Cl
channels in basolateral renal medullary
membranes XII. Anti-rbClC-Ka antibody blocks MTAL Cl
channels
Christopher J.
Winters,
Ludwika
Zimniak,
W. Brian
Reeves, and
Thomas E.
Andreoli
Division of Nephrology, Department of Internal Medicine, University
of Arkansas College of Medicine; and John L. McClellan Veterans
Affairs Hospital, Little Rock, Arkansas 72205
 |
ABSTRACT |
Cl
channels
in the medullary thick ascending limb (MTAL) studied by either
patch-clamp technique or reconstitution into lipid bilayers are
activated by increases in intracellular Cl
concentrations. rbClC-Ka, a ClC Cl
channel, may
represent this channel. We therefore evaluated the role of rbClC-Ka in
transcellular MTAL Cl
transport in two separate ways.
First, an antibody was raised against a fusion protein containing a
153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney
sections with the antibody was localized to basolateral regions of MTAL
and cortical thick ascending limb (CTAL) segments and also to the
cytoplasm of intercalated cells in the cortical collecting duct.
Second, Cl
uptake and efflux were measured in
suspensions of mouse MTAL segments. Cl
uptake was
bumetanide sensitive and was stimulated by treatment with a combination
of vasopressin + forskolin + dibutyryl adenosine 3',5-cyclic
monophosphate (DBcAMP). Cl
efflux was also increased
significantly by vasopressin + forskolin + DBcAMP from 114 ± 20 to
196 ± 36 nmol · mg
protein
1 · 45 s
1
(P = 0.003). Cl
efflux was inhibited by the
Cl
channel blocker diphenylamine-2-carboxylate
(154 ± 26 vs. 70 ± 21 nmol · mg
protein
1 · 45 s
1,
P = 0.003). An anti-rbClC-Ka antibody, which inhibits the
activity of MTAL Cl
channels in lipid bilayers, reduced
Cl
efflux from intact MTAL segments (154 ± 28 vs.
53 ± 14 nmol · mg
protein
1 · 45 s
1,
P = 0.02). These results support the view that rbClC-Ka is
the basolateral membrane Cl
channel that mediates
vasopressin-stimulated net Cl
transport in the MTAL
segment.
medullary thick ascending limb; chloride channel; vasopressin; rbClC-Ka; immunohistochemistry
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INTRODUCTION |
THE PURPOSE of the experiments reported here is to
provide support for the view (24, 25) that a member of the ClC family of Cl
channels, termed rbClC-Ka, mediates net salt
absorption in intact medullary thick ascending limb (MTAL)
segments. The rbClC-Ka protein is encoded by rbClC-Ka, a cDNA
derived from a rabbit outer medullary library (24). An antibody
specific for rbClC-Ka suppressed the activity of Cl
channels fused from cultured mouse MTAL cells into planar lipid bilayers (25) and an antisense oligonucleotide to
rbClC-Ka decreased Cl
channels in
cultured mouse MTAL cells (25). To evaluate the role of rbClC-Ka in
transepithelial net Cl
absorption in the MTAL, we used
two parallel approaches.
The first set of studies was immunohistochemical. In these experiments,
we used a guinea pig polyclonal antibody (25), which reacted
specifically on Western blots with a 75-kDa band, that is, with a
protein having the predicted molecular size (75,201 Da) for the gene
product of rbClC-Ka (24). This antibody stained basal regions
of rabbit cortical thick ascending limb (CTAL) and rabbit MTAL, both
identified explicitly using simultaneous staining with antibodies to
Tamm-Horsfall protein. The gene product of rbClC-Ka was also
found in intercalated cells of cortical collecting tubules (CCT), in
accord with prior studies (24) on the intrarenal localization of the
rbClC-Ka message in rabbit kidney using reverse transcription-polymerase chain reaction (RT-PCR) technology.
In functional studies, we found that 36Cl
uptake by suspensions of immunodissected mouse MTAL segments was
augmented by antidiuretic hormone (ADH) and inhibited by bumetanide.
