [Cl - ] i is dependent on only extracellular Cl - concentration. Observed values of the cellular fluorescence/EV ratio in the 0, 20, 40, 60, 80, and 100 mM Cl - buffers were applied as fluorescent intensity ( F ) for the Stern-Volmer equation as follows. The Stern-Volmer equation is
where F 0 is the fluorescence at [Cl - ] i = 0 mM, F is the fluorescent intensity of the cells equilibrating with a [Cl - ] i, and K SV is the Stern-Volmer constant. K SV was determined by performing a linear regression on a plot of F 0 /F vs. [Cl - ] i ( Fig. 1 ).
Fig. 1. Calibration of MQAE fluorescent intensity and [Cl - ] i in A6 cell. A6 cells were loaded with MQAE (10 mM) in a culture medium for 12 h at 27°C. Calibration was performed in situ by coapplication of 5 µM nigericin, 5 µM valinomycin, 5 µM carbonyl cyanide 3-chloropenylhydrazone (CCCP), and 10 µM tributyltin chloride with [Cl - ] i calibrating solutions. Fluorescence without Cl - divided by fluorescence in presence of Cl - ( F 0 /F Cl ) is plotted against the known Cl - concentration. F Cl is the mean ratio of FL/EV for various values of [Cl - ] i and F 0 is the ratio of FL/EV at [Cl - ] i = 0. A typical Stern-Volmer plot is shown and the regression line gives the Stern-Volmer constant ( K SV; slope of the line) of 21.7 M -1 ( r 2 = 0.99).
Measurement of MQAE leakage during RVD. For MQAE leakage measurement, A6 cells were cultured in a Cl - -free NCTC-109 medium for 24 h to displace intracellular Cl - with NO 3 -, a permeable anion to Cl - channels. Cells were then loaded with 10 mM MQAE in a Cl - -free NCTC-109 medium for 12 h. Cell volume and fluorescence were then measured by Quanta at 1, 5, 10, 15, 20, and 30 min after exposure to a Cl - -free hypotonic solution. The fluorescent intensity obtained from the cells under this condition indicated the content of MQAE remaining in the intracellular space, since MQAE is not quenched by NO 3 -. Furthermore, in this time period, the intracellular water content of the cells incubated in the Cl - -free medium was not significantly different from that of the cells incubated in the control medium. Therefore, the intracellular MQAE content was not affected by incubation with the Cl - -free medium.
Measurement of [Cl - ] i in A6 cells after hypotonic shock. A6 cells were cultured in a culture medium containing MQAE (10 mM) for 12 h at 27°C, in a CO 2 incubator. MQAE-loaded cells were detached from culture flask. The cell suspension was centrifuged and resuspended in 120 mM NaCl isotonic buffer (1.5 ml). After 15-min incubation at 27°C, the cell suspension of 0.3 ml was mixed with the same volume of 120 mM NaCl isotonic buffer. This cell suspension was used as a sample without hypotonic shock (0 min). The remainder (1.0 ml) was diluted twofold by distilled water to provide hypotonic shock with 50% osmolality. Cell volume and fluorescent intensity were measured at 1, 5, 10, 15, 20, and 30 min after hypotonic shock by using Cell Lab Quanta. The [Cl - ] i was estimated by using the Stern-Volmer equation with the determined value of K SV.
Application of NPPB (a Cl - channel blocker) and quinine (a K + channel blocker) at measurements of RVD and [Cl - ] i. We studied the effect of a Cl - channel blocker, NPPB, or a K + channel blocker, quinine, on cell volume and [Cl - ] i of A6 cells during RVD. First, we determined the K SV and the MQAE leakage rate in the presence of these inhibitors. These inhibitors were added to all media for measurements of K SV and the MQAE leakage rates. Effects of NPPB or quinine on the change of cell volume and [Cl - ] i after exposure to the hypotonic solution were studied as follows. A6 cells were incubated in a culture medium containing MQAE (10 mM) for 12 h at 27°C in a CO 2 incubator. MQAE-loaded cells were detached from culture flask. The cell suspension was centrifuged and resuspended in 1.5 ml 120 mM NaCl isotonic buffer. Then, cells were incubated with 1.0 ml 120 mM NaCl buffer containing 100 µM NPPB or 1 mM quinine for 15 min at 27°C. After this treatment, the cell volume and the fluorescent intensity were measured before and after hypotonic shock by using the solution containing the inhibitor.
