Mechanisms of chloride transport in thymic lymphocytes

Donatas Stakisaitis1, Michael S. Lapointe1,2, and Daniel Batlle1,2

1 Division of Nephrology and Hypertension, Department of Medicine, Northwestern University Medical School, and 2 Veterans Affairs Chicago Health Care System, Lakeside Division, Chicago, Illinois, 60611


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined mechanisms of Cl- transport in rat lymphocytes under a variety of conditions. Basal intracellular Cl- concentration ([Cl-]i) was not different between cells assayed in the presence of HCO3- or its absence (HEPES). Removal of external Cl- resulted in a fall in [Cl-]i and a rapid rise in intracellular pH (pHi). Both Cl- efflux and the rise in pHi were blocked by DIDS or removal of external Na+ but were unaffected by furosemide. The mechanisms governing Cl- influx were assessed in cells that had been Cl- depleted for 1 h. Reexposure to Cl- resulted in a rapid rise in [Cl-]i that was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked Cl- influx. Cl- depletion caused a marked increase in pHi that rapidly declined toward normal when the cells were reexposed to Cl-. Preincubation with DIDS completely blocked this decrease in pHi. In contrast, neither removal of Na+ nor preincubation with furosemide affected the decline in pHi when the cells were reexposed to Cl-. We conclude that, in thymic lymphocytes, Cl-/HCO3- (or Cl-/base exchange) regulates both Cl- influx and efflux. Cl- efflux is totally inhibited by DIDS and is mediated by a Na+-dependent Cl-/HCO3- exchanger. Cl- influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a Na+-independent Cl-/HCO3- exchanger and by a Na+-K+-2Cl- cotransporter.

chloride/bicarbonate exchange; sodium-potassium-2 chloride cotransport; stilbenes; furosemide; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid


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INTRACELLULAR CHLORIDE CONCENTRATION ([Cl-]i) is maintained at a higher level than its electroneutral value, as calculated by the Nernst equation, indicating that it is maintained by mechanisms other than simple diffusion (17). [Cl-]i must therefore be regulated by the relative activities of plasma membrane Cl--influx and Cl--efflux pathways. In previous studies, we showed that rat thymic lymphocytes possess both Na+-dependent and Na+-independent Cl-/HCO3- exchangers and examined the roles they play in the regulation of intracellular pH (pHi) (3, 41). With the availability of a chloride-sensitive dye, we now explore the role of these and other potential exchangers on the pathways for Cl- entry and exit.

Two classes of Cl-/HCO3- exchangers have been identified in mammalian cells. The band 3 family of exchangers [erythroid (AE1, AE2, and AE3) isoforms] is sodium independent. Band 3 protein-mediated anion exchange has been previously demonstrated in lymphocytes (17, 27, 28), which, as in most other cell types, express the AE2 isoform (1, 2, 27). AE2 is expressed in all regions of the kidney (6). In contrast, alpha -intercalated cells in the kidney, like erythrocytes, express the AE1 isoform (13). The activity of this family of transporters is governed by the relative concentration gradients of Cl- and HCO3- across the cell membrane. Under normal physiological conditions this transporter is said to be inactive (17). It is activated by cellular alkalinization and normally acts as a Cl--influx mechanism (the exchange of intracellular HCO3- for extracellular Cl-). This protein is capable of transporting Cl- or mediating pHi changes that reflect Cl-/HCO3- exchange (27, 28). Thus in lymphocytes, AE2 is the likely candidate for regulation of pHi and [Cl-]i (23). A second class of Cl-/HCO3- exchanger that has been identified is a Na+-dependent Cl-/HCO3 (41). This antiporter acts as a Cl--efflux mechanism (intracellular Cl- exits the cell in exchange for HCO3-) (33).

Another family of transport proteins capable of regulating [Cl-]i is the Na+-K+-2Cl- transporters (38). Two isoforms of the Na+-K+-2Cl- transporter have been identified to date. NKCC1 (also dubbed BSC-2) is a widely distributed isoform. In renal cells, it is located in basolateral membranes and has been named a "secretory" isoform. This transporter is involved in maintenance of cell volume, and its activity is greatly increased on cell shrinkage (29, 39). A distinct isoform, NKCC2, (also dubbed BSC-1) exists only in the apical membrane of the thick ascending limb in the kidney where it is involved in the reabsorption of sodium chloride. It is sometimes referred to as the "reabsorptive" isoform. The Na+-K+-2Cl- transporters are sensitive to inhibition by the loop diuretics furosemide and bumetanide. Studies that examined the role of Na+-K+-2Cl- transport in the regulation of [Cl-]i in lymphocytes have resulted in conflicting findings (14, 17). One study suggested that a bumetanide sensitive and Na+- and Cl--dependent influx of 86Rb, a measure of K+ transport, accounted for roughly 75% of the 86Rb uptake (14). In contrast, others reported that Cl- uptake in lymphocytes was largely unaffected by omission of extracellular Na+ and K+ or by the addition of bumetanide.

Functional characterization of the Cl-/HCO3- exchangers for the most part has been limited to experiments that evaluated fluorimetric measurements of pHi changes during the removal and restoration of extracellular chloride or to experimentally driven alterations in unidirectional Cl- fluxes, as measured by using radiolabeled Cl-. Similarly, studies examining the activity of Na+-K+-2Cl- transporters have centered largely on cell volume. There is little information on how the interplay of these Cl--transport mechanisms impact on [Cl-]i. In the present study, we examined the mechanisms involved in Cl- influx and efflux, by using the fluorimetric indicators N(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), to determine net changes in [Cl-]i and pHi in lymphocytes during the acute exposure to Cl--free media and during reexposure to external Cl- after Cli- depletion. We show that Cl- efflux is totally inhibited by DIDS and is mediated by a Na+-dependent Cl-/HCO3- exchanger. Cl- influx, which is partially DIDS sensitive and partially furosemide sensitive, is mediated by both a Na+-independent Cl-/HCO3- exchanger and by a Na+-K+-2Cl- cotransporter.


