Transport characteristics of the apical anion exchanger of
rabbit cortical collecting duct
-cells
Cheryl
Emmons
Departments of Internal Medicine, University of Cincinnati and
Cincinnati Veterans Affairs Medical Center, Cincinnati, Ohio
45267-0585
 |
ABSTRACT |
To functionally characterize transport properties of the apical
anion exchanger of rabbit
-intercalated cells, the mean change in
anion exchange activity,
dpHi/dt
(where pHi is intracellular pH), was measured in response to lumen
Cl
replacement with
gluconate in perfused cortical collecting ducts (CCDs).
-Cell apical
anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS
in the presence of 115 mM
Cl
. In contrast, apical
anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the
absence of Cl
.
-Cell
apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM
phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However,
-cell
apical anion exchange was inhibited by
-cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM.
Substitution of either sulfate or gluconate for lumen
Cl
resulted in a similar
rate of alkalinization. Conversely,
pHi was unchanged by substitution
of sulfate for lumen gluconate, confirming the lack of transport of
sulfate on the
-cell apical anion exchanger. Taken together, the
results demonstrate a distinct "fingerprint" of the rabbit CCD
-cell apical anion exchanger that is unlike that of other known
anion exchangers.
intercalated cell; anion exchange; intracellular pH; acid-base
transport
 |
INTRODUCTION |
THE CORTICAL SEGMENT of the kidney collecting duct
(CCD) plays an important role in the maintenance of acid-base balance. CCD transepithelial bicarbonate transport is mediated by intercalated cells.
-Intercalated cells are thought to effect transepithelial bicarbonate secretion via an apical
Cl
/HCO
3
exchanger, whereas
-intercalated cells are thought to function in
bicarbonate absorption via a basolateral Cl
/HCO
3
exchanger (27, 28). Three anion exchanger isoforms have been
identified, with the prototype being AE1 (or band 3) of the erythrocyte
(1, 19). Immunocytochemical studies have identified the basolateral
anion exchanger of
-cells as AE1 (4, 27); however, the protein
identity of the apical anion exchanger remains uncertain. No antibodies
to any of the anion exchange isoforms have ever been demonstrated to
bind to the CCD
-cell apical membrane (4, 27). In addition, the apical anion exchanger of CCD
-cells is not inhibited by
4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid
(H2-DIDS), a derivative
of the stilbene drugs that inhibit all three anion exchange isoforms in
micromolar concentrations (12). Similarly, CCD bicarbonate secretion is
insensitive to stilbenes (26). A recent study of a transformed rabbit
kidney
-cell line suggested that the apical anion exchanger was AE1, the same protein as the basolateral exchanger (33). However, using
primary
-cell cultures, Fejes-Toth and co-workers (13) found that
AE1 was expressed differentially in
- and
-cells, suggesting that
AE1 does not function as both the apical and the basolateral anion
exchanger of intercalated cells. Such studies are hindered by the lack
of any cell culture line with stable transport characteristics
consistent with those demonstrated for
-cells from in vitro
perfusion experiments and the lack of markers for distinguishing
-
and
-cells, combined with the lack of concordance of such markers
(i.e., apical peanut lectin binding for
-cells) to functional
properties of the cells. The present studies were done to functionally
characterize transport properties of the apical anion exchanger of
-cells of in vitro perfused rabbit CCDs relative to known properties
of the three anion exchange isoforms, specifically with regard to
1) effect of
Cl
on stilbene sensitivity,
2) sensitivity to nonstilbene
inhibitors, and 3) transport of sulfate.
 |
MATERIALS AND METHODS |
In vitro tubule perfusion. Rabbit CCDs
were isolated and perfused in vitro as described previously (11, 12).
Male New Zealand White rabbits, 1.5-2 kg, with free access to
rabbit chow (Agway Prolab, Syracuse, NY) and water were killed by
cervical dislocation after pentobarbital anesthesia. The left kidney
was removed, cut in coronal sections, and placed in room temperature solution 1 (see Table
1). CCDs ~0.4 mm in length were dissected from the
outer one millimeter of cortex, with the superficial end
starting at the most distal connecting tubule arcade into the
collecting duct. The tubule was transferred to a 100-µl laminar flow
chamber and cannulated, and the peritubular bathing solution was
exchanged at a rate of 2 ml/min. For the experiments demonstrating the
ability to measure intracellular pH
(pHi) changes due to sulfate transport on AE1 in
-cells, outer medullary collecting ducts were
dissected from the inner stripe
(OMCDis).
Solutions. Table 1 lists the
compositions of the solutions used in this study.
Solution 1 was
Na+ free (tetramethylammonium
replacement) and HEPES buffered and was used for dissection.
Solution 2 was used for dye loading
and, in combination with solution 3,
for identification of anion exchange. These two solutions were
Na+ free and
HCO
3 buffered, differing only in the presence (solution 2) or absence of
Cl
(equimolar gluconate
replacement in solution 3). In
solution 4, equimolar sulfate replaced
Cl
.