Data presentation and statistical analysis. Results are expressed as means ± SE. Statistical analysis was carried out using Student's t -test. Differences were considered significant when the P value was <0.05.
RESULTS
Intracellular water volume of A6 cells. First, we determined a cellular water space, which was the MQAE-accessible space in the A6 cell, to measure the exact change of [Cl - ] i during RVD. The putative water volume was calculated from the water content in A6 cells that was obtained by subtracting the dry weight from the wet weight of A6 cells in the isotonic solution. A6 cells ( 5.0 x 10 7 cells) were detached from flasks and then centrifuged. The wet weight was determined by weighing pellets of A6 cells after discarding supernatants as possible, and the dry weight was measured after drying the pellets for 24 h in a 90°C oven. The wet and the dry weights were 141.5 ± 4.1 and 16.0 ± 0.5 mg, respectively ( n = 14). Assuming that the difference between the wet and dry weights indicated the intracellular water volume with its specific gravity nearly equal to 1.0, the intracellular water space in A6 cell was 88.7 ± 0.2%. This datum was used for correction of MQAE concentration change caused by a cell volume change in all subsequent measurements of [Cl - ] i.
Relationship between MQAE fluorescence and [Cl - ] i in A6 cells. To convert the fluorescent intensity into the [Cl - ] i, the relationship between intracellular MQAE fluorescence and [Cl - ] i was determined using the ionophore combination technique. The Stern-Volmer plot showing the fluorescent intensity against [Cl - ] i appears in Fig. 1, indicating a K SV of 21.7 M -1.
MQAE leakage rate after hypotonic shock. To exactly correct the MQAE content in A6 cells after exposure to the hypotonic solution, we also measured the MQAE leakage rate. A6 cells were incubated in a Cl - -free NCTC-109 medium for 24 h and were loaded with 10 mM MQAE in a Cl - -free NCTC-109 medium for 12 h. Then, we analyzed MQAE leakage curves during a 30-min hypotonic period and applied curve-fitting techniques by measuring changes of the fluorescent intensity ( n = 14). Figure 2 shows a representative result on MQAE fluorescence and the fitted equation under the condition. The MQAE leakage rate of A6 cells during 30-min hypotonic period was 1.08% per minute. By use of these data, we determined the exact content of MQAE remaining in A6 cells before (0 min) and after hypotonic shock.
Fig. 2. Estimation of MQAE leakage after hypotonic shock. A6 cells were cultured in a Cl - -free (Cl - replacement with NO 3 - ) NCTC-109 medium for 24 h to displace [Cl - ] i. Cells were then loaded with 10 mM MQAE in a Cl - -free NCTC-109 medium for 12 h. After hypotonic stress (osmolality was reduced to 50%), cell volume and fluorescence were measured by Quanta as described in MATERIALS AND METHODS. Under this condition, the fluorescence indicated MQAE content, because MQAE was not quenched by NO 3 -. A typical plot of MQAE fluorescence is shown and the regression line gives a rate of MQAE leakage.
Changes of [Cl - ] i in A6 cells during RVD. To investigate whether the [Cl - ] i changes during RVD, we simultaneously measured cell volume and MQAE fluorescence during a 30-min incubation period after exposure of the cells to the hypotonic solution using the K SV value of 21.7 M -1 described above. When extracellular osmolality was lowered, A6 cells swelled quickly due to water influx ( Fig. 3 ); i.e., within 1 min after hypotonic shock from 268 to 134 mosmol/kgH 2 O the cell volume was rapidly increased to 152% of its initial volume ( Fig. 3 ). After swelling, the cell volume of A6 cell was gradually decreased, returning toward its initial volume. The cell volume went back to 130% of its initial volume 30 min after application of hypotonic shock. The [Cl - ] i in the isotonic medium was 43.6 ± 3.1 mM before application of hypotonic shock ( Fig. 3 ). One minute after hypotonic shock, the [Cl - ] i was drastically reduced by 49% (22.5 ± 2.4 mM) according to the hypotonicity-induced swelling (a simple dilution due to water influx; Fig. 3 ). Then, the [Cl - ] i was gradually decreased according to the progression of RVD. The [Cl - ] i was reduced to 10.8 ± 2.1 mM (75.2% decline) 30 min after application of hypotonic shock ( Fig. 3 ), indicating that the [Cl - ] i was decreased during RVD.