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Preparation of cells. Thymic lymphocytes were isolated from Sprague-Dawley rats, 6-9 wk of age, as previously described (4, 41). Briefly, the rats were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg body wt), and the chest cavity was opened by cutting the ribs along the sternum. The thymus was removed with forceps and cleared of blood vessels. Contaminating blood was removed by rinsing with RPMI-1640. The thymus was then minced and pipetted vigorously with a syringe several times. Large cell aggregates and connective tissue were removed by passage through four layers of surgical gauze. The resulting suspension of lymphocytes was washed free of red blood cell contamination, by centrifugation three times in RPMI-1640 at 150 g for 10 min each.

Loading of lymphocytes with MQAE, a chloride-sensitive dye. After the final wash, the lymphocytes were resuspended in RPMI-1640 containing 5 mM MQAE (Molecular Probes, Eugene, OR) and incubated at 37°C for 120 min. After loading, the cell suspension was centrifuged for 10 min to remove the external dye solution. The cell pellet was then split and the cells resuspended in either a bicarbonate containing physiological salt solution (solution 1, Table1), in a Cl--free buffer solution (solution 2, Table 1), or their bicarbonate-free buffer equivalents. All solutions were kept at 37°C by using a water bath. Bicarbonate-containing buffers were preequilibrated with 5% CO2-95% O2 by directly bubbling the gas mixture through the buffer solution for at least 30 min before use. A constant stream of CO2-O2 over the cuvette was used to keep the solutions equilibrated with CO2 during fluorometer readings. The composition of the standard buffer solutions used for this study is listed in Table 1. All solutions were filtered through a 0.2-µm filter before use to reduce autofluorescence of solutions.

                              
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Table 1.   Solution compositions

Fluorescence measurements. Fluorescence measurements were obtained with an SLM DMX-1000 spectrofluorometer at an excitation wavelength of 352 nm and an emission wavelength of 450 nm (49). The dye-loaded cells were kept under constant magnetic stirring in a thermostatically controlled cuvette (37°C).

MQAE fluorescence was calibrated against [Cl-]i by using a double ionophore technique in high-K+ media (8). The calibration solutions were made by mixing a high-KCl buffer (solution 5, Table 1) and a high-KNO3 buffer (solution 6, Table 1) to generate final Cl- concentrations of 60, 30, and 0 mM. Nitrate, an anion with high-cell permeability and minimal MQAE quenching, was found to be an ideal substitute for Cl- for the calibration of MQAE (30). Nigericin (6 µM), a K+/H+ antiporter (47), and tributyltin (10 µM), a OH-/Cl- antiporter (45), were used to equalize the Cl- gradient across the cell membrane. Under these calibration conditions, [Cl-]i and extracellular Cl- concentration ([Cl-]o) were assumed to be equal.

The autofluorescence of each sample was determined at the end of each experiment by the addition of 150 mM KSCN to the cuvette to quench the MQAE fluorescence. SCN- has a much higher affinity for MQAE than Cl- and therefore quenches most of the MQAE fluorescence, resulting in the cellular autofluorescence. The percentage of the total fluorescence signal due to autofluorescence was 14.6 ± 0.76% for the initial readings obtained for the efflux studies. Because the MQAE fluorescence signal is inversely related to the [Cl-], this would be the point of the lowest signal-to-noise ratio. This background fluorescence was subtracted from all readings before calculation of the calibration curve and subsequent [Cl-]i determinations. After correction for background fluorescence, the calibration curve was generated by plotting the ratio of the total quenchable signal (Ft; in the total absence of Cl-) to the fluorescence at each Cl- concentration used at each calibration point (FCl; the fluorescence at the given Cl- concentration) against the [Cl-]i (i.e., Ft/FCl vs. [Cl-]i) as described below. The use of the ratio of Ft to FCl for each individual sample was necessary to normalize the data for the absolute amount of indicator present in the cuvette. The relationship between the normalized fluorescence intensity of intracellular MQAE and[Cl-]i was linear over the range used in this study.

Calculation of [Cl-]i. [Cl-]i was calculated by using a modification of the procedure described by others (8, 11, 12, 30, 32). The Ft was obtained by subtracting the autofluorescence (after addition of KSCN) from the Fmax, which was obtained as fluorescence signal in Cl--depleted cells assayed in a Cl--free solution. Cl- depletion was accomplished by incubation of the cells in a Cl--free solution for 60 min before use.
F<SUB>t</SUB><IT>=</IT>F<SUB>max</SUB><IT>−</IT>F<SUB>KSCN</SUB>
The FCl was determined by the equation
F<SUB>Cl</SUB><IT>=</IT>F<IT>−</IT>F<SUB>KSCN</SUB>
where F was fluorescence signal of cells assayed in a normal physiological buffer solution.

Determination of Cl- efflux and influx. Basal [Cl-]i was determined from the first reading of fluorescence of efflux curves. Chloride efflux rates were determined as the initial decline in [Cl-]i after suspending the cells in a chloride-free media. The initial rate of Cl- efflux was defined as change in [Cl-]i during the first 90 s after exposure to the Cl--free solution. For these experiments isethionic acid (2-hydroxyethanesulfonic acid; sodium salt) was used to replace extracellular chloride (solution 2, Table 1).