Solution 5, with addition of 10 µM
nigericin, was used for calibration of
pHi. The
HCO
3-containing solutions were bubbled with 6.5% CO2 and 93.5%
O2, whereas the HEPES-containing
solutions were bubbled with 100%
O2. Tetramethylammonium
bicarbonate was made by bubbling tetramethylammonium hydroxide with
100% CO2. Glass tubing surrounded
by heated water jackets connected the solution reservoirs and the
perfusion chamber to minimize any CO2 or temperature loss. The pH of
solutions 1,
2, 3,
and 4 was adjusted to 7.40 with
tetramethylammonium hydroxide or gluconic acid lactone, and all
experiments were performed at 37°C.
pHi
measurements/confocal imaging.
To measure pHi, 5 µM of the
acetoxymethyl ester of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF-AM) was added to the perfusate (solution
2) for 5 min, resulting in selective dye loading of
only the intercalated cells (34). The fluorescent measurements were
done with a dual-excitation laser-scanning inverted confocal
fluorescent microscope, as previously described (11, 12). Briefly, a
laser-scanning system (model MRC600; Bio-Rad, Hercules, CA) was coupled
to the side port of a Diaphot inverted microscope (Nikon, Melville,
NY). Excitation at 488 nm was provided by an argon laser
(model 5424A; Ion Laser Technology, Salt Lake City, UT), and a
helium-cadmium laser (model 4214NB; Liconix, Santa Clara, CA) was used
for the 442-nm excitation. A ×40 fluorite objective (Nikon, NA
0.8) was used for all experiments.
pHi was measured in single
intercalated cells from an area of cytoplasm ~2-4 µm in
diameter in up to three cells along a 250-µm length of tubule (SOM
software, Bio-Rad) using gray scale image analysis. Tubule images were
obtained using a zoom factor of 2.5, for a final magnification of
×800. The duration of laser exposure was limited to 1 s per ratio
by a computer-driven electronic shutter (Vincent Associates, Rochester,
NY). pHi measurements were taken every 5-10 s after a solution change. At the end of every
experiment, an in vitro BCECF calibration curve was performed for each
cell using nigericin and high-K+
solutions. Earlier studies using these solutions with a pH of 6.00, 6.30, 6.80, 7.35, and 7.80 for calibration points have shown that the
488/442 nm fluorescence excitation
ratio-pHi relationship between
6.80 and 7.35 is linear in this experimental system (9). So for the
present study, the pH of solution 5 was adjusted to only 7.35 and 6.80, values that bracketed the
pHi changes occurring in the
experiments, with KOH or HCl. The maximum decrease in the absolute
intensity from the 442-nm excitation from the start to the end of any
experiment was <10%.
Intercalated cell subtyping.
Complexities exist in the functional phenotyping of rabbit CCD
intercalated cells. By measuring pHi of CCD intercalated cells in
response to sequential replacement of lumen and basolateral
Cl
with gluconate, Emmons
and Kurtz (12) first functionally identified rabbit outer CCD
intercalated cells with both apical and basolateral anion exchange,
noting that 57% of rabbit outer CCD intercalated cells had both apical
and basolateral anion exchange (called
-cells), whereas 39% had
exclusively apical anion exchange (
-cells) and 4% had only
basolateral anion exchange (
-cells). Concerned that intracellular
Cl
depletion with such a
protocol altered the ability to detect basolateral anion exchange,
Weiner et al. (35) found that all female rabbit CCD intercalated cells
with apical anion exchange also possessed basolateral anion exchange
when the apical anion exchanger was immobilized by removal of both
Cl
and
HCO
3 simultaneously from the lumen. In contrast, in a preliminary report, Emmons (10) found 55% of intercalated cells from male CCDs were identified as possessing exclusively apical anion exchange with either a 0 Cl
, 25 mM
HCO
3 or a 0 Cl
, 0 HCO
3 perfusate, whereas 45% cells
demonstrated both apical anion exchange and basolateral with either
perfusate, suggesting the existence of discrete
- and
-subtypes
in the male rabbit CCD at a given point in time.
In the present study, tubules were perfused and bathed in
Na+-free,
HCO
3-buffered solution
(solution 2). The perfusate was then
changed to a dye-free solution (solution 2) for 5 min before any measurements were recorded.
All cells were initially subtyped according to the location of anion
exchange. To functionally identify anion exchange,
Cl
was replaced with
gluconate in the perfusate (solution
3). Resultant intracellular alkalinization identified
the presence of apical Na+-independent anion exchange
(11, 12). After a new steady-state pHi was obtained (~1 min),
Cl
was then replaced with
gluconate in the bathing solution. Additional intracellular
alkalinization identified the presence of basolateral Na+-independent anion exchange.