Thus we reasoned that this 36Cl
uptake
occurred via the apical membrane
Na+-K+-2Cl
triporter. We also
found that 36Cl
efflux from these MTAL
segments was, as in microperfused mouse MTAL segments (5, 11),
augmented by ADH and unaffected by bumetanide. This
36Cl
efflux was also inhibited strikingly by
the antibody described above.
These results thus provide direct evidence for the contention (25) that
rbClC-Ka, the gene product of rbClC-Ka (24), mediates ADH-stimulated net Cl
absorption across basolateral
membranes of intact MTAL segments. Preliminary accounts of some of the
data presented in this study have been previously reported in abstract
form (23).
 |
METHODS |
Immunohistochemistry
Kidneys removed from New Zealand White rabbits were fixed in 10%
neutral-buffered Formalin. Paraffin-embedded specimens were cut into
5-µm sections. The sections were deparaffinized with xylene, followed
by decreasing gradient washes of ethanol. Endogenous peroxidase
activity was quenched by incubation with 0.3%
H2O2 in methanol for 30 min.
The polyclonal antibodies against rbClC-Ka used in these experiments
were obtained from two different guinea pigs. As in our previous
studies (25), the polyclonal antibodies were prepared against a fusion
protein containing a 153-amino acid COOH-terminal fragment of rbClC-Ka.
As reported previously (25), the first guinea pig antiserum recognized
a 75-kDa protein present in basolaterally enriched rabbit outer
medullary vesicles and in cultured mouse MTAL cells. To confirm the
specificity of the polyclonal antibody obtained from the second guinea
pig, we used this antibody in a Western blot analysis of basolaterally
enriched membranes from rabbit outer medulla. The membranes were
prepared from rabbit medulla, as described previously (19-25),
except that a mixture of protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 0.2 mM phenylmethylsulfonyl fluoride) was
present throughout the isolation procedure. A comparison of lanes
2 and 3 in Fig. 1 shows that
preincubation with the fusion protein used as an antigen for antibody
production blocked almost completely antibody interactions with the
75-kDa protein.

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Fig. 1.
Western blot of basolaterally enriched membrane vesicles from rabbit
outer medulla. Lane 1: preimmune serum, 1:100 dilution.
Lane 2: anti-rbClC-Ka antiserum, 1:100 dilution. Lane
3: anti-rbClC-Ka antiserum, 1:100 dilution, preincubated for 30 min
with 3.2 µg/ml of fusion protein used as antigen for antibody
production. Antibody reactivity was visualized using an alkaline
phosphatase-coupled secondary antibody followed by color reaction with
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate
as described in METHODS.
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It is pertinent to place these results in quantitative perspective. We
have carried out 28 such Western blot analyses on ~26 different
membrane preparations. First, in 27 of the 28 Western blots, we have
detected the 75-kDa protein illustrated in Fig. 1. In all instances
tested, preincubation of polyclonal antibody with fusion protein
virtually abolished detection of the protein (e.g., Fig. 1), whereas
isolation of membranes with the mixture of protease inhibitors
described above did not affect detection of the 75-kDa protein on
Western blots. Moreover, as noted previously (25), preincubation of
cultured mouse MTAL cells with an antisense oligonucleotide specific
for the rbClC-Ka cDNA abolishes uniquely the density of this
75-kDa band on Western blots. Second, in 16 of the 28 Western blots,
the polyclonal antibody also recognized a 110-kDa protein (e.g., Fig. 3
and Ref. 25). But the antisense nucleotide specific for the
rbClC-Ka cDNA did not affect (using densitometry tracings) the
quantitative detection of this protein on Western blots (25). Finally,
in 16 of the 28 Western blots, we also observed a 50-kDa protein not
detected when fusion protein was preincubated with polyclonal antibody.
The presence of the 50-kDa protein on Western blots was abolished by
preincubating the membranes with the mixture of protease inhibitors
described above (e.g., Fig. 1).