For the chloride influx studies, the cells were chloride depleted by incubation in a chloride-free media (solution 2, Table 1) for 1 h before study. The cells were then resuspended in a physiological salt solution (solution 1, Table 1) containing ~100 mM chloride. The initial rate of Cl- influx was determined as the change in [Cl-]i during the first 90 s of exposure to the normal physiological salt solution.

All solutions used were isotonic to the control solutions. For experiments examining the bicarbonate dependence on the rate of chloride influx, NaHCO3 was replaced with sodium isethionate and, when appropriate, KHCO3 was replaced with potassium gluconate. For experiments examining the sodium dependence of chloride influx and efflux, sodium was substituted isotonically with choline and N-methyl-D-glucamine (solutions 3 and 4, Table 1) (52).

Measurement of pHi. The pH-sensitive fluorescent probe, BCECF was used to measure pHi (4, 41). The cells were loaded with 1 µg/ml BCECF-AM (Molecular Probes) for 30 min at 37°C. After loading, the cells were washed three times by centrifugation with the assay buffer and then kept for at least 30 min before proceeding. Samples of cells were washed again immediately before use to remove any external dye.

BCECF fluorescence was measured at excitation wavelengths of 500 nm and 440 nm and an emission wavelength of 520 nm. Calibration of the 500/440-nm ratio to pHi was done at the beginning of each experiment by using 6 µg/ml nigericin in a 120 mM KCl solution as previously described (4, 35).

Chemicals. MQAE, ethylisopropylamiloride (EIPA), and H2DIDS were purchased from Molecular Probes, Eugene, OR. All other chemicals were purchased from Sigma or Aldrich, St. Louis, MO.

Statistical analysis. The n for each set of experiments was determined as average from duplicate readings from each animal used in the study. All data are expressed as means ± SE. Statistical differences between groups were determined by using a Student's t-test for paired or unpaired data when appropriate. Differences were considered significant when P <=  0.05.


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Intracellular Cl-. There was no difference in the basal [Cl-]i between lymphocytes kept for 1 h in HCO3--containing and those maintained in a HCO3--free solution [HEPES buffered; 24.3 ± 1.3 vs. 26.2 ± 1.5 mM, respectively, n = 11, not significant (NS)]. On the basis of an external [Cl-] of 101 mM (Table 1) and a resting membrane potential of -55 mV for lymphocytes (17), the electrochemical equilibrium for [Cl-]i is 13 mM as calculated by the Nernst equation. Thus [Cl-]i is maintained at a higher level than its electroneutral value indicating that it is regulated by mechanisms other than simple diffusion.

Cl- efflux. After the removal of extracellular chloride (solution 2, Table 1), the initial decline in [Cl-]i was used to estimate the net chloride efflux rate. In cells maintained in a HCO3--containing solution, this rate was markedly faster than that of paired cells kept in HCO3--free solution ([Cl-]i -2.7 ± 0.36 vs. -1.1 ± 0.29 mM/90 s, respectively, n = 11, P < 0.01). (Fig. 1).


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Fig. 1.   Chloride efflux from cells assayed in the presence or nominal absence of HCO3-/CO2. Thymic lymphocytes were isolated and loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) for the measurement of intracellular Cl- concentration ([Cl-]i)as described in METHODS. Paired aliquots of cells were kept in either a HCO3-/CO2 (open circle , n = 11) solution or one that was nominally HCO3- free (HEPES) (, n = 11). Injection of cells into a Cl--free solution resulted in a faster decline in [Cl-]i in cells assayed in the presence of HCO3-/CO2 than in cells assayed in its nominal absence.

Pretreatment of cells for 25 min with 125 µM DIDS, a compound known to inhibit Cl-/HCO3- exchange, had no effect on basal [Cl-]i compared with paired controls (24.4 ± 1.3 vs. 24.5 ± 1.8 mM, respectively, n = 6, NS). Pretreatment with DIDS, by contrast, resulted in the complete blockade of net Cl- efflux when the cells were placed in a Cl--free HCO3--containing solution (Fig. 2A). These data suggest that the decline in [Cl-]i observed in control cells exposed to a Cl--free buffer was due to cell exit via a Cl-/HCO3- exchanger.


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Fig. 2.   Effect of DIDS on net Cl- efflux and increase in intracellular pH (pHi). Cells were either pretreated for 25 min with DIDS (125 µM; ) or maintained in a control HCO3-/CO2 solution (open circle ) before injection into a Cl--free HCO3- containing solution. A: there was a rapid decline in [Cl-]i (net Cl- efflux) when cells were injected into a Cl--free solution. Preincubation with DIDS (n = 6) had no effect on the initial [Cl-]i compared with controls (n = 6), but resulted in the complete inhibition of net Cl- efflux that was observed in control cells that were exposed to a Cl--free solution. B: there was a rapid increase in pHi when the cells were injected into a Cl--free solution. Preincubation with DIDS (n = 5) had no effect on the initial pHi, but almost completely blocked the rise in pHi that was observed in control cells (n = 5) that were exposed to a Cl--free solution.

