Functional Characterization of a Cloned Human Kidney Na+:HCO3minus Cotransporter*

Hassane AmlalDagger , Zhaohui WangDagger , Charles BurnhamDagger , and Manoocher SoleimaniDagger §

From the Dagger  Department of Medicine, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267-0585 and the § Veterans Affairs Medical Center, Cincinnati, Ohio 45267

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Functional properties of a cloned human kidney Na+:HCO3- cotransporter (NBC-1) were studied in cultured HEK-293 cells that were transiently transfected with NBC-1 cDNA. The Na+:HCO3- cotransporter activity was assayed as the Na+ and HCO3-dependent pHi recovery from intracellular acidosis with the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In acid-loaded cells and in the presence of amiloride (to block Na+/H+ exchange), switching to a Na+-containing solution (115 mM) resulted in rapid pHi recovery only in the presence of HCO3-. This recovery was completely abolished by 300 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Replacing the Na+ with Li+ (115 mM) caused significant HCO3--dependent, DIDS-sensitive pHi recovery from intracellular acidosis, with Li+ showing lower affinity than Na+. Potassium (K+) had no affinity for the Na+:HCO3- cotransporter. The Na+-dependent HCO3- cotransport was abolished in the presence of 0.2 mM harmaline. The Na+:HCO3- cotransporter could also function in Na+:OH- cotransport mode, although only at high external pH (7.8). Based on functional similarities with the mammalian kidney experiments, we propose that NBC-1 is the proximal tubule Na+:HCO3- cotransporter.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The majority of the filtered load of HCO3- is reabsorbed in the kidney proximal tubule via the luminal Na+/H+ exchanger NHE-3 (1-4). The exit of HCO3- across the basolateral membrane of proximal tubule is via the Na+:HCO3- cotransporter (5-8). The Na+:HCO3- cotransporter (NBC)1 mediates an electrogenic process with an stoichiometry of 3 eