1 Department of Medical Biochemistry, 2 Department of Cell Biology, Institute of Anatomy, and 3 Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark; and 4 Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520
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ABSTRACT |
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An electroneutral
Na-HCO3 cotransporter (NBCN1) was
recently cloned, and Northern blot analyses indicated its expression in
rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and
electron microscopic level. A peptide-derived antibody was raised
against the COOH-terminal amino acids of NBCN1. The
affinity-purified antibody specifically recognized one band, ~180
kDa, in rat kidney membranes. Peptide-N-glycosidase F
deglycosylation reduced the band to ~140 kDa. Immunoblotting of
membrane fractions from different kidney regions demonstrated strong
signals in the inner stripe of the outer medulla (ISOM), weaker signals
in the outer stripe of the outer medulla and inner medulla, and no
labeling in cortex. Immunocytochemistry demonstrated that
NBCN1 immunolabeling was exclusively observed in the
basolateral domains of thick ascending limb (TAL) cells in the outer
medulla (strongest in ISOM) but not in the cortex. In addition,
collecting duct intercalated cells in the ISOM and in the inner medulla
also exhibited NBCN1 immunolabeling. Immunoelectron
microscopy demonstrated that NBCN1 labeling was confined to
the basolateral plasma membranes of TAL and collecting duct type A
intercalated cells. Immunolabeling controls were negative. By using
2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients
were measured in kidney slices from ISOM and from mid-inner medulla.
The results revealed DIDS-sensitive, Na- and HCO3
-dependent net acid extrusion only in the ISOM
but not in mid-inner medulla, which is consistent with the
immunolocalization of NBCN1. The localization of
NBCN1 in medullary TAL cells and medullary collecting duct
intercalated cells suggests that NBCN1 may be important for
electroneutral basolateral HCO3
transport in these cells.
bicarbonate handling; immunohistochemistry; immunoelectron microscopy; intracellular pH; ion transport
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INTRODUCTION |
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IN THE PROXIMAL TUBULES, reabsorption
of HCO3 by electrogenic Na-HCO3
cotransport is well documented (4, 5). With the cloning of
an electrogenic Na-HCO3
cotransporter (NBC1) from the
kidney (6, 27, 28), it was possible to produce antibodies
to disclose the cellular and subcellular distribution of NBC1.
Immunohistochemistry demonstrated the presence of NBC1 in the
basolateral parts of proximal tubule segments S1 and S2 in rat
(29) and the entire proximal tubule in rabbit (1), albeit less in segment S3. Immunoelectron microscopy
has recently demonstrated that NBC1 immunogold labeling is exclusively found associated with basal and lateral plasma membranes of rat proximal tubule cells (21). However, there was no labeling
in other segments of the rat kidney (21, 29). There is,
however, HCO3
reabsorption also in the distal part of
the nephron (see, e.g., 10) and, although
Na+/H+ and
Cl
/HCO3
exchange are thought to be
important (8), the precise role of these and, potentially,
of Na-HCO3
cotransport, is not clear
(25). In this context, it is of interest that
Na-HCO3
cotransport activity has been suggested in
the mouse medullary thick limb (14) and in rat inner
medullary collecting duct cells (12) as well as in rat
cortical thick ascending limb (16), whereas in rat outer
medulla an electroneutral K+-HCO3
cotransport has been suggested in the thick ascending limb
(17). Furthermore, HCO3
-dependent,
DIDS-sensitive Na transport has recently been demonstrated in rat
medullary thick ascending limb (18), consistent with Na-HCO3
cotransport activity here. The situation with
respect to a role for Na-coupled HCO3
transport
outside the proximal tubules is therefore confusing. Recently, several
NBC1-like proteins have been identified, including NBC2 isolated from
human retina (13) and NBC3 from human skeletal muscle
(26). Also, a NBC-like partial clone was reported
(3), which was also called NBC3. Recently, we
(7) cloned a novel electroneutral
Na-HCO3
cotransporter (NBCN1) from rat
aorta (GenBank accession no. AF070475), which has a high degree of
homology with the NBC3 cloned from a human muscle library
(26). Northern blot analysis suggested a wide tissue
distribution of NBCN1, including its presence in the
kidney. In the present study, we therefore wanted to establish the
cellular and subcellular distribution of NBCN1 in rat
kidney by using immunoblotting, immunohistochemistry, and
immunoelectron microscopy as well as providing a functional assay for
Na-HCO3
cotransport activity.