Two classes of Cl-/HCO3- exchangers have been identified in mammalian cells. The band 3 family of exchangers (AE1, AE2, and AE3) is sodium independent, and under normal physiological conditions act as a HCO3--efflux mechanism (the exchange of intracellular HCO3- for extracellular Cl-). However, under experimental conditions of external Cl- removal, these antiporters can be reversed. A second class of Cl-/HCO3- exchangers that has been identified is a Na+-dependent Cl-/HCO3- exchanger. This antiporter acts as a Cl--efflux mechanism both physiologically and under experimental conditions of external Cl- removal. To distinguish which of these transporters could account for the decline in [Cl-]i when lymphocytes were exposed to a Cl--free solution, the sodium dependence of net chloride efflux was examined. For these experiments N-methyl-D-glucamine was used as the major cation (solution 3, Table 1). The decline in [Cl-]i seen in control cells was completely blocked when cells were exposed to a Na+-free, Cl--free solution (Fig. 3A). These data indicate that in lymphocytes assayed in a HCO3--containing buffer solution, removal of external Cl- results in net Cl- efflux from the cell due to a Na+-dependent Cl-/HCO3- exchanger.


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Fig. 3.   Effect of acute Na+ removal on net Cl- efflux and increase in pHi. Cells were maintained in a sodium-containing HCO3-/CO2 solution and then either injected into a Na+-free, Cl--free, HCO3--containing solution () or injected into a control Cl--free HCO3- containing solution (open circle ). A: removal of external Na+ (n = 7) resulted in the complete inhibition of net Cl- efflux that was observed in control cells (n = 7) that were exposed to a Cl--free solution. B: removal of external Na+ (n = 3) resulted in a transient cell acidification followed by only a slight rise in pHi that failed to alkalinize beyond the initial value.

If a Na+-dependent Cl-/HCO3- exchanger was responsible for the net Cl- efflux, then the experimental conditions used in the above experiments should have similar, but opposite effects on pHi, as was observed with [Cl-]i. To examine this, cells were loaded with BCECF, and the alterations in pHi during exposure to a Cl--free solution were determined.

Exposure of the cells to a Cl--free media resulted in a rapid increase in pHi. Pretreatment with DIDS completely blocked the rise in pHi observed when the cells were exposed to a Cl--free media (Fig. 2B). In the absence of external Na+, Cl- removal does not result in a large increase in pHi (Fig. 3B). Under this condition, there is a slight fall in pHi, likely due to the acute inhibition of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange by the removal of external Na+. The pHi then gradually returns to basal values. The cells do not develop an alkalosis (pHi 7.29 ± 0.05 vs. 7.32 ± 0.06, NS) despite the absence of external Cl-, as was observed in cells assayed in the presence of external Na+.

The removal of sodium from the external media does not specifically inhibit the Na+-dependent Cl-/HCO3- exchanger. Sodium removal would also inhibit the Na+/H+ antiporter. This could potentially result in acid accumulation and thereby prevent the rise in pHi during exposure to a Cl--free solution, through a mechanism that does not involve a Na+-dependent Cl-/HCO3- exchanger. To rule out this possibility, the Na+/H+ antiporter was inhibited by EIPA (20 µM). Treatment of the cells with EIPA had no effect on the rise in pHi when the cells were exposed to a Cl--free media (pHi 0.13 ± 0.02 vs. 0.14 ± 0.01, respectively, n = 5, NS). Similarly, EIPA had no effect on the initial rate of net Cl- efflux during exposure to a Cl--free solution compared with paired controls ([Cl-]i -5.7 ± 0.93 vs. -4.2 ± 1.02 mM/90 s, respectively, n = 5, NS). Taken together, these data are consistent with our previous work demonstrating that in the presence of HCO3-/CO2, a Na+-dependent Cl-/HCO3- exchanger regulates pHi (41).

Because [Cl-]i was not different between cells assayed in either the presence or absence of HCO3-/CO2 in the media, we sought to determine whether this mechanism of net Cl- efflux required external HCO3- to operate. In the nominal absence of external HCO3-, pretreatment with DIDS completely abolished the initial decline in [Cl-]i when the cells were exposed to a Cl--free solution ([Cl-]i +0.75 ± 0.64 vs. -1.19 ± 0.58 mM/90 s, respectively, n = 5, P < 0.01). When the cells were exposed to a Cl--free solution in the absence of external Na+, there was a slight initial decline in [Cl-]i over the first 45 s that then failed to decline further, whereas paired [Cl-]i in control cells steadily declined over time (data not shown). This initial decline in [Cl-]i is likely due to residual Na+ associated with the cell pellet before injection into a Na+-free Cl--free solution. Thus in the nominal absence of HCO3-, net Cl- efflux appears to be mediated through a Na+-dependent Cl-/base exchanger that is sensitive to DIDS as well.

In cells maintained in a HCO3--containing solution, pretreatment of cells with 200 µM furosemide for one h resulted in a 38% decline in [Cl-]i compared with paired controls (14.7 ± 0.2 vs. 23.7 ± 1.5 mM, respectively, n = 3, P < 0.025). The lower basal [Cl-]i in cells pretreated with furosemide is likely due to inhibition of net Cl- influx (see below). Pretreatment with furosemide also resulted in a slower initial rate of net Cl- efflux than controls when the cells were exposed to a Cl--free solution, ([Cl-]i -2.2 ± 0.44 vs. -4.2 ± 1.02 mM/90 s, respectively, n = 5, P < 0.05) (Fig. 4A). This effect on net Cl- efflux is likely due to the lower basal [Cl-]i in cells pretreated with furosemide, because the rate of net Cl- efflux at any given [Cl-]i was not different between cells pretreated with furosemide and controls. Pretreatment of the cells with furosemide also had no affect on the rapid increase in pHi that is seen when the cells are exposed to a Cl--free solution (Fig. 4B).