-Cells were identified as those intercalated cells that demonstrated
exclusively apical anion exchange. In the present study,
-cells
represented 48% of the outer CCD intercalated cells. Anion exchange
activity was measured as the
dpHi/dt,
in the initial 30 s after lumen
Cl
replacement with
gluconate. All measurements of anion exchange activity were completed
within 30 min of initiation of perfusion due to the time-dependent
decay in HCO
3 transport by in vitro
perfused rabbit CCDs (26).
Statistics. Results are reported as
means ± SE. A paired t-test was
used to compare results of anion exchange activity for the same cell in
the protocols before and after exposure to a specific inhibitor and in
the protocols designed to study sulfate transport. Statistical
significance was accepted at the P
0.05 level.
Materials. Tetramethylammonium
hydroxide was from Fisher. BCECF-AM was purchased from Molecular
Probes. All other chemicals were from Sigma.
 |
RESULTS |
Effect of stilbenes on apical anion exchange of
rabbit CCD
-intercalated cells.
Prior studies had demonstrated a lack of effect of 0.5 mM
H2-DIDS on the apical anion
exchanger of rabbit CCD
-cells (12). However,
H2-DIDS is known to react more
slowly with erythroid AE1 than DIDS (23). In addition, DIDS is the most
potent stilbene antagonist of AE1 and is the stilbene derivative that
has been most extensively studied for the three anion exchange isoforms (7). To determine the sensitivity of the apical anion exchanger of CCD
-cells to DIDS, apical anion exchange activity was measured in
single
-intercalated cells in paired experiments before and after
~15 min addition of DIDS to the perfusate. Figure
1 shows a representative trace of this
protocol with 0.2 mM lumen DIDS. In this tracing, baseline
pHi was ~7.0. Replacement of
lumen Cl
with gluconate at
point A resulted in a brisk
intracellular alkalinization. Neither replacement of bath
Cl
with gluconate nor
subsequent return of bath
Cl
caused any further
pHi change. Then,
Cl
was returned to the
lumen in addition to 0.2 mM lumen DIDS. After about 15 min, lumen
Cl
was again replaced with
gluconate in the ongoing presence of 0.2 mM DIDS
(point B). An intracellular
alkalinization similar to that in the absence of DIDS resulted. There
was no difference in baseline pHi
prior to the measurement of initial anion exchange (point A) between the groups of
tubules (6.90 ± 0.06 in the 0 mM DIDS group, 6.94 ± 0.08 in the 0.1 mM DIDS group, and 6.88 ± 0.05 in the
0.2 mM DIDS group). Mean baseline anion exchange activity
(dpHi/dt
at point A) was no different between
the groups of tubules (1.28 ± 0.08 pH/min in the 0 DIDS group, 1.22 ± 0.10 pH/min in the 0.1 mM DIDS group, and 1.16 ± 1.12 pH/min
in the 0.2 mM DIDS group). There was also no difference in baseline
pHi prior to the measurement of
anion exchange activity in the absence (point
A) and the presence of lumen DIDS
(point B) in each group of tubules
(data not shown). In the presence of
Cl
, the mean percent
inhibition of apical anion exchange was 5.0 ± 2.2% with 0.1 mM
lumen DIDS (n = 15 cells, 6 CCDs) and 4.3 ± 2.8% with 0.2 mM lumen DIDS
(n = 27 cells, 9 CCDs). These values were no different than the 4.7 ± 2.6% mean inhibition found in control experiments (n = 38 cells, 12 CCDs). Thus
-cell apical anion exchange was not affected by either
0.1 or 0.2 mM lumen DIDS applied in the presence of 115 mM
Cl
.

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Fig. 1.
Representative trace showing effect of 0.2 mM lumen DIDS exposure in
presence (+) and absence ( ) of lumen
Cl on apical anion exchange
activity in a -cell. Rate of intracellular alkalinization in
response to replacement of lumen
Cl
(solution 2, Table 1) with gluconate
(solution 3), used as a measure of
apical anion exchange activity, is unchanged before
(point A) and after
(point B) 14-min perfusion with 0.2 mM lumen DIDS. In presence of 115 mM
Cl , 0.2 mM DIDS did not
inhibit -cell apical anion exchange.
pHi, intracellular pH.