Fig. 3. Changes of cell volume and [Cl - ] i after hypotonic shock. Cell volume ( ) is represented as a relative value (%) to that at 0 min. The [Cl - ] i ( ) was calculated from the Stern-Volmer constant ( K SV ) shown in Fig. 1 with the compensation of MQAE leakage shown in Fig. 2. Results are expressed as means ± SE ( n = 9). When the error bars are not seen, the bars are smaller than the symbols.
Effects of NPPB and quinine on [Cl - ] i during the hypotonicity-induced RVD. During RVD, KCl release, in general, occurs through the volume-sensitive K + and Cl - channels that are, respectively, highly sensitive to quinine and NPPB. To clarify the role of RVD in regulation of [Cl - ] i, we studied effects of NPPB and quinine on the hypotonicity-induced change in [Cl - ] i. First, we measured K SV of MQAE by Cl - in the 120 mM NaCl buffer containing 100 µM NPPB or 1 mM quinine. The values of K SV were 15.9 or 16.7 in 100 µM NPPB and 1 mM quinine, respectively. The rate of MQAE leakage in an NPPB- or a quinine-containing buffer was also measured. In the NPPB-containing buffer, the rate of MQAE leakage was 0.17%/min, whereas the rate of MQAE leakage was 0.66%/min in a quinine-containing buffer. Then, we measured the cell volume and [Cl - ] i that were immersed in a hypotonic buffer containing NPPB or quinine. As shown in Fig. 4, preincubation with 100 µM NPPB or 1 mM quinine for 15 min before the hypotonic shock drastically inhibited RVD that was observed in hypotonic shock without these channel blockers. In the absence of these channel blockers, the [Cl - ] i was reduced by 50% (from 43.6 to 22.5 mM) at 1 min after hypotonic shock. This initial reduction of [Cl - ] i in the presence of NPPB (from 63.0 to 32.2 mM) was almost identical to that under the normal condition (without channel blockers). However, the following reduction of [Cl - ] i during RVD observed in control was suppressed by NPPB treatment ( Fig. 4 A ). The [Cl - ] i at 30 min after exposure to the hypotonic solution in NPPB-treated cells was 23.7 mM, which was 62.3% reduction from its initial value under the isotonic condition. This reduction of [Cl - ] i in NPPB-treated cells was significantly lower than in control cells (75.2%; P < 0.01). The [Cl - ] i in quinine-treated cells was also decreased from 45.7 to 20.4 mM at 1 min after hypotonic shock similar to control. A similar suppression in reduction of [Cl - ] i was also observed in quinine-treated cells ( Fig. 4 B ). The [Cl - ] i at 30 min after exposure to the hypotonic solution in quinine-treated cells was 23.6 mM, which was 48.4% reduction from isotonic condition. This reduction of [Cl - ] i in quinine-treated cells was also significantly lower than in control cells (75.2%; P < 0.001). These results indicate that the blockade of RVD by applying NPPB or quinine suppressed the further loss of [Cl - ] i. Therefore, we can conclude that one of the physiological functions of RVD is the reduction of [Cl - ] i by activating Cl - efflux through the NPPB-sensitive Cl - channel, which is most likely a volume-activated Cl - channel.
Fig. 4. Effects of ion channel blockers on hypotonicity-induced changes in cell volume and [Cl - ] i. A : effects of NPPB (100 µM), a Cl - channel blocker, on regulatory volume decrease (RVD) and a change of [Cl - ] i after hypotonic shock. Cell volume ( ) is represented as a relative value (%) to that at 0 min. Cells were preincubated with 100 µM NPPB for 15 min before hypotonic shock. Hypotonic shock was applied by adding the same amount of distilled water containing 100 µM NPPB as that of the incubating solution (osmolality was reduced to 50%). The [Cl - ] i ( ) was calculated from the Stern-Volmer constant ( K SV ) of NPPB-treated cells. RVD and the reduction of [Cl - ] i after hypotonic shock were completely inhibited by NPPB treatment. Results are expressed as means ± SE ( n = 13). When the error bars are not seen, the bars are smaller than the symbols. B : effects of quinine (1 mM), a K + channel blocker, on RVD and [Cl - ] i after hypotonic shock. Cell volume ( ) is represented as a relative value (%) to that at 0 min. Cells were preincubated with 1 mM quinine for 15 min before hypotonic shock and then the incubating solution was diluted by adding the same amount of distilled water containing 1 mM quinine as that of the incubating solution (osmolality was reduced to 50%). The [Cl - ] i ( ) was calculated from the K SV of quinine-treated cells. Results are expressed as means ± SE ( n = 11). When the error bars are not seen, the bars are smaller than the symbols.