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METHODS |
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NBCN1 Antibodies
Rabbits were immunized with keyhole limpet hemocyanin conjugated with synthetic peptide, corresponding to the predicted COOH terminal of the NBCN1 (NH2-EDEPSKKYMDAETSL-COOH). Anti-NBCN1 IgG was affinity purified by a two-step strategy by using a protein A Sepharose CL-4B column (Pharmacia Biotech, Uppsala, Sweden) as the first step and an epoxy-activated amino hexane Sepharose 4B column (Pharmacia Biotech) as the second (31).Membrane Fractionation for Immunoblotting
The kidneys from normal Munich-Wistar rats were divided into cortex, outer stripe and inner stripe of the outer medulla (ISOM), and inner medulla. These tissues were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) by using an ultra-turrax T8 homogenizer (IKA Labortechnik) at maximum speed for 30 s, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles (19). Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet.Electrophoresis and Immunoblotting
Samples of membranes from rat kidney cortex, outer stripe and inner stripe of the outer medulla, and inner medulla were run on 6-16% gradient polyacrylamide minigels (Bio-Rad Mini Protean II). After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20 (PBS-T), pH 7.5, for 1 h and incubated overnight at 4°C with anti-NBCN1 diluted 1:300. To control for nonspecific reactions, blocking experiments were performed by adding 10 µg of synthetic peptide to the anti-NBCN1 (~50-fold molar excess) and incubated overnight before immunoblotting. The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P448 or P217, DAKO, Glostrup Denmark, diluted 1:3,000) by using the enhanced chemiluminescence system (Amersham International). The blots shown in Fig 1 represent one of three experiments, each with different rats.
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Deglycosylation
For N-glycosidase F digestion, 100-µl membrane fractions from the inner stripe of the outer medulla were incubated at room temperature for 6 h in the presence of 5 U of peptide-N-glycosidase F (PNGase F) obtained from Boehringer Mannheim (Mannheim, Germany). Boiling the suspensions in Laemmli sample buffer stopped the enzymatic reactions, and samples were analyzed by immunoblotting.Immunohistochemistry and Immunocytochemistry
Kidneys from normal Munich-Wistar rats were fixed by retrograde perfusion via the aorta with periodate-lysine-paraformaldehyde (0.01 M NaIO4, 0.075 M lysine, 2% paraformaldehyde, in 0.0375 M Na2HPO4 buffer, pH 6.2). Tissue blocks prepared from cortex, outer stripe of outer medulla and ISOM, and inner medulla were cryoprotected with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen (24). For preparation of cryostat sections, tissue was cryoprotected in 25% sucrose. Cryostat sections (10 µm) and semithin sections (0.8-1 µm, Reichert Ultracut S Cryoultramicrotome, Leica, Vienna) were incubated overnight at 4°C with anti-NBCN1 (diluted 1:50), and labeling was visualized with horseradish peroxidase-conjugated secondary antibody (P448, 1:100, DAKO) (33).The results, shown in Fig 2, represent the labeling seen in one of three rats.
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Immunoelectron Microscopy
For immunoelectron microscopy, two different methods were used. The frozen samples were either used directly for cryosectioning (Reichert Ultracut S Cryoultramicrotome, Leica) or freeze-substituted in a Reichert AFS freeze-substitution unit (Leica) (20, 22, 23, 33). In brief, the samples for freeze substitution were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at temperatures gradually raised fromIntracellular pH Measurements
Kidneys from 13-wk-old male Wistar rats were quickly removed after the rats were killed with CO2. An ~1-mm slice of the kidney (sliced perpendicular to the polar axis) was fixed above a coverslip, which formed the floor in a 10-ml organ bath. The bathing solution was physiological salt solution (PSS; for composition, see below). The slice was incubated for ~40 min with 5 µM of the acetoxymethyl form of 2,7-bis-carboxyethyl-5,6-caboxyfluorescein (BCECF-AM) at 37°C. The temperature was thereafter reduced to ~21°C, and the organ bath was placed on the stage of an inverted microscope for either time-resolved fluorescence measurements or confocal fluorescence measurements. For time-resolved measurements, the preparation was excited alternately via a monochromator with 435- and 488-nm light. The emission from the preparation was collected through a band-pass filter (520-560 nm), and the ratio of the emissions at the two excitation wavelengths was determined after subtraction of the background fluorescence, which was always <10% of the signal. The equipment used was a PTI Deltascan fitted to a Leica DM IRB microscope with a Leica ×40/0.55 objective. The confocal