|
|
Stilbenes are known to compete with
Cl
for the external binding
site of erythroid AE1 (7, 23). To determine whether the lack of DIDS
sensitivity of the
-cell apical anion exchanger was due, at least in
part, to the presence of
Cl
, additional experiments
were performed with luminal DIDS exposure in the prolonged absence of
Cl
. A representative trace
of this protocol is seen in Fig. 2. After initial removal and return of lumen
Cl
to identify apical anion
exchange (point A), lumen
Cl
was again replaced with
gluconate but with either 0.1 or 0.2 mM DIDS added to the perfusate
(point B). The tubule was perfused with DIDS in the prolonged absence of lumen
Cl
for ~15 min. Then,
Cl
was returned to the
lumen, in the ongoing presence of DIDS. Finally, lumen
Cl
was again replaced by
gluconate to assess apical anion exchange activity
(point C). In contrast to the lack
of effect of DIDS on
-cell apical anion exchange activity in the
presence of Cl
, apical
anion exchange activity was significantly decreased by lumen DIDS
exposure in the absence of
Cl
, as can be seen at
point C in Fig. 2. There
was no difference in baseline pHi
prior to the measurement of initial anion exchange (point A) between the groups of
tubules (6.88 ± 0.06 in the 0 DIDS group, 6.92 ± 0.05 in the
0.1 mM lumen DIDS group, and 6.90 ± 0.06 in the 0.2 mM lumen DIDS
group). There was no difference in baseline anion exchange activity
regardless of whether it was measured at either
point A or point
B for any tubule (data not shown). Mean baseline anion
exchange
(dpHi/dt
at point A) was no different in the
three groups of tubules (1.26 ± 0.10 pH/min in the 0 DIDS group,
1.28 ± 0.14 pH/min in the 0.1 mM DIDS group, and 1.18 ± 0.12 pH/min in the 0.2 mM DIDS group). There was also no difference in
baseline pHi prior to measurement
of anion exchange activity in the absence (points
A and B) and
presence (point C) of lumen DIDS for
any group of tubules (data not shown).

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Fig. 2.
Representative trace showing effect of 0.2 mM lumen DIDS exposure in
absence of lumen Cl on
apical anion exchange activity in a -cell. After initial measurement
of apical anion exchange at point A
(as described for Fig. 1 and in the text), 0.2 mM DIDS was added to a
Cl -free luminal solution
(solution 3) at
point B, and perfusion continued for
~15 min. Remeasurement of apical anion exchange at
point C revealed a significantly
decreased rate of alkalinization, indicating inhibition of apical anion
exchange by 0.2 mM DIDS in absence of
Cl .
|
|
Figure 3 summarizes the mean
percent inhibition of
-cell apical anion exchange due to lumen DIDS
exposure in the absence of
Cl
. In control experiments
with this protocol,
-cell apical anion exchange was not altered
after a 15-min perfusion without lumen Cl
(n = 27
-cells, 9 CCDs).
In contrast, after 0.1 mM lumen DIDS exposure in the absence of
Cl
, there was a 36%
inhibition of apical anion exchange (n = 8
-cells, 4 CCDs, P < 0.05 vs.
control). Similarly, after 0.2 mM lumen DIDS exposure in the absence of
Cl
, there was a 67%
inhibition of apical anion exchange (n = 24
-cells from 9 CCDs, P < 0.001 vs. control). Thus, when applied in the absence of
Cl
, both 0.1 and 0.2 mM
lumen DIDS caused significant inhibition of
-cell apical anion
exchange.

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Fig. 3.
Mean percent inhibition of -cell apical anion exchange due to a
15-min exposure to lumen DIDS in absence of lumen
Cl . When applied in absence
of Cl , both 0.1 and 0.2 mM
lumen DIDS caused significant inhibition of -cell apical anion
exchange. * P < 0.05 vs. 0 DIDS; ** P < 0.001 vs. 0 DIDS;
n indicates number of cells.
|
|
Effect of nonstilbene anion exchange inhibitors on
the apical anion exchange of rabbit CCD
-intercalated
cells.
To determine the sensitivity of the CCD
-cell apical anion exchanger
to other nonstilbene anion exchange inhibitors, apical anion exchange
activity was measured in single
-intercalated cells in paired
experiments before and after ~15 min of luminal addition of known
anion exchange inhibitors. An example of this protocol for 10 mM
phenylglyoxal is shown in Fig. 4. At
point A, baseline apical anion
exchange was assessed by replacement of lumen
Cl
with gluconate. Then
Cl
was returned to the
lumen with the addition of 10 mM phenylglyoxal for ~14 min. At
point B, apical anion exchange was
remeasured in the ongoing presence of phenylglyoxal. As is evident,
there was no inhibition of apical anion exchange activity. Table
2 lists the different nonstilbene
inhibitors tested in paired experiments with 15-min exposure to the
particular inhibitor. In separate paired experiments, neither 15 mM
maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM
ethacrynic acid, 1 mM furosemide, 1 mM probenecid, nor 0.1 mM
diphenylamine-2-carboxylate (DPC) inhibited
-cell apical anion
exchange activity.

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Fig. 4.
Representative trace showing effect of 10 mM lumen phenylglyoxal on
apical anion exchange of a -cell. Apical anion exchange was
unchanged before (point A) and after
(point B) 12 min of perfusion with
10 mM phenylglyoxal.