DISCUSSION
In the present study, we established a novel method to measure the [Cl - ] i during cell volume changes by using a high-resolution flow cytometer, Cell Lab Quanta, and a halide-sensitive dye, MQAE. The [Cl - ] i has been measured by X-ray analysis, radioisotope, Cl - -sensitive electrode, or Cl - -sensitive fluorescent dye in previous studies (e.g., Ref. 18 ). In general, halide-specific indicators (Cl - -sensitive fluorescent dyes) such as SPQ, MEQ, and MQAE are more accurate than other legacy methods such as an X-ray analysis, a radioisotope, or a Cl - -sensitive electrode method. However, the [Cl - ] i measurement with MQAE is performed by using a single excitation wavelength and is not inherently normalized for the dye concentration in cells that are affected by cell volume changes. The change in dye concentration associated with no actual change in [Cl - ] i only due to cell volume changes influencing fluorescent intensity of dye misleads us to an incorrect conclusion as if [Cl - ] i itself changed. This means that the usage of MQAE is unsuitable for [Cl - ] i measurements during RVD without measurements of the dye concentration (i.e., cell volume), since the intracellular concentration of MQAE is significantly changed according to cell volume changes during RVD. In the present study, we attempted to solve this problem by using a high-resolution flow cytometer, Cell Lab Quanta. We used a cellular fluorescence/EV ratio to normalize the change in the dye concentration due to cell volume change. This normalization of the dye concentration based on cell volume is necessary, since no Cl - indicators with a fluorescence ratio technique are available. Using Cell Lab Quanta, we can simultaneously measure the fluorescent intensity and accurate cell volume (as EV) of intact cells. The measurement of EV by Coulter principle is a reliable, accurate method for determination of cell volume. Thus we can normalize the intracellular MQAE concentration by using the EV, even if the cell volume is changed during [Cl - ] i measurement period. Until now, MQAE is reported to be used to monitor [Cl - ] i changes in CHO cells by using a fluorescence/forward angle light scatter (FALS) to normalize the dye content ( 2 ). The data from FALS are obtained in a laser flow cytometry for identification of cell populations based on their approximate size and granularity. Earlier reports showed that light scatter is not an accurate technique measuring the cell volume or diameter despite its utility and widespread use ( 28, 30 ). Therefore, our novel method reported in the present study enables us to solve the problem caused by cell volume change. However, we should have further considered the MQAE soluble space of the cell, since the MQAE concentration in the cell should be determined by the water space of the cell. Therefore, we estimated the intracellular water volume by measuring dry/wet weight ratio. The cellular dry/wet weight ratio under control conditions was 89%. Under the condition, the 50% reduction of the extracellular osmolality should increase the cellular volume to 189% of the initial cell volume, if no osmolytes are released from the cell and the part of 11% (100-89%) does not function as an intracellular osmolyte. However, the actual cellular volume was increased to only 152%. The difference between the expected value (189%) and the observed one (152%) suggests to us two possibilities; 1 ) unstirred layers are not negligibly small in osmotic water movements, and 2 ) some osmolytes are released from the intracellular space before the cell volume reaches its peak value. Regarding the former possibility, unstirred layers decrease the effective osmotic gradient across a barrier ( 3 ). The unstirred layer would cause a phenomenon that the actual cellular volume increment after hypotonic shock is smaller than the increment in the putative cellular volume. In the present study, we indicate that the intracellular water volume of A6 cells under the isotonic condition was 89% of cell volume. On the other hand, Grosse et al. ( 18 ) reported that the mean value of the intracellular water volume in A6 cells is 80% by calculating dry/wet weight ratio, suggesting that the intracellular water volume of A6 cells reported in the present study may be overestimated. However, even if the value of intracellular water space reported in the present study, 89%, was overestimated and it would be 80%, the conclusion of the present study does not essentially alter. In fact, if the intracellular water space is 80% in the isotonic condition, the [Cl - ] i at 30 min after hypotonic stress is estimated to be 12.8 ± 2.5 mM. This was not significantly different ( P value = 0.39) from [Cl - ] i at 30 min after hypotonic stress, 10.8 ± 2.1 mM, which was calculated by using the value of intracellular water space used in this study, 89%. Therefore, even if the estimation of the intracellular water space reported in the present study (89%) is overestimated, the conclusion of the present study does not change.