fluorescence measurements were made with a laser scanning confocal microscope (Odyssey XL, Noran), with excitation set at 488 nm (Ar/Kr laser), and emission was collected through a 520-nm-long pass filter. Sixteen frames (640 × 480 pixels) collected at 33 Hz were averaged to produce one image. To reduce dye bleaching to a minimum, an image was generally only obtained immediately before and after a solution change. With this protocol, little dye bleaching was seen. For image analysis, the fluorescent images were represented as 256 intensity levels by using the programs Intervision (Noran) and Imagespace (Molecular Dynamics). The PSS contained (in mM) 119 NaCl, 4.7 KCl, 1.8 KH2PO4, 1.17 MgSO4, 25 NaHCO3, 1.6 CaCl2, 0.026 EDTA, 10 HEPES, and 5.5 glucose. The solution was gassed with 5% CO2 in air, and pH was 7.45-7.5. In Na-free PSS, NaCl was substituted with N-methyl-D-glucamine (NMDG) and NaHCO3 with cholinebicarbonate on an equimolar basis, and the pH was titrated to 7.45 with HCl. In HCO3 ![]() |
RESULTS |
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Anti-NBCN1
After purification of the antibody, immunoblotting was performed by using membrane fractions from different regions of rat kidney. As demonstrated in Fig. 1A, a strong band of NBCN1 was observed, corresponding to ~180 kDa. The strongest signal was obtained in membranes from the outer stripe of the outer medulla and ISOM, whereas no specific labeling was encountered in membranes from kidney cortex (Fig. 1A). By using higher membrane loading, an anti-NBCN1 signal was also observed in the inner medulla (Fig. 1B). Immunolabeling controls performed by using affinity-purified antibody preabsorbed with the immunizing peptide exhibited no labeling (Fig. 1C).To determine whether the protein was extensively glycosylated, we determined its sensitivity to PNGase F treatment, which cleaves N-linked saccharides. As demonstrated in Fig. 1D, treatment of membranes from kidney with PNGase F caused a significant reduction in the molecular mass to ~140 kDa.
Immunohistochemical Localization of NBCN1
To determine the cellular localization of NBCN1, we performed immunohistochemistry by using cryostat sections from perfusion-fixed rat kidneys (Fig. 2). In ISOM strong labeling was associated with thick ascending limbs (Fig. 2B), and the labeling was exclusively present in the basolateral domains. In contrast, apical domains were unlabeled, which is especially apparent in supranuclear regions, which are completely devoid of NBCN1 labeling (arrowheads in Fig. 2B). In the outer stripe of outer medulla, only sparse labeling was associated with thick ascending limbs (not shown), whereas in cortex no detectable labeling was found (Fig. 2A). In addition to the labeling of thick ascending limb cells, distinct labeling was also observed of intercalated cells in the inner medulla (Fig. 2, C and D). Parallel sections immunolabeled for aquaporin-2, known to be present in collecting duct principal cells, confirmed the presence of NBCN1 in intercalated cells and not in principal cells (Fig. 2D). Immunoelectron microscopy documented this labeling pattern (see below). Only weak NBCN1 labeling was associated with intercalated cells in the ISOM (arrows in Fig. 2B). As demonstrated in Fig. 2, C and D, the NBCN1 immunolabeling was exclusively associated with basolateral plasma membrane domains (arrows in Fig. 2, C and D). Immunolabeling controls, by using peptide-preabsorbed anti-NBCN1, revealed no labeling (not shown). At the immunolabeling conditions used, no detectable immunolabeling was seen associated with glomeruli (Fig. 2A), proximal tubules (Fig. 2A), collecting duct principal cells (Fig. 2, B-D), descending or ascending thin limbs, or vascular structures (Fig. 2, B-D). Thus NBCN1 immunolabeling in rat kidney is associated with basolateral domains of thick ascending limb cells and medullary collecting duct intercalated cells.Immunoelectron Microscopic Localization of NBCN1
Immunoelectron microscopy using ultrathin cryosections of rat kidney confirmed the presence of NBCN1 in thick ascending limb cells. As demonstrated in Fig. 3, NBCN1 labeling was exclusively associated with the basolateral plasma membrane of thick ascending limb cells (arrows in Fig. 3). Figure 4A shows a survey ultrathin cryosection of a collecting duct from kidney inner medulla. Figure 4B shows a higher magnification of the area indicated by the rectangle in Fig. 4A. Immunogold labeling of NBCN1 was exclusively associated with the basolateral plasma membranes of intercalated cells (arrows in Fig. 4B), whereas no labeling was seen of the apical plasma membrane domains or of collecting duct principal cells. Identical labeling patterns were also observed in ultrathin Lowicryl HM20 sections (not shown).
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Intracellular pH Measurements
Time-resolved measurements.