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|
In contrast to the above agents that had no effect on
-cell apical
anion exchange, 6 mM
-cyano-4-hydroxycinnamic acid (CHC) almost
totally inhibited apical anion exchange activity. Additional experiments with CHC found that the degree of inhibition of apical anion exchange for a given CHC concentration was no different after
either a 1-min or a 15-min exposure to luminal CHC (data not shown).
So, because of the nonspecificity of this inhibitor, a 1-min luminal
CHC exposure was used in subsequent experiments to determine a
dose-response curve. An example of this protocol is seen in Fig.
5. Baseline
pHi in this
-cell was ~6.95.
At point A, lumen
Cl
was replaced with
equimolar gluconate, resulting in a brisk alkalinization, which
returned to baseline with return of lumen
Cl
. After a few minutes, 6 mM CHC was added to the perfusate for 1 min, and then, at
point B, lumen
Cl
was again replaced with
gluconate in the ongoing presence of CHC.
pHi was essentially unchanged with
this maneuver. As is also seen in Fig. 5, apical anion exchange
inhibition by CHC was reversible (point
C). The mean percent inhibition of
-cell apical anion exchange for various concentrations of CHC is
listed in Table 2 and reveals an
IC50 of 2.4 mM.

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Fig. 5.
Representative trace showing the effect of a 1-min exposure to 6 mM
lumen -cyano-4-hydroxycinnamic acid (CHC) on apical anion exchange
of a -cell. Compared with initial apical anion exchange activity in
this cell (point A), apical anion
exchange is significantly inhibited after 1-min exposure to 6 mM
luminal CHC (point B). This also
demonstrates the reversibility of this inhibition of apical anion
exchange after 5-min perfusion without CHC (point
C).
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|
Transport of sulfate on the
-cell
apical anion exchanger.
One of the substrates band 3 is known to transport in addition to
Cl
and
HCO
3 is sulfate. In the first protocol designed to investigate the substitution of sulfate on the apical anion
exchanger of CCD
-cells, pHi
was measured as lumen Cl
was acutely replaced by either equimolar gluconate or sulfate in random
order. Figure 6 shows a representative
tracing for this protocol. Baseline
pHi was ~7.0. Replacement of
lumen Cl
with equimolar
sulfate (point A) resulted in a
brisk alkalinization. Return of lumen
Cl
resulted in return to a
similar baseline pHi. Then,
replacement of lumen
Cl
with gluconate
(point B) resulted in an
alkalinization that was of a rate and magnitude similar to that which
occurred with sulfate perfusion. Results of this protocol from 35
-cells are summarized in Table 3. Return
of lumen Cl
between anion
substitutions resulted in similar baseline
pHi for both sulfate and gluconate
(data not shown). Both the rate of
pHi increase and the
pH were
similar regardless of whether either gluconate or sulfate replaced
lumen Cl
.

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Fig. 6.
Representative trace showing effect of replacement of lumen
Cl by equimolar sulfate
(solution 4) or gluconate
(solution 3) on -cell
intracellular pH (pHi). Similar
intracellular alkalinizations result from substitution of either
sulfate (point A) or gluconate
(point B) for luminal
Cl .
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|
In a second protocol designed to investigate transport of sulfate on
the apical anion exchanger, lumen
Cl
was initially replaced
with gluconate. After a new steady-state pHi was reached, lumen gluconate
was then replaced with either equimolar sulfate or chloride in a random
order. Figure 7 demonstrates a typical
tracing for this protocol. Baseline
pHi was ~6.95. Replacement of
lumen Cl
by gluconate
resulted in a brisk alkalinization, with a new steady-state pHi of ~7.25. At
point A, equimolar sulfate was
substituted for gluconate. pHi was
essentially unchanged over the next 8 min. However, substitution of
Cl
for lumen sulfate
(point B) resulted in a brisk
acidification, back to the baseline
pHi. Table
4 summarizes the results of this protocol
for 41
-cells. Neither the rate of
pHi change nor the
pH was
significantly different from zero when sulfate replaced lumen
gluconate. In contrast, brisk acidification was always seen when
Cl
was returned to the
lumen in place of gluconate. Taken together, these data indicate that
sulfate is transported similarly to gluconate on the apical anion
exchanger of rabbit CCD
-cells.

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Fig. 7.
Representative trace showing effect of replacement of lumen gluconate
(solution 3) by equimolar sulfate
(solution 4) or
Cl
(solution 2) on -cell
pHi. At point
A, -cell pHi is
unchanged when sulfate is substituted for lumen gluconate; however, a
brisk intracellular acidification occurs at point
B when Cl
is substituted for lumen gluconate.