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Fig. 4.   Effect of furosemide on net Cl- efflux and increase in pHi. Cells were either pretreated for 60 min with furosemide (200 µM; ) or maintained in a control HCO3-/CO2 solution (open circle ) before injection into a Cl--free HCO3--containing solution. A: preincubation with furosemide (n = 5) resulted in a significantly lower initial [Cl-]i than controls (n = 5), and a slower initial rate of net Cl- efflux than controls. When normalized for [Cl-]i, net Cl- efflux was similar between the 2 groups of cells. B: preincubation with furosemide (n = 5) had no effect on either the initial pHi or the increase in pHi that occurred after injection into a Cl--free HCO3- containing solution.

Cl- influx. The mechanisms involved in Cl- entry into lymphocytes were examined by chloride depleting the cells for 1 h (solution 2, Table 1) and then reexposing them to a Cl--containing solution (solution 1, Table 1) . This protocol resulted in initial levels of Cl-i close to 0 (0.54 ± 0.11 mM, n = 39). Resuspension of Cl--depleted cells in a Cl--containing solution resulted in a rapid increase in [Cl-]i. The increase in [Cl-]i was significantly faster in cells resuspended in a bicarbonate buffer than in those exposed to a bicarbonate-free (HEPES) buffer ([Cl-]i 10.4 ± 1.29 vs. 5.8 ± 0.62 mM/90 s, respectively, n = 10, P < 0.01) (Fig. 5).


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Fig. 5.   Cl- influx in cells assayed in the presence or nominal absence of HCO3-/CO2. Paired aliquots of cells were kept in either a HCO3-/CO2 solution (open circle , n = 10) or one that was nominally HCO3- free (HEPES) (, n = 10). The cells were Cl- depleted for 60 min and then injected into a Cl--containing solution. Injection of cells into a Cl--containing solution resulted in a faster increase in [Cl-]i in cells assayed in the presence of HCO3-/CO2 than in cells assayed in its nominal absence.

Because net Cl- efflux was completely inhibited by preincubation of the cells with DIDS, we sought to determine if Cl- influx was also completely DIDS sensitive. In cells maintained in a HCO3--containing solution, pretreatment of Cl--depleted cells with 125 µM DIDS for 25 min caused only a partial inhibition of net Cl- influx (57 ± 6%) compared with paired controls cells not pretreated with DIDS ([Cl-]i 5.1 ± 0.78 vs. 11.9 ± 1.07 mM/90 s, respectively, n = 6, P < 0.001) (Fig. 6A). Similar results were obtained by using cells preincubated with 125 µM H2DIDS, a stilbene derivative that causes less interference with MQAE fluorescence ([Cl-]i 6.8 ± 1.99 vs. 14.5 ± 0.45 mM/90 s, respectively, n = 3, P < 0.05).


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Fig. 6.   Effect of DIDS on net Cl- influx and decline in pHi. Cl--depleted cells were either pretreated for 25 min with DIDS (125 µM; ) or maintained in a control Cl--free HCO3-/CO2 solution (open circle ) before injection into a Cl--containing, HCO3--containing solution. A: there was a rapid rise in [Cl-]i in control cells (n = 6) that began to plateau after ~2 min. In cells pretreated with DIDS (n = 6), the rise in [Cl-]i was nearly linear and did not show any apparent saturation. The initial rate of Cl- influx, at 90 s, was decreased in cells pretreated with DIDS by ~60% compared with controls. B: the initial pHi was increased compared with cells that had not been Cl- depleted (see Fig. 4B). Reexposure to Cl- resulted in a rapid decline in pHi in control cells (n = 5). Preincubation with DIDS (n = 5) had no effect on the initial pHi, but completely blocked the decline in pHi that was observed in control cells.

To determine the sodium dependence of chloride entry, Cl- influx was measured in sodium-free bicarbonate buffer (solution 4, Table 1). The chloride influx rate was only partially (46 ± 7%), but significantly, reduced in cells exposed to a sodium-free solution, compared with paired controls ([Cl-]i 5.9 ± 0.97 vs. 11.3 ± 1.93 mM/90 s, respectively, n = 5, P < 0.025) (Fig. 7A). Thus unlike Cl- efflux that was completely blocked by DIDS or by removal of external sodium, Cl- influx was only partially sensitive to DIDS and to removal of external sodium.


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Fig. 7.   Effect of acute Na+ removal on net Cl- influx and decline in pHi. Cells were Cl- depleted in a Na+-containing HCO3-/CO2 solution for a total of 60 min and then either injected into a Na+ free, Cl--containing solution () or injected into a control Na+-containing, Cl--containing solution (open circle ). A: removal of external Na+ (n = 5) resulted in ~50% inhibition of the net Cl- influx that was observed in control cells (n = 5). Note that the shapes of the 2 curves were similar. B: removal of external Na+ (n = 3) has no effect on either the initial pHi or the decline in pHi that occurred in control cells (n = 3) that were reexposed to Cl-.

To examine the possibility that Na+-K+-2Cl- cotransport contributed to chloride influx, cells were pretreated with 200 µM furosemide for 15 min (Fig. 8A) or for 1 h. Pretreatment of cells with 200 µM furosemide for 15 min caused a partial reduction in net Cl- influx (45 ± 8%) compared with paired control cells not pretreated with furosemide ([Cl-]i 6.6 ± 1.34 vs. 11.6 ± 1.23 mM/90 s, respectively, n = 5, P < 0.005). Pretreatment of the cells with 200 µM furosemide for 1 h resulted in a similar level of inhibition in net Cl- influx (65 ± 7%, n = 3, data not shown). Of note, the shape of the net Cl--influx curve in cells pretreated with furosemide was similar to that seen in cells acutely exposed to a Na+-free media (compare Figs. 7A and 8A), whereas in cells pretreated with DIDS the net Cl--influx curve was essentially linear with time.