Thus, as in previous studies (25), we consider the 75-kDa protein shown
in Fig. 1 to be the putative basolateral MTAL Cl
channel
in basolateral rabbit medullary vesicles and cultured mouse MTAL cells
(14, 19, 25). And, as in previous studies (25), we term this protein
rbClC-Ka and the guinea pig antibody as anti-rbClC-Ka. We consider the
50-kDa protein described above, although not present in Fig. 1, to be a
degradation product of rbClC-Ka. Finally, we as yet cannot provide an
explanation for the 110-kDa protein described previously (Fig. 3 and
Ref. 25) but not seen in Fig. 1.
The kidney sections were double labeled with anti-rbClC-Ka and then
with either Tamm-Horsfall protein antibody or peanut agglutinin. The
anti-rbClC-Ka antiserum was used at a 1:100 dilution. Antibody binding
was detected by the avidin-biotin complex method (8, 10), using
reagents from the ImmunoPure Peroxidase Staining Kit (Pierce, Rockford,
IL). 3,3'-Diaminobenzidine (DAB) tetrahydrochloride was used as the
peroxidase substrate. In some experiments, antibody specificity was
tested by preincubating the serum for 30 min with 3.2 µg/ml of the
fusion protein used as antigen for antibody production. The depleted
serum was then used for immunohistochemistry as described above. After
completion of staining for anti-rbClC-Ka, sections were incubated
either with a 1:100 dilution of goat anti-human Tamm-Horsfall protein
antibody followed by incubation with anti-goat immunoglobulin G (IgG)
conjugated with fluorescein isothiocyanate (FITC) or with a 1:500
dilution of FITC-labeled peanut agglutinin. Slides were mounted with
Gel Mount medium (Biomeda) and examined using an Olympus BH2 microscope
with an epifluorescence attachment and photographed with an Olympus
PM-10ADS automatic photomicrographic system.
36Cl
Fluxes in MTAL Tubule
Suspensions
Suspensions of mouse MTAL segments were prepared as described
previously (21). This method yields a tubule suspension containing
95% pure MTAL segments (9, 21).
The isotope flux experiments were carried out by rapid filtration using
HAWP Millipore filters (pore size, 0.45 µm). In each experiment,
36Cl
flux was terminated at timed intervals
by pipetting aliquots of the reaction mixture directly onto the filter
followed by three 2 ml washes with a 4°C wash solution containing (in
mM) 116 sodium isethionate, 5.4 KCl, 26.2 NaHCO3, and 1 NaH2PO4. The experiments were carried out at
37°C.
To measure 36Cl
uptake, the MTAL tubule
suspensions were incubated in a modified Earle's balanced salt
solution (EBSS) containing (in mM) 116 NaCl, 5.4 KCl, 26.2 NaHCO3, 1 MgCl2, 1 CaCl2, 1 NaH2PO4, and 10 glucose (pH 7.4). Aliquots were
preincubated at 37°C for 15 min. Where indicated, a solution
containing 0.2 IU/ml vasopressin, 20 µM forskolin, and 0.5 mM
dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) (final
concentrations) was added 5 min prior to uptake studies. Bumetanide,
when present, was added to the solution in a final concentration of 0.1 mM. 36Cl
uptake was initiated by the
addition of 36Cl
in a final concentration of
4.5 mM. One-hundred-microliter aliquots of the reaction mixtures were
applied to the filters to terminate Cl
uptake.
To measure 36Cl
efflux from MTAL tubule
suspensions, MTAL tubules were loaded for 15 min by incubation at
37°C in a modified EBSS containing 4.5 mM
36Cl
. Where indicated, the loading solutions
also contained 0.2 IU/ml vasopressin, 20 µM forskolin, and 0.5 mM
DBcAMP. When present, bumetanide was added at a final concentration of
0.1 mM, and diphenylamine-2-carboxylate (DPC) was added at a final
concentration of 3 mM. When present, anti-rbClC-Ka serum (Fig. 1) or
preimmune serum was added during the final 5 min of the
36Cl
loading period in a dilution of 1:100
and was also present in the 2.5 ml of modified EBSS in the same
concentration. After the 15-min loading period,
36Cl
efflux was initiated by diluting
100-µl aliquots of the loaded tubule suspensions into 2.5 ml of EBSS.