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|
Although sulfate is thought to have the same binding affinity for
erythroid AE1 as Cl
, it is
transported at a rate ~3 orders of magnitude slower than Cl
(16). To make certain
that the reason for the apparent lack of sulfate transport on the
apical anion exchanger of
-cells was not due to inability of the
experimental system used for these studies to detect sulfate-induced
pHi changes, experiments were done
to measure pHi of single
-cells
(known to have basolateral AE1) from rabbit
OMCDis in response to basolateral
ionic changes. For each OMCDis
intercalated cell, the absence of an apical anion exchanger in the cell
was initially demonstrated by the lack of any
pHi change resulting from
replacement of lumen Cl
with gluconate. Then, pHi of
single OMCDis
-cells was
measured as bath Cl
was
acutely replaced by either equimolar gluconate or sulfate in random
order. Replacement of bath
Cl
with equimolar gluconate
resulted in an alkalinization at a rate of 1.53 ± 0.19 pH/min in 14
-cells from 6 OMCDis, similar
to that seen for CCD
-cells. However, in contrast to the results in
CCD
-cells (Table 3; Fig. 6), after return of bath
Cl
and restoration of
baseline pHi, replacement of bath
Cl
with equimolar sulfate
in the same cells caused an intracellular acidification at a rate of
1.01 ± 0.20 pH/min.
For erythroid AE1, sulfate transport by the basolateral anion exchanger
occurs by sulfate-proton cotransport (16). Although the mode of sulfate
transport by kidney AE1 has not yet been proved, given the presence of
E681 (the proton binding site for sulfate-proton cotransport in human
red cells) in kidney AE1, it is very likely that sulfate-proton
cotransport occurs in the kidney also. When bath
Cl
is replaced by equimolar
sulfate, the ion concentration gradients acutely change, with a 25 mM
Cl
concentration gradient
from the cell to the bath and an ~115 mM sulfate concentration
gradient from the bath into the cell. Thus the intracellular
acidification can be explained by
Cl
leaving the cell down
its concentration gradient in exchange for sulfate entry down its
concentration gradient from the bath into the cell.
To confirm the ability to identify sulfate-induced
pHi changes in
-cells, separate
experiments were done where bath
Cl
was initially changed to
equimolar gluconate, resulting in alkalinization. After establishment
of a new steady-state pHi, the
bath solution was changed to either equimolar
Cl
or sulfate, in a random
order. OMCDis
-cell
pHi decreased at a rate of
1.45 ± 0.14 pH/min in 11 cells from 4 tubules in response to
Cl
substitution for bath
gluconate, not different from the response demonstrated in CCD
-cells. However, in contrast to the response in CCD
-cells (Table
4; Fig. 7), OMCDis
-cell pHi decreased at a rate
of
1.12 ± 0.10 pH/min when sulfate was substituted for bath
gluconate in the same 11 cells.
One technical point worthy of note concerns the differences in
effective osmotic coefficients between anions and the
possibility that increased bath osmolarity with sulfate substitution
altered cell volume. That cell volume did not change in the experiments with bath sulfate substitution was evidenced by the lack of any directional change in the 442-nm signal (within the overall <10% decrease that occurs over the time course of any experiment due to
bleaching and dye loss). In addition, neither cell area based on
two-dimensional tracings of individual cell outlines from saved images
taken along the longitudinal axis of the tubule 1 min after each ion
substitution nor the inner or outer diameters of the tubule from the
same images were altered in either bath chloride, gluconate, or sulfate
(data not shown). Finally, intracellular dye uptake in the cells was
homogeneous, so it is unlikely that the measured signal came mostly
from an intracellular osmotically isolated compartment. This indicates
that no volume changes occurred with the bath anion substitutions.
Thus the pHi changes seen in the
bath sulfate substitution protocols identify functional sulfate
transport on the basolateral surface of
OMCDis
-cells. This could be
accomplished by exchange of intracellular chloride for extracellular
sulfate, but might be masked by exchange of intracellular bicarbonate
for extracellular sulfate. These could well be mediated by AE1.
However, another sulfate/anion exchanger might also be contributing,
since at pH 7.0, erythroid AE1 has never been observed to transport
sulfate so rapidly in comparison to chloride.
 |
DISCUSSION |
There are at least three members of the AE anion exchanger gene family.
AE1, the prototype anion exchanger, comprises ~25% of the protein
mass of the erythrocyte membrane and has also been identified as the
basolateral anion exchanger of CCD
-intercalated cells. In addition,
two nonerythroid anion exchange isoforms (AE2 and AE3) have been
identified. AE2 has been shown to be a basolateral anion exchanger in
non-
-intercalated cells, and at least one other AE has been reported
in the kidney in abstract publications (2, 3).
Although the three isoforms have ~85% amino acid sequence homology
of their transmembrane domains, each has a distinctive pattern of
tissue expression, and differences in both transport function and
regulation between the isoforms have also been demonstrated (1, 19).
The present studies were done to functionally "fingerprint" transport properties of the apical anion exchanger of
-intercalated cells of in vitro perfused rabbit CCDs relative to known properties of
the three anion exchange isoforms, specifically with regard to
1) effect of
Cl
on stilbene sensitivity,
2) sensitivity to nonstilbene
inhibitors, and 3) transport of sulfate.