The recovery from an intracellular acidosis induced by washout of 20 mM
NH4Cl is demonstrated Fig. 5,
and mean values are shown in Table 1. In
slices from ISOM (Fig. 5A), the recovery in the presence of
HCO3 and 1 mM amiloride was inhibited by removal of
Na [difference in recovery rate with and without Na (
rate): 62 ± 6 ratio units/s]. In the absence of HCO3
, there
was no recovery from acidosis in the presence of amiloride (Fig.
5A), and omission of Na had little effect (
rate: 4.5 ± 1.8 ratio units/s). Even though buffering power was not determined, these findings strongly suggest the presence of net influx of HCO3
coupled to Na in ISOM. Figure 5A also
shows that 1 mM amiloride affected intracellular pH in a way consistent
with the presence of Na+/H+ exchange in ISOM.
To further substantiate the presence of HCO3
transport under these conditions, the effect of anion transport inhibitor DIDS was evaluated. In these experiments, the tissue was
preincubated with DIDS for ~30 min. In the last 15 min, 20 mM
NH4Cl was present. DIDS (which washes out from the tissue
very slowly) and NH4Cl were then washed out, and 1 mM
amiloride was washed in (if DIDS and amiloride are present in solution
simultaneously, precipitation forms). The rate of recovery from the
acidosis in four experiments was inhibited 76 ± 7% in the
presence of amiloride and DIDS relative to the recovery rate in the
absence of inhibitors. Without preincubation with DIDS (only amiloride
present), the recovery from the acidosis was inhibited 33 ± 9%
relative to the recovery rate in the absence of amiloride in four
experiments. The inhibition in the presence of DIDS was significantly
(P < 0.01) larger than in the absence of DIDS.
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Confocal Measurements
With the confocal microscope it was possible to visualize the individual segments of the nephron in the ISOM (Fig. 6). It was therefore possible to analyze the intracellular pH transients in the individual segments by choosing appropriate regions of interest. With this approach, it was confirmed that the NH4Cl-induced intracellular pH transients seen in the time-resolved measurements were indeed reflecting changes in thick tubules in the inner stripe (Fig. 6, A-C). The data further confirmed that the Na-dependent recovery from acidosis in the presence of HCO3
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DISCUSSION |
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With the cloning of the kidney form of NBC1 (6, 27,
28), the tissue distribution of electrogenic
Na-HCO3 cotransport activity (4) could
be determined at the cellular and subcellular level. These experiments
confirmed the presence of NBC1 in the proximal tubule and demonstrated
that NBC1 is present in the basolateral plasma membranes of the rat
proximal tubule segments S1 and S2 (21, 29). Bicarbonate
transport is also of significant importance in other nephron and
collecting duct segments of the rat kidney where
Cl/HCO3
exchange is known to play a significant role
(2, 18, 30). The role of Na-dependent
HCO3
transport outside the proximal tubule is,
however, uncertain (25). In the present study we
determined the cellular localization in the kidney of an electroneutral
Na-HCO3
cotransporter. Our results demonstrate that
NBCN1 is present in the basolateral plasma membranes of
thick ascending limb cells and medullary collecting duct intercalated
cells. Thus the results suggest that NBCN1 may participate
in electroneutral Na-dependent HCO3
transport in
these cells.
Immunoblot analyses with anti-NBCN1 revealed an ~180-kDa protein in membrane fractions from rat kidney. The labeling was ablated after preabsorption of the antibody with the immunizing peptide, indicating specific labeling. The expected molecular mass of NBCN1 is ~136 kDa (7). The 180 kDa seen here is therefore higher than expected. However, deglycosylation by PNGase F treatment reduced the molecular size to ~140 kDa, which is close to the predicted size for the gene product. Thus the immunoblotting data strongly support the view that the antibody recognizes NBCN1.
Immunoblotting demonstrated that NBCN1 is abundant in ISOM and less abundant in the outer stripe of the outer medulla and inner medulla. Very little labeling was observed in membrane fractions from cortex. Consistent with immunoblotting, immunohistochemistry demonstrated that NBCN1 is present in thick ascending limb cells. The labeling was exclusively associated with basolateral domains. This finding was substantiated by high-resolution immunoelectron microscopy,
Surprisingly, NBCN1 labeling was also found associated with intercalated cells of the inner medulla and faintly in collecting ducts in the ISOM. The cell type was documented by the use of immunoelectron microscopy. The labeling was exclusively present in the basolateral plasma membranes.
