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
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ABSTRACT |
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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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INTRODUCTION |
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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,
-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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METHODS |
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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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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 [ClCalculation 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.
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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).
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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RESULTS |
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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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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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DISCUSSION |
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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.
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FOOTNOTES |
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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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