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Fig. 8.   Effect of furosemide on net Cl- influx and decline in pHi. Cells were Cl- depleted for a total of 60 min. The cells were either pretreated for 15 min with furosemide (200 µM; ) or maintained in a control Cl--free HCO3-/CO2 solution (open circle ) before injection into a Cl--containing, HCO3--containing solution. A: pretreatment with furosemide (n = 5) resulted in ~50% inhibition of the net Cl- influx that was observed in control cells (n = 5). Note that the shapes of the 2 curves were similar. B: pretreatment with furosemide (n = 5) had no effect on either the initial pHi or the decline in pHi that occurred in control cells (n = 5) that were reexposed to Cl-.

Pretreatment of lymphocytes with both furosemide and H2DIDS together completely inhibited Cl- influx (Fig. 9). These data indicate that Cl- entry is due to two mechanisms, Na+-independent Cl-/HCO3- exchange and Na+-K+-2Cl- cotransport.


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Fig. 9.   Effect of furosemide and H2DIDS on net Cl- influx. Cells were Cl- depleted for a total of 60 min. The cells were either pretreated with furosemide (200 µM) and H2DIDS (125 µM) together (, n = 9) or maintained in a control Cl--free HCO3-/CO2 solution (open circle , n = 9) before injection into a Cl--containing, HCO3--containing solution. Preincubation with furosemide and H2DIDS together almost completely inhibited the net Cl- influx that was observed in control cells.

To further characterize the Cl--influx mechanisms, cells were loaded with BCECF to monitor pHi under conditions identical to those used to monitor Cl-i.. Chloride depletion for 1 h caused a marked increase in pHi. When the cells were reexposed to a Cl--containing solution, the pHi rapidly declined toward control values. Preincubation of the cells with DIDS completely blocked this decrease in pHi (Fig. 6B). In contrast, neither removal of Na+ from the Cl--containing solution (Fig. 7B) nor preincubation of the cells with furosemide (Fig. 8B) had any affect on the decline in pHi when the cells were reexposed to a Cl--containing solution. Taken together with the Cl- influx data these data indicate that net Cl- influx is mediated by both a Na+-independent Cl-/HCO3- exchanger and Na+-K+-2Cl- cotransporter, whereas the decline in pHi (HCO3- efflux or H+ influx) can be completely accounted for by the action of a DIDS-sensitive, Na+-independent Cl-/HCO3- exchanger. Moreover, the finding that furosemide does not alter changes in pHi during Cl- repletion (Fig. 8B), but DIDS completely prevented the fall in pHi (Fig. 6B), indicates that furosemide at the concentration used in this study did not inhibit Cl-/HCO3- exchangers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In thymic lymphocytes, pHi is regulated by both Na+-independent and Na+-dependent Cl-/HCO3- exchangers (41) as well as the NHE-1 isoform of the Na+/H+ exchange family (34, 43). Evidence for Cl-/base exchange has accumulated largely on the basis of either flux studies or indirectly by measurements of pHi during the removal and readdition of Cl- from the media. [Cl-]i, however, was not measured in those studies. The present study was undertaken to examine the pathways involved in Cl- entry and exit into thymic lymphocytes. Examination of Cl- efflux pathways was performed by acutely exposing the cells to a Cl--free media and determining the change in [Cl-]i and pHi under various experimental conditions. Similarly, examination of Cl- influx pathways was performed by Cl- depleting the cells for 1 h and then acutely exposing the cells to a Cl--containing media. Our data show that, in thymic lymphocytes, [Cl-]i is regulated by a balance of Cl- efflux via a Na+-dependent Cl-/HCO3- exchanger and Cl- influx via a Na+-independent Cl-/HCO3- exchanger and a furosemide-sensitive mechanism consistent with a Na+-K+-2Cl- cotransporter.

The basal [Cl-]i reported in the present study (24.3 ± 1.3 mM) by using thymic lymphocytes was similar to values reported for other cell types (25-36 mM) including tonsillar B lymphocytes (20), astrocytes (5, 50), and vascular smooth muscle cells (10, 30), but lower than some other studies on lymphocytes (17, 25, 40). These differences are likely due to the methods used to measure [Cl-]i or the assay conditions under which they were determined. Methods that determine total Cl- content in the cells, photometrically, with electrodes, or via radiolabeled Cl- dilutions generally yield higher estimates of [Cl-]i than fluorimetric techniques that determine free [Cl-]i .

Interestingly, in the present study the basal [Cl-]i was not different between cells maintained in a HCO3-/CO2-buffered solution and those maintained in the absence of HCO3-/CO2, indicating that the presence of HCO3-/CO2 in the external media is not an absolute requirement for the maintenance of normal [Cl-]i. pHi, is also similar under both conditions (4) although it can be shown to be significantly lower in the nominal absence of HCO3-. A number of possibilities that could account for this observation include 1) HCO3--independent mechanisms predominate the regulation of [Cl-]i in these cells; 2) Cl-/HCO3- exchangers do not have an absolute requirement for HCO3- but rather can use other counteranions to exchange for Cl-; and 3) both Cl- influx and Cl- efflux are equally affected by the removal of HCO3- from the media, thus resulting in no net alteration in [Cl-]i . Evidence from our study and others (18) suggests that all of these possibilities may explain the observed phenomenon.