The relatively strong labeling of NBCN1 in thick ascending
limbs suggests that Na-dependent Na-HCO3 cotransport
activity should be detectable in ISOM. To test this, we measured
time-resolved intracellular pH transients with BCECF in kidney slices
obtained from ISOM and in slices from mid-inner medulla. The
observation that a DIDS-sensitive and Na- and
HCO3
-dependent recovery from acidosis was seen in
slices from the ISOM is consistent with the presence of a
Na-HCO3
cotransporter. In time-resolved measurements
it is not possible to know from which cells the fluorescence is
obtained, which weakens the interpretation. We therefore obtained
measurements of spatially resolved intracellular pH transients by using
confocal microscopy. The dominating structures in the images were the
relatively thick tubular structures, of which the majority are thick
ascending limbs with thick walls. It was also possible to recognize
thin structures, which are likely to be thin limbs, although we cannot exclude that they could be vascular structures. When imaging the structures, we took great care to focus them so they had the greatest possible diameter. The dye did not distribute in a uniform way. A
patchy distribution of BCECF has previously been reported for rabbit
isolated cortical collecting tubules (32) under certain loading conditions. In the study by Weiner and Hamm (32),
this dye distribution was reflecting uptake into different cell
populations. Whether this is the case here or whether it reflects a
nonuniform subcellular distribution of BCECF cannot be determined. In a
preliminary analysis we therefore analyzed the bright spots and the
more weakly stained areas separately. We found no qualitative
differences in this analysis, and the basis for the data in Fig. 6 is
therefore an analysis of the entire tubular structure. One concern with the use of the NH4Cl technique to acid load the cells of
the thick ascending limb is that the effect of NH4Cl
depends on whether luminal or basolateral application is used
(15). Because we found alkalinization on addition of
NH4Cl and acidification with washout of NH4Cl,
it seems likely that the application in this setup is predominantly
basolateral, which is consistent with lumen of the tubules running
parallel with the tissue surface and being at least partly collapsed.
The confocal data therefore strongly support the time-resolved
measurements and are furthermore consistent with the lack of labeling
of the thin limbs in the immunohisotochemical experiments. The
functional data thus provide support for the immunohistochemical
observation that the electroneutral Na-dependent Na-HCO3
cotransporter NBCN1 is present in
thick ascending limb cells in
ISOM.1
In the inner medulla no Na- and HCO3-dependent
recovery from acidosis could be demonstrated. This also supports the
immunohistochemical data, which showed no NBCN1
immunolabeling in this part of the kidney (mid-inner medulla).
Immunolabeling of NBCN1 was only present in the collecting
duct intercalated cells, which are present only in the proximal part of
the kidney inner medulla.
Electroneutral Na-HCO3 cotransport activity has not
previously been demonstrated in the rat medullary thick ascending limb. However, a recent paper (18) provided evidence for
HCO3-dependent, DIDS-sensitive Na transport in basolateral
vesicles from medullary thick ascending limb. Although the magnitude of
the transport was small compared to a similar transport in cortical
tubules, this may be partly explained by the fact that an electrical
driving force was used in addition to a pH gradient, which will favor the electrogenic NBC1 in the proximal tubules. These findings are
therefore consistent with our findings. Because NBCN1 is
present in the basolateral membranes and probably mediates influx of
HCO3
, unless the Na and HCO3
gradients under physiological conditions are very unusual, it is
unlikely to take part in the reabsorption of HCO3
demonstrated here (9, 11), which is likely mediated
through Cl/HCO3 exchange (18). Also, its
localization to the basolateral site of the intercalated cells in the
inner medulla, which are of the acid-secretion type, with
Cl/HCO3
exchange (AE1) at the basolateral membrane
(2), is puzzling, and it will be a challenge to find out
what role NBCN1 plays in HCO3
handling.
Future studies including potential development of NBCN1 transgene mice may help elucidate this.
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ACKNOWLEDGEMENTS |
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The authors thank Kirsten Peterslund, Zhila Nikrozi, and Else-Merete Løcke for expert technical assistance.
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FOOTNOTES |
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* H. Vorum and T.-H. Kwon contributed equally to this study.
Support for this study was provided by the Danish Medical Research Council, the Karen Elise Jensen Foundation, the University of Aarhus Research Foundation, the Velux Foundation, and the University of Aarhus.
Address for reprint requests and other correspondence: C. Aalkjær, Dept. of Physiology, or S. Nielsen, Institute of Anatomy, Univ. of Aarhus, DK- 8000 Aarhus C, Denmark.
1
The effect of amiloride on the intracellular pH
in slices from ISOM (in both the absence and presence of
HCO3) is consistent with the presence also of an
amiloride-sensitive Na/H exchange here.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 August 1999; accepted in final form 30 June 2000.
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