One distinguishing characteristic of the three anion exchange isoforms
is their sensitivity to stilbene inhibitors. Stilbenes can cause
inhibition of an anion exchanger either reversibly by competing with
extracellular anions for the external anion binding site or
irreversibly by binding covalently to one of two conserved lysines.
Thus the particular stilbene agent used, as well as its concentration
and the duration of exposure, the presence of other anions, and the
temperature affect the degree of inhibition of anion exchange (7, 16,
23). DIDS is the most potent stilbene antagonist of AE1 (7). Of the
three isoforms, the erythroid AE1 isoform demonstrates the highest
degree of inhibition due to DIDS, with an
IC50 of 0.08 µM in the presence
of Cl
(7). Although still
sensitive in the micromolar range, the nonerythroid anion exchangers
require higher DIDS concentrations for inhibition. Humphreys and
coworkers (15) found an IC50 of 13 µM in 131 mM Cl solutions, but 0.53 µM in 0 Cl solutions for murine
AE2 expressed in oocytes. Similarly, He and coworkers (14) found an
IC50 of DIDS of 4 µM for rat AE2
expressed in SF9 cells in 0 Cl medium. In contrast, Lee and co-workers
(21) found a DIDS IC50 of 142 µM
for mouse AE2 expressed in human 293 cells. It is
noteworthy that the IC50 for
Cl
flux on the anion
exchanger in K562 cells, known to express AE2 mRNA, is 1.5 µM (20).
The reasons for the higher AE2 inhibitory DIDS concentration
demonstrated in the study by Lee et al. (21) is not clear but may be
reflective of the different expression systems used in the two studies,
either due to differences in membrane composition of the specific
expression system or differences in the surface density of the
expressed anion exchanger. For murine AE3, Lee and co-workers (21)
found the DIDS IC50 was 0.43 µM in 0 Cl solutions at 37°C for this isoform expressed in human 293 cells.
The present studies demonstrate that the apical anion exchanger of
rabbit CCD
-cells demonstrates significantly less sensitivity to
DIDS than is known for the three anion exchange isoforms. It should be
noted that many of the studies in erythrocytes or of expressed anion
exchangers involve significantly longer exposures to the inhibitors
than used in the present study. In the present studies, the exposure to
DIDS was limited to 15 min because of the time required to load the
cells with BCECF and complete the baseline anion exchange measurement
coupled to the known time-dependent decay in CCD bicarbonate transport,
and likely reflects a combination of reversible and irreversible
interactions. In the presence of Cl
, 0.2 mM DIDS had no
effect on the
-cell apical anion exchanger, whereas in the absence
of Cl
, the 0.2 mM DIDS
significantly inhibited the
-cell apical exchanger. It is unlikely
that depletion of intracellular
Cl
is responsible for the
effect of Cl
removal on
DIDS sensitivity, given the similar alkalinization seen in the control
tubules with this protocol without DIDS. Although the higher
concentrations of DIDS required to inhibit the
-cell apical
exchanger might reflect differences in membrane composition of the
-cell, this characteristic could also be due to structural differences in the stilbene-binding site compared with the known anion
exchange isoforms.
In addition to stilbenes, several other agents inhibit anion exchange
at relatively low concentrations (16). Of the three anion exchange
isoforms, nonstilbene inhibitor potency has been studied most
extensively for AE1. Motais and Cousin (24) found an instantaneous and
reversible inhibition of ox erythrocyte anion exchange to ethacrynic
acid with an IC50 of 7 µM.
Cousin and Motais (8) found that niflumic acid caused an instantaneous,
reversible, and noncompetitive inhibition with an
IC50 of 0.6 µM in human erythrocytes. Obaid et al. (25) showed that 15 mM maleic anhydride, an
agent known to react with amino groups of proteins, caused a 40%
decrease in Cl flux in human erythrocytes. Motais and Cousin (24) found
an IC50 of 40 µM to probenecid
in ox erythrocytes. Brazy and Gunn (6) found a rapid inhibition of
human erythrocyte anion exchange by furosemide, with an
IC50 of 150 µM. Carbantchnik and Greger (7) reported an
IC50 of 70 µM for DPC for anion exchange in human erythrocytes. Although phenylglyoxal is usually thought of as an arginine-specific reagent, this directed inhibition occurs under an alkaline extracellular pH and is
competitive with Cl
. In
conditions different from these, phenylglyoxal can also cause a
reversible non-arginine-specific inhibition and has been
shown to inhibition Cl
self-exchange in human erythrocytes with an
IC50 of 2 mM, studied at pH 7.2 (17). In contrast, under the perfusion conditions in this study,
-cell apical anion exchange was not altered by 1 mM ethacrynic acid,
0.2 mM niflumic acid, 15 mM maleic anhydride, 1 mM probenecid, 1 mM
furosemide, 0.1 mM DPC, or 10 mM phenylglyoxal.