Pretreatment of the cells with furosemide for 1 h resulted in a 38% decline in [Cl-]i but had no effect on pHi. In contrast, pretreatment with DIDS had no effect on either [Cl-]i or pHi. These findings indicate that basal [Cl-]i, but not pHi, is highly dependent on the activity of a Na+-K+-2Cl- cotransporter, whereas inhibition of the Cl-/HCO3- exchangers had no net effect on the basal [Cl-]i. This is not to say that the sodium-dependent and -independent Cl-/HCO3- exchangers are not active under basal conditions. The finding that their coinhibition had no discernible affect on [Cl-]i is likely due to their counterbalancing each other. These findings are consistent with previous studies. In rabbit aorta, it was found that [Cl-]i is decreased in the presence of furosemide but not in the presence of DIDS or in HCO3--free media (18). It had also been shown that the reduction in Cl- transport observed in the rectal gland cells of Squalus acanthias after administration of furosemide is due to the fall in the [Cl-]i and not a direct effect on the chloride current (19).

Under control conditions, in the presence of a HCO3-/CO2 solution, acute exposure of the cells to an isotonic Cl--free solution resulted in a rapid decline in [Cl-]i that was accompanied by a rise in pHi (increase in [HCO3-]i). When the cells were pretreated with furosemide and then subsequently exposed to a Cl--free solution, the rate of decline in [Cl-]i (net Cl- efflux) was reduced compared with control cells, whereas furosemide pretreatment had no effect on the change in pHi. As stated above, however, the control cells had a higher initial [Cl-]i than cells pretreated with furosemide. When the rate of decline in [Cl-]i for control cells and those pretreated with furosemide was compared at similar [Cl-]i, there was no difference, suggesting that the decrease in net Cl- efflux was caused by the lower [Cl-]i because of reduced Cl- influx, rather than a direct effect of furosemide on Cl- efflux.

To determine whether preincubation with furosemide reduced Cl- influx, the rate of Cl- influx was measured in cells that had been Cl- depleted and then reexposed to a Cl--containing solution. Preincubation of the cells with furosemide for 15 min resulted in a 45% decrease in net Cl- influx, while having no effect on the pHi. Similarly, blockade of Na+-K+-2Cl- cotransport by the acute removal of external Na+ resulted in a 46% inhibition of net Cl- influx, while having no effect on the pHi. Our findings that inhibition of Na+-K+-2Cl- cotransport reduces [Cl-]i and Cl- influx into thymic lymphocytes, but does not directly affect Cl- efflux, are in agreement with other studies that examined the effects of Na+-K+-2Cl--cotransport inhibition in lymphocytes (14) or other cell types (18, 19, 30). In contrast to these studies, others were unable to demonstrate any effect of bumetanide on [Cl-]i or Cl- influx in rat lymphocytes (17).

Evidence for the existence of a Na+-dependent Cl-/HCO3- exchanger comes from our studies examining the effects of DIDS or Na+ removal on net Cl- efflux and pHi. Pretreatment of the cells with DIDS before exposure to a Cl--free solution completely inhibited both the decline in [Cl-]i and the rise in pHi that were observed in control cells, suggesting that a Cl-/HCO3- exchange mechanism was responsible for Cl- efflux in these cells. These findings are consistent with what others have shown, examining the effects of DIDS and SITS on pHi and radiolabeled Cl- fluxes in lymphocytes (37).

It is not likely that differences in the orientation of the inhibitor binding site accounts for the different inhibitor effects on Cl- efflux and influx, but rather our data are best explained by the presence of two different transporters. Studies indicate that the stilbene binding site is located on the outer surface of the membrane rather than buried within the pocket formed by the tertiary complex of the protein (7, 44). It is also distinct from the anion binding site(s). The best model for explaining the transport of anions by the AE exchangers is the ping pong model, in which the anion binding site changes its orientation. To our knowledge, however, present models do not predict a change in the orientation of the inhibitor binding site.

Two classes of Cl-/HCO3- exchangers have been identified in mammalian cells. The band 3 family of exchangers (AE1, AE2, and AE3) is sodium independent. The activity of this family of transporters is governed by the relative concentration gradients of Cl- and HCO3- across the cell membrane. Under normal physiological conditions, this transporter is said to be inactive (17). It is activated by cellular alkalinization and normally acts as a Cl--influx mechanism (the exchange of intracellular HCO3- for extracellular Cl-). However, under experimental conditions of external Cl- removal, this antiporter can be reversed (17, 33, 37). A second Cl-/HCO3- exchanger that has been identified is a Na+-dependent Cl-/HCO3- exchanger. This antiporter acts as a Cl--efflux mechanism (the exchange of intracellular Cl- for extracellular Na+ and HCO3-) both physiologically and under experimental conditions of external Cl- removal (33). Ion flux studies suggest that the Na+-dependent Cl-/HCO3- exchanger transports one Cl- out of the cell in exchange for one Na+ and two base equivalents into the cell. The functional mode of the exchanger may be HCl extrusion in exchange for NaHCO3, or Cl- extrusion in exchange for 1 Na+ and 2 HCO3- ions (42). In either case, the transporter is electroneutral. To distinguish which of these transporters could account for the decline in [Cl-]i when the lymphocytes were exposed to a Cl--free solution, the sodium dependence of net chloride efflux was examined.

Our data show that the net Cl- efflux and rise in pHi were both completely blocked by the acute removal of Na+ from the external media, indicating that Cl-/HCO3- exchange was due to a sodium-dependent mechanism. To rule out the possibility that these effects were caused by inhibition of the Na+/H+ antiporter, additional experiments were performed with EIPA, an inhibitior of the Na+/H+ antiporter. EIPA had no effect on net Cl- efflux or the rise in pHi. Taken together, these data support the contention that in thymic lymphocytes, Cl- efflux is totally mediated by a Na+-dependent Cl- base exchanger.