Efflux was terminated by applying 500-µl aliquots of the mixture to
filters with the amount of 36Cl
remaining in
the tubules determined by scintillation spectrophotometry.
Materials
Fluorescein peanut agglutinin was purchased from Biomeda, and
goat anti-human Tamm-Horsfall protein antibody was purchased from
Organon. DPC was obtained from Fluka Chemie, and collagenase A was
obtained from Boehringer-Mannheim. 36Cl
had
a specific activity of
0.4 mCi/mol and was obtained from DuPont
Chemical. All other chemicals were purchased from Sigma.
 |
RESULTS |
Immunolocalization of rbClC-Ka
Outer renal medulla.
The rbClC-Ka message is kidney specific, more concentrated in
rabbit renal medulla than in rabbit renal cortex, and, in RT-PCR experiments, detectable in glomeruli, CCT, CTAL, and MTAL (24). Figure
2 shows a section of rabbit renal medulla
stained either with anti-rbClC-Ka (Fig. 2A) or with preimmune
serum (Fig. 2C). The anti-rbClC-Ka serum stained certain
tubules in the outer medulla. The stain was not present on apical
membranes but rather appeared to be predominantly in on basal regions.
A comparison of Fig. 2, A and B, indicates that the
medullary tubules stained with anti-rbClC-Ka were also stained with
anti-Tamm-Horsfall antibody, thus confirming the fact that the stained
tubules in Fig. 2A were MTAL segments. It is interesting to
note in this regard that Vandewalle et al. (18) recently found that
rClC-K proteins were also localized to diffuse basal regions of rat
MTAL and CTAL.

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Fig. 2.
Immunohistochemical localization of rbClC-Ka in rabbit kidney medulla.
A: immunoreactivity with anti-rbClC-Ka antibody. B:
double labeling of same section in A with anti-Tamm-Horsfall
protein antibody visualized by fluorescence microscopy. C:
adjacent section incubated with 1:100 dilution of preimmune serum.
D: immunoreactivity of same section in C with
anti-Tamm-Horsfall protein antibody visualized by fluorescence
microscopy. Magnification, ×145.
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As indicated in Fig. 2C, staining was completely absent when
preimmune serum was used as a control. Likewise, a comparison of the
staining patterns in Fig. 3A (using
anti-rbClC-Ka alone) and in Fig. 3B [using anti-rbClC-Ka
preincubated with the fusion protein used as the antigen for antibody
production (see METHODS)] shows clearly that preincubation
of anti-rbClC-Ka with the fusion protein also abolished staining of
these medullary tubules.

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Fig. 3.
Competition for staining of rabbit kidney medulla by fusion protein.
A: immunoreactivity of a section of rabbit kidney medulla with
anti-rbClC-Ka antibody. B: lack of immunoreactivity of an
adjacent section using anti-rbClC-Ka antibody preincubated with fusion
protein. Magnification, ×145.
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The double-label experiments presented in Fig. 4,
A and B, indicate that
anti-rbClC-Ka antiserum did not stain outer medullary collecting ducts,
identified in Fig. 4B by apical membrane staining of
intercalated cells with fluorescein isothiocyanate peanut agglutinin (1). The positive controls for the experiments shown in Fig. 4B
are provided by the medullary segments in Fig. 4A: those that appeared identical to those in Fig. 2A were stained intensely with anti-rbClC-Ka antiserum (Fig. 4A).

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Fig. 4.
Double staining of rabbit kidney medulla with anti-rbClC-Ka antibody
and with peanut agglutinin. A: immunoreactivity with
anti-rbClC-Ka antibody. B: reactivity of same section in A with
fluorescein isothiocyanate-labeled peanut agglutinin visualized by
fluorescence microscopy. Magnification, ×145.
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Renal cortex.