In this study, hypotonic shocks were imposed by diluting the extracellular solution with an equal volume of distilled water. This would clearly cause a 50% reduction in the concentrations of all ions, which were critical for maintenance of cellular homeostasis such as K +, Ca 2+, and Mg 2+ in the external fluid. For example, the reduction of [K + ] o from 3.5 to 1.75 mM would shift the K + equilibrium potential ( E k ) from -90 to -108 mV (assuming that [K + ] i is kept constant at 120 mM). If the membrane potential ( V m ) is largely dependent on the E k, the dilution of [K + ] o may induce the hyperpolarization in the cell membrane. These raise a possibility that the decline of K +, Ca 2+, and Mg 2+ may influence the change of [Cl - ] i during RVD. Therefore, to clarify this point, we applied hypotonic shocks by reducing only the concentration of NaCl with keeping the concentrations of other components constant; i.e., we applied a hypotonic buffer containing 3.5 mM KCl, 1.0 mM CaCl 2, 1.0 mM MgCl 2, 5.0 mM glucose, and 10 mM HEPES (pH 7.40) instead of distilled water for the hypotonic stimulation. To reduce the osmolality by 50%, the cell suspension was diluted 2.26 times by the hypotonic buffer containing 3.5 mM KCl, 1.0 mM CaCl 2, 1.0 mM MgCl 2, 5.0 mM glucose, and 10 mM HEPES. Under this experimental condition, the cell volume of A6 cells was rapidly increased to 146% of its initial volume at 1 min after application of hypotonic shock. After swelling, the cell volume went back to 115% of its initial volume at 30 min after application of hypotonic shock. The [Cl - ] i in the isotonic solution was 41.5 ± 1.1 mM ( n = 5). The [Cl - ] i was reduced to 18.3 ± 0.5 and 8.3 ± 0.3 mM at 1 and 30 min after application of hypotonic shock, respectively ( n = 5). Changes of cell volume and [Cl - ] i under this experimental condition were almost identical to those caused by application of hypotonic shock with the distilled water, strongly suggesting that the reduction of [Cl - ] i after RVD was not affected by the reduction of extracellular K +, Ca 2+, and Mg 2+ concentrations. Therefore, the conclusion of the present study does not essentially alter.
Our results demonstrated that the [Cl - ] i of A6 cell was 43.6 mM in the isotonic solution. In amphibian distal nephron epithelia, the [Cl - ] i has been reported to be 20-50 mM depending on the source of the cells and techniques used for measurements as described below. The value of [Cl - ] i reported in the present study fits well within this range. In other cases, the intracellular electrolytes of A6 cells have been demonstrated by measuring the Cl - content in polarized cells by the X-ray analysis (28.2 ± 1.5 mmol/kg wet wt) ( 18 ) and the intracellular Cl - activity by MQAE (27.0 ± 2.0 mM) ( 10 ). However, it is difficult to compare these data with the value in the present study, since the Cl - contents are given in millimoles per kilogram of wet weight by X-ray analysis ( 18 ) and the Cl - measurements by using MQAE were obtained from preparations in which the extracellular Cl - concentration of the initial incubating solution was considerably lower (89.5 mM) ( 10 ) than in the present study (127.5 mM). In the present study, the [Cl - ] i was drastically reduced from 43.6 to 22.5 mM according to the cell swelling at 1 min after hypotonic shock when the cell volume reached its maximum value. Then, the [Cl - ] i was further reduced to 10.8 mM during the time period of RVD progression. The reduction of [Cl - ] i according to the initial cell swelling after hypotonic stress is also observed in another experiment by the X-ray analysis ( 18 ); at 2 min after applying hypotonic stress, the [Cl - ] i was drastically reduced from 28.2 to 12.1 mmol/kg wet wt (57% reduction). However, they could not detect the further decline of [Cl - ] i following the initial reduction of [Cl - ] i after exposure to the hypotonic medium. This is primarily due to technical difficulties in estimation of dry and wet weights, resulting in incorrect estimation of [Cl - ] i. Based on these observations, we strongly suggest that the [Cl - ] i measurement system established by using Cell Lab Quanta in the present study has a high sensitivity to Cl - with high accuracy and that we can determine the [Cl - ] i more easily by using the system represented in the present study.