Our study shows that, in the nominal absence of HCO3-/CO2 in the media, Cl- efflux is still mediated by a Na+-dependent, DIDS-sensitive mechanism. The nature of this mechanism has not been elucidated, but the evidence suggests that it may be a different functional mode of the Na+-dependent Cl-/HCO3- exchanger. To date, this transporter has not been isolated and cloned. However, if one were to extrapolate the findings on other anion exchangers to that of the Na+-dependent Cl-/HCO3- exchanger then it would be likely that this transporter would be capable of using a number of different anions to exchange for Cl-i.

Studies have shown that the band 3 protein family of Cl-/HCO3- exchangers is capable of transporting a number of different anions and working in a number of different modes in addition to Cl-/HCO3- exchange (e.g., Cl-/Cl-exchange, SO42-/SO42- exchange, etc.). AE1 has a higher affinity for nitrite than for Cl- (15) and has also been shown to transport SO4-2 (9, 26, 46, 48) and divalent and monovalent forms of phosphate (16). Transfection studies with AE2 have also shown that it is capable of transporting a number of different anions (24). Cl- efflux via AE2 is maximally stimulated by external Cl- or nitrite, with the Michaelis-Menten constant for external Cl- (Cl-/Cl- exchange) of 5.6 mM. The rank order for activation of AE2 by other external anions is bromide > isethionate >=  gluconate > iodine (24). Thus substitution of Cl- with gluconate and isethionate, as was done in our present study, would greatly decrease the activity of AE2 but would not be expected to completely inhibit it. It is also possible that in the nominal absence of HCO3-/CO2 in the external media, moderate levels of Cl- flux could still be driven by ambient HCO3-. It has been estimated that at pHi 7.4, ambient atmospheric CO2 would result in [HCO3-]i of ~0.18 mM, and that this could help drive Cl- exchange (37). It has also been suggested that band 3 protein may also be able to act as a Cl-/OH- exchanger (22).

When Cl--depleted cells were reexposed to a Cl--containing solution, there was a rapid rise in [Cl-]i and a fall in pHi. Furosemide partially inhibited the initial rise in [Cl-]i (45%) but had no effect on pHi. Removal of Na+ from the Cl--containing solution also partially blocked Cl- influx but had no affect on the decline in pHi when the cells were reexposed to a Cl--containing solution. Thus Cl- influx was partially due to a sodium-dependent, furosemide-sensitive mechanism, whereas the decline in pHi was due to a sodium-independent, furosemide-insensitive mechanism. Preincubation with DIDS also partially inhibited the initial rise in [Cl-]i (56%) but, in contrast to furosemide, completely inhibited the fall in pHi. Preincubation of the cells with furosemide and DIDS together completely inhibited Cl- influx. Taken together, these findings indicate that after Cl- depletion, Cl- influx is mediated by both a Na+-K+-2Cl- cotransporter and a Na+-independent Cl-/HCO3- exchanger (band 3 protein).

In control cells that had been Cl- depleted, on reexposure to a Cl--containing solution, the [Cl-]i continued to increase after the decline in pHi had waned. This suggests that either the activity of the band 3 protein turns off at a lower [Cl-]i than the Na+-K+-2Cl- cotransporter or that other pHi regulatory mechanisms are turned on to help maintain the pHi. Experimental evidence and theoretical considerations suggest that the activity of the band 3 protein is driven by the pHi (or [HCO3-]) rather than the [Cl-] (21, 36, 37). Thus once the pHi approaches the basal level the activity of the band 3 protein would be expected to greatly diminish, whereas the Na+-K+-2Cl- cotransporter would still be activated by the favorable net ionic driving force. This lack of activity of band 3 protein near basal pHi is not unique. The kinetic profile of the Na+/H+ antiporter is such that it is also minimally active near the resting pHi in lymphocytes (43). Thus near resting pHi, Cl- entry may depend on Na+-K+-2Cl--cotransport activity and be unassociated with changes in pHi.

Band 3 protein-mediated anion exchange has been previously demonstrated in lymphocytes (17, 27, 28). The band 3 anion exchangers, AE1, AE2, and AE3, are sequence analogs of the erythroid (AE1) isoforms. They share ~80% homology in their membrane spanning COOH-terminus domain (2, 31). AE2 has a shorter cytoplasmic sequence but additional extracellular mass compared with AE1 and displays a lower affinity for DIDS compared with AE1 (36). Lymphocytes, as do most other cell types, express the AE2 isoform (1, 2, 27). Three AE2 variants, which are caused by alternative splicing of the gene, have been found in the rat. AE2a is expressed in all tissues examined, whereas AE2b is more restricted, with highest levels in the stomach, and AE2c only expressed in the stomach (51). Thus AE2 activity is likely responsible for ~50-60% of the Cl- entry in thymic lymphocytes in the presence of HCO3-/CO2.

In summary, our study shows that DIDS-sensitive mechanisms of Cl-/HCO3- or Cl-/base exchange regulate both Cl- influx and efflux. Net Cl- efflux is totally DIDS sensitive and is mediated by a Na+-dependent, Cl-/HCO3- exchanger. Net Cl- influx, by contrast, is only partially DIDS sensitive and partially furosemide sensitive. Cl- influx, unlike Cl- efflux, occurs via a Na+-independent, Cl-/HCO3- exchanger and Na+-K+-2Cl- cotransport.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Batlle, Northwestern Univ. Medical School, Div. of Nephrology and Hypertension, 320 East Superior, Searle 10-475, Chicago, IL 60611 (d-batlle{at}northwestern.edu).

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.

Received 26 May 2000; accepted in final form 19 October 2000.


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Am J Physiol Renal Fluid Electrolyte Physiol 280(2):F314-F324
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