Figure 5 shows the results of experiments
with rabbit renal cortex identical in format to those presented in Fig.
2 for rabbit outer medulla. The anti-rbClC-Ka serum clearly stained
basal regions of CTAL segments (Fig. 5, A and B), and
staining did not occur using preimmune serum (Fig. 5, C and
D). In occasional experiments not indicated in Fig. 5, we
also observed faint glomerular staining with anti-rbClC-Ka, in accord
with results from earlier RT-PCR experiments detecting the
rbClC-Ka message (24). Finally, Fig. 6, A and
B, show that rbClC-Ka was also
present in intercalated cells of CCT and that, in the latter,
immunoreactive staining was diffusely cytoplasmic rather than
basolateral (Fig. 6A).

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Fig. 5.
Immunohistochemical localization of rbClC-Ka in rabbit kidney cortex.
A: immunoreactivity with anti-rbClC-Ka antibody. B:
immunoreactivity of same section in A with
anti-Tamm-Horsfall protein antibody visualized by fluorescence
microscopy. C: adjacent section incubated with 1:100 dilution
of preimmune serum. D: immunoreactivity of same section in
C with anti-Tamm-Horsfall protein antibody visualized by
fluorescence microscopy. Magnification, ×145.
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Fig. 6.
Double staining of rabbit kidney cortex with anti-rbClC-Ka antibody and
with peanut agglutinin. A: immunoreactivity with anti-rbClC-Ka
antibody. B: reactivity of same section in A with
fluorescein isothiocyanate-labeled peanut agglutinin visualized by
fluorescence microscopy. Magnification, ×145.
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36Cl
Influx and Efflux Studies
Anti-rbClC-Ka, when applied to solutions facing extracellular surfaces
of basolateral Cl
channels incorporated vesicles from
cultured mouse MTAL cells into bilayers, reduces dramatically the open
time probability of these channels (25). Those results show that
anti-rbClC-Ka blocked rbClC-Ka channels in bilayers but do not indicate
that the rbClC-Ka channel mediates normally occurring net
Cl
absorption across basolateral MTAL segments. The
present experiments were designed to test this possibility directly.
The line of reasoning is as follows.
In all mammalian MTAL and CTAL species studies to date (2, 3, 5, 15,
16), apical NaCl entry involves the bumetanide-sensitive Na+-K+-2Cl
triporter. In the
mouse MTAL, the adenylate cyclase cascade increases the rate of apical
Cl
entry (4) by altering the stoichiometry of apical
salt entry from Na+-Cl
to
Na+-K+-2Cl
(17) and,
simultaneously, by activating the K+ conductance of apical
membranes (13). This ADH-dependent increase in apical salt entry raises
cytosolic Cl
concentrations, which, in turn, augment the
time-average conductance of Cl
channels in basolateral
membranes (14, 19, 20, 22). Thus, in the present studies, we evaluated
whether both 36Cl
uptake and
36Cl
efflux in suspensions of mouse MTAL
segments had the properties enumerated previously in individual
microperfused mouse MTAL segments (3, 5, 6, 15) and, if so, whether
anti-rbClC-Ka suppressed 36Cl
efflux.
36Cl
uptake.
The principal results are presented in the paired experiments shown in
Fig. 7 and Table
1. The results presented in Fig. 7 show
that 0.1 mM bumetanide reduced by nearly two-thirds both the initial
and near-steady-state rates of 36Cl
uptake.
The paired results presented in Table 1 show that adding a cocktail
containing ADH + forskolin + DBcAMP produced an ~55% increase in the
rate of 36Cl
uptake.

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Fig. 7.
36Cl uptake into mouse medullary thick
ascending limb (MTAL) tubule suspensions in paired observations
measured in the presence ( ) or absence ( , control) of 0.1 mM
bumetanide. In all experiments, solutions contained vasopressin + forskolin + dibutyryl cAMP. Results are expressed as means ± SE.
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36Cl
efflux.