Less is known about the effects of nonstilbene inhibitors on the AE2
and AE3 isoforms. Most information about nonstilbene inhibitor
potencies for AE2 comes from the work of Humphreys and coworkers (15),
who measured
36Cl
influx mediated by the murine isoform expressed in
oocytes. In separate studies, 25 µM niflumic acid, 500 µM
probenecid, and 1 mM CHC caused only minimal nonsignificant decreases
in
36Cl
flux, and 50 µM dipyridamole was without effect (15). Whether the
higher inhibitor concentrations used in the present study would have
altered AE2 transport is not known. Unfortunately, the nonstilbene
inhibitors have not been studied for the AE3 isoform.
Although the physiological function of the anion exchange proteins is
to effect
Cl
/HCO
3
exchange, they can transport other anions, including sulfate. Sulfate
is thought to have the same binding affinity for erythroid AE1 as
Cl
but is transported at a
rate ~3 orders of magnitude slower than Cl
(16). Although the
sulfate flux is slower than that of
Cl
on AE1, given the
rapidity of the AE1-mediated
Cl
flux, it is possible to
measure an erythrocyte sulfate flux that is greater than baseline
sulfate permeability, and the potency of inhibitors has been
demonstrated to be practically identical for both sulfate and
Cl
tracer flux on AE1 (16).
Sulfate transport has been demonstrated in AE2 and AE3 expression
systems (29). One difficulty in comparing substrate fluxes between
experiments is that the relative fluxes are affected by specific
conditions of the experiments, such as temperature, anion
concentrations, and pH. Nonetheless, the present studies were unable to
identify any transport of sulfate on the
-cell apical anion
exchanger over that of gluconate. This lack of demonstrable transport
of sulfate in
-cells is consistent with the finding of Boyer and
Burg (5) who noted that replacement of lumen chloride with sulfate in
perfused rabbit CCDs inhibited unidirectional bicarbonate secretion.
Apart from the three anion exchange isoforms, there are two anion
exchangers that bear particular similarities to the
-cell transporter. First, the turtle bladder, thought to be the
homolog of the mammalian collecting duct, possesses an apical anion
exchanger that is thought to mediate bicarbonate secretion. Neither the molecular nor the protein identity of the turtle bladder apical anion
exchanger is known at present. It is interesting that, similar to the
lack of transport of sulfate on the
-cell apical anion exchanger
described in the present studies, substitution of sulfate for
Cl
caused the bicarbonate
flux to decrease to 13% in the turtle bladder (18). However, unlike
the characteristics demonstrated for the
-cell apical anion
exchanger presented here, bicarbonate flux in turtle bladders was
unchanged by the presence of 9 mM CHC, but decreased slightly (14%)
but significantly with 1 mM furosemide (14). In further contrast to the
-cell apical anion exchanger, bicarbonate flux in the turtle bladder
was significantly inhibited (32%) by 100 µM DIDS in the presence of
102 mM Cl
(32). Some
substrate differences appear to exist between the turtle bladder and
the
-cell apical anion exchanger as well. Substitution of
Br
for
Cl
caused the turtle
bladder bicarbonate flux to decrease to less than 25% of control flux
(18). In contrast, a study of halide transport on the
-cell apical
anion exchanger was unable to distinguish Br
transport from that of
Cl
(11).
Neutrophils possess an anion exchanger that is relatively insensitive
to DIDS. The neutrophil anion exchanger can be inhibited by CHC, with a
Ki of 9 mM, but
is insensitive to 1 mM furosemide and 1 mM ethacrynic acid (30).
Interestingly, the neutrophil anion exchanger, similar to the
-cell
apical anion exchanger, does not transport sulfate. However, there are
some apparent substrate differences between the neutrophil and the
-cell transporters. Whereas the neutrophil anion exchanger
transports F
with greater
affinity than I
, no
transport of F
could be
detected on the
-cell apical anion exchanger (11, 30). As for the
-cell apical anion exchanger, the molecular/protein identity of the
neutrophil anion exchanger is also not known.
The transport "fingerprints" of the
-cell apical anion
exchanger described here are distinct from those demonstrated for any
of the three anion exchange isoforms or from those described for anion
exchange transporters in other tissues. Yet unrecognized features
of the
-cell apical anion exchanger must account for these distinctive transport characteristics.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Veterans Affairs Career Development
Research Award and a Veterans Affairs Merit Review Award.
 |
FOOTNOTES |
Portions of this work were published in abstract form
(J. Am. Soc. Nephrol. 5: 252, 1994;
and J. Am. Soc. Nephrol. 6: 308, 1995).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Emmons, Univ.
of Cincinnati, PO Box 670585, Cincinnati, OH 45267-0585.
Received 2 March 1998; accepted in final form 22 December 1998.
 |
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