The reduction of [Cl - ] i according to the progression of RVD agrees with the observation that hypotonic shock applied to A6 cells causes immediate increases of K + and Cl - effluxes ( 12, 40 ). In general, volume reduction during RVD is accompanied in parallel with substantial loss of cellular K + and Cl -. In cells of higher vertebrates, three broad classes of KCl efflux mechanisms have been implicated in RVD; i.e., K + -Cl - cotransporter ( 27 ), parallel K + /H + and Cl - /HCO 3 - exchanger ( 7 ), and parallel conductive pathways for K + and Cl - such as K + and Cl - channels ( 17, 21 ). However, the KCl efflux via K + -Cl - cotransporter and parallel K + /H + and Cl - /HCO 3 - exchangers seem to have no contribution to the KCl efflux in A6 cells during RVD ( 40 ). The KCl efflux during RVD in A6 cells would occur mainly via the K + and Cl - conductive pathways. In fact, the progression of RVD after hypotonic shock was significantly inhibited by the treatment with a Cl - channel blocker, NPPB, or a K + channel blocker, quinine ( Fig. 4 ). In general, the [Cl - ] i is correlated to cell volume ( 31, 32, 38 ). When RVD occurs, the [Cl - ] i decreases because the major membrane-permeable anion is Cl -, and a large number of membrane-impermeable anions, such as proteins, are located in the intracellular space ( 29 ). Therefore, when the same amounts of Cl - and K + are released from the cytosol, the [Cl - ] i has to be changed substantially without a proportionally large change in the intracellular K + concentration ( 29 ). In this study, we could not simultaneously measure the changes of [Cl - ] i and [K + ] i during RVD because of technical difficulties. However, the previous report ( 18 ) demonstrated that the reduction of [Cl - ] i (45.5% decline) after hypotonic stimulation is larger than that of [K + ] i (23.8% reduction), supporting an idea that the [Cl - ] i would be changed substantially without a proportionally large change in [K + ] i.
Acute osmoregulation of Na + reabsorption has been reported in renal ( 10, 33, 42 ) and other ( 11, 16 ) epithelia. We also reported that hypotonicity stimulated Na + reabsorption mainly by increasing the expression of epithelial Na + channels ( -ENaC) via a pathway dependent on protein tyrosine kinases ( 33, 34 ). Furthermore, we hypothesized that the stimulatory action of the hypotonicity on -ENaC mRNA expression in A6 cells might be caused via reduction of [Cl - ] i ( 33 ). In the present study, we showed that the effect of a 50% dilution (-132.5 mosmol/kgH 2 O) of the extracellular medium induced a huge reduction of [Cl - ] i (from 43.6 to 10.8 mM) during RVD. This large reduction of [Cl - ] i may play a crucial role in many physiological responses after hypotonic stimulation. Gordon ( 16 ) demonstrated that small changes in serosal osmolality (±24 mosmol/kgH 2 O), which might induce small changes of [Cl - ] i, were needed to alter the rate of Na + absorption in the toad urinary bladder; however, the reduction of [Cl - ] i acts as an intracellular signal not only for the induction of Na + absorption, but also for many other cellular functions. In other cells, the large change in [Cl - ] i has been reported to modify gene expression in renal cells ( 8, 44 ) and the activity of Na + -K + -2Cl - cotransporter (NKCC) in dog tracheal cells ( 19, 20 ) and in human trabecular meshwork cells ( 35 ). These results strongly suggest that the large change of [Cl - ] i is able to act as an intracellular signal regulating cellular functions. In the present study, we confirmed that the drastic reduction of [Cl - ] i was elicited by RVD in A6 cells. Therefore, this significant decrease of [Cl - ] i during RVD supports the hypothesis that RVD acts as a signal via reduction of [Cl - ] i playing important roles in regulation of cell function as described above.
As a conclusion, we indicate that this novel method of [Cl - ] i measurement will provide investigators with important data confirming physiological functions of intracellular chloride ions.
GRANTS
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (17790154, 18659056) and from Japan Society of The Promotion of Science (17390057, 17590191), a Grant-in-Aid from The Salt Science Research Foundation (0241), and a Leading Project for Biosimulation from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Fuji Foundation for Protein Research.
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作者单位：Departments of 1 Molecular Cell Physiology and 2 Surgery, Division of Digestive Surgery, 3 Department of Respiratory Molecular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan