Clminus channels in basolateral renal medullary membranes XII. Anti-rbClC-Ka antibody blocks MTAL Clminus 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
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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 approx 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 approx 0.4 mCi/mol and was obtained from DuPont Chemical. All other chemicals were purchased from Sigma.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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.

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.

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 (bullet ) or absence (black-square, 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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Table 1.   Effect of vasopressin + forskolin + DBcAMP on initial rates of 36Cl- uptake in MTAL tubule suspensions

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 (bullet ) or absence (black-square, control) of 0.1 mM bumetanide. Vasopressin + forskolin + DBcAMP were present in all experiments. Results are expressed as means ± SE.

                              
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Table 2.   Effect of vasopressin + forskolin + DBcAMP on initial rates of 36Cl- efflux in MTAL tubule suspensions

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 (bullet ) or absence (black-square, 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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Table 3.   Effect of DPC and anti-rbClC-Ka antiserum on initial rates of 36Cl- efflux in MTAL tubule suspensions

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 (bullet ) or preimmune serum (black-square, control) in paired observations. Unpaired mean controls from Figs. 7 and 8 and Table 2 are shown ( square , dashed line) for comparison. All solutions contained vasopressin + forskolin + DBcAMP. Results are expressed as means ± SE.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Renal Physiol 273(6):F1030-F1038
0363-6127/97 $5.00 Copyright © 1997 the American Physiological Society




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