In intact mouse MTAL segments, ADH (or forskolin or DBcAMP) augments
the rate of net Cl
absorption across basolateral
Cl
channels (5, 11, 16). Peritubular furosemide has no
effect on this process (5). The data presented in Fig.
8 and Table 2
show that the initial rates of 36Cl
efflux
from tubule suspensions preloaded with isotope had rather similar
properties. Specifically, the results presented in Fig. 8 indicate
that, in paired experiments, the addition of bumetanide to the
incubation media had no perceptible effect on the initial rates of
36Cl
efflux. However, as indicated in Table
2, the addition of ADH + forskolin + DBcAMP to the incubation media
produced an ~70% increase in the initial rate of
36Cl
efflux.

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Fig. 8.
36Cl efflux from mouse MTAL tubule
suspensions in paired observations measured in the presence ( ) or
absence ( , control) of 0.1 mM bumetanide. Vasopressin + forskolin + DBcAMP were present in all experiments. Results are expressed as means ± SE.
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It was pertinent to evaluate the effects of the Cl
channel blocker DPC and of anti-rbClC-Ka on the initial rates of
36Cl
efflux. The data shown in Fig.
9 and Table 3
indicate that, in paired experiments, 3 mM DPC suppressed by ~50%
the initial rate of 36Cl
efflux from tubule
suspensions preincubated with ADH + forskolin + DBcAMP.

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Fig. 9.
36Cl efflux from mouse MTAL tubule
suspensions in paired observations measured in the presence ( ) or
absence ( , control) of 3 mM diphenylamine-2-carboxylate. Vasopressin + forskolin + DBcAMP were present in all experiments. Results
are expressed as means ± SE.
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The paired and unpaired results with anti-rbClC-Ka (Fig.
10, Table 3) were striking. In paired
experiments with tubules preincubated with ADH + forskolin + DBcAMP,
anti-rbClC-Ka antibody reduced the rate of
36Cl
efflux by ~65%. In two of the paired
experiments shown in Fig. 10, anti-rbClC-Ka antibody was from the
guinea pig, as described previously (25). In the three remaining paired
experiments shown in Fig. 10, we used the anti-rbClC-Ka antiserum
described in METHODS (Fig. 1). In other words, reproducible
data on Cl
channel blockade obtained using two different
antisera prepared in the same manner.

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Fig. 10.
36Cl efflux from mouse MTAL tubule
suspensions incubated with anti-rbClC-Ka ( ) or preimmune serum ( ,
control) in paired observations. Unpaired mean controls from Figs. 7
and 8 and Table 2 are shown ( , dashed line) for comparison. All
solutions contained vasopressin + forskolin + DBcAMP. Results are
expressed as means ± SE.
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 |
DISCUSSION |
The experiments reported in this study were intended to provide direct
evidence that rbClC-Ka channels mediated net Cl
absorption in mouse MTAL suspensions. Several results are noteworthy in
this context.
The 36Cl
influx and efflux experiments
reported in Figs. 7-10 and Tables 1-3 share certain
characteristics common to net Cl
absorption in intact
microperfused mouse MTAL segments (3, 5, 6, 11, 15, 16). Specifically,
like apical Cl
entry in microperfused mouse MTAL
segments (5, 11, 16), 36Cl
uptake in these
mouse MTAL segments was activated by ADH + forskolin + DBcAMP and
suppressed by bumetanide (Fig. 7, Table 1). Moreover, the initial rates
of 36Cl
efflux from these mouse MTAL
suspensions (Table 2) were augmented by ADH + forskolin + DBcAMP, as in
intact microperfused segments (5-7, 11, 16), where increased
intracellular Cl
concentrations attendant on increased
apical Cl
entry activate basolateral Cl
channels (4, 11, 14). This activation of basolateral MTAL Cl
channels by increasing cytosolic Cl
levels also occurs when the channels are fused into bilayers (14,
19-22) or studied by patch clamping basolateral membranes of
cultured mouse MTAL cells (14). Finally, as in microperfused mouse MTAL
segments (5), bumetanide did not affect 36Cl
efflux in the present experiments with mouse MTAL suspensions (Fig. 8).
It is evident from the bumetanide data (Figs. 7 and 8) that, in these
mouse MTAL suspensions, 36Cl
influx and
36Cl
efflux involved different pathways. And
given the similarities between 36Cl
uptake
in these MTAL suspensions and apical Cl
entry in
microperfused mouse MTAL segments (5, 11, 16, 17), it is reasonable to
argue that 36Cl
uptake in the MTAL tubule
suspensions involved apical
Na+-K+-Cl
triporters. Likewise,
given the similarities between 36Cl
efflux
in mouse MTAL suspensions (Fig. 8, Table 2) and in intact microperfused
MTAL segments (4, 5, 11), it is reasonable to infer that the
36Cl
efflux measured in the present
experiments (Figs. 8-10; Tables 2 and 3) involved the
Cl
channels mediating net Cl
absorption
in intact microperfused mouse MTAL segments (4, 5, 11).
We note in this context that the Cl
channel blocker DPC,
which inactivates basolateral MTAL Cl
channels fused
into bilayers (12), also blocked 36Cl
efflux
in these mouse MTAL suspensions (Fig. 9, Table 3). Finally, anti-rbClC-Ka antibodies obtained from two separate guinea pigs, which
recognize specifically the rbClC-Ka protein (Fig. 1, Ref. 25) and which
block Cl
channels incorporated from basolateral
membranes of cultured mouse MTAL cells into bilayers (25), also blocked
36Cl
efflux from mouse MTAL suspensions in
the present experiments (Fig. 10, Table 3). Thus we conclude that, in
the intact mouse MTAL, rbClC-Ka is the basolateral Cl
channel mediating net Cl
absorption.
Additional support for this view obtains from the immunolocalization
experiments. The anti-rbClC-Ka antiserum, which interacted with the
75-kDa rbClC-Ka protein (Fig. 1) showed, as expected from previous
studies (24), a high degree of localization for CTAL and MTAL segments.
This immunostaining was principally in basal regions and not in apical
membranes (Figs. 2 and 5), did not occur with control preimmune serum
(Figs. 2 and 5), and could be blocked by preincubating anti-rbClC-Ka
antibody with the fusion protein used as the antigen for antibody
production (Fig. 3). It should also be stressed in this context that
MTAL and CTAL segments contain extensive basolateral infoldings.
Consequently, the basal region staining observed in Figs. 2-5 may
represent rbClC-Ka localization to basolateral membranes, although we
cannot exclude the possibility that some of this staining was
cytoplasmic.
Finally, we emphasize that the present data provide no insight into the
mechanism for anti-rbClC-Ka blockade of rbClC-Ka channels. Nor is it
clear what functional significance rbClC-Ka channels have in
intercalated cells of rabbit CCT (Fig. 6) or in glomeruli (see
RESULTS). But the present data do provide, when viewed in the context of earlier studies (14, 24, 25), reasonable evidence that
rbClC-Ka mediates net Cl
absorption across basolateral
membranes of the MTAL.
 |
ACKNOWLEDGEMENTS |
C. J. Winters and L. Zimniak contributed equally to this work.
 |
FOOTNOTES |
We are very grateful to Drs. Patrick D. Walker (Department of
Pathology) and Charlotte A. Peterson (Department of Geriatrics) for
skilled aid and advice in carrying out the immunofluorescence experiments. We greatly appreciate the technical assistance provided by
Anna Grace Stewart and the secretarial assistance provided by
Clementine Whitman.
This work was supported by National Institutes of Health Grant
5-RO1-DK25540) and a Veterans Administration Merit Review Grant (to T. E. Andreoli). W. B. Reeves is an Established Investigator of the
American Heart Association (Grant no. 95-1450).
Address for reprint requests: T. E. Andreoli, Dept. of Internal
Medicine, University of Arkansas College of Medicine, 4301 W. Markham
St., Slot 640, Little Rock, AR 72205.
Received 31 March 1997; accepted in final form 14 August 1997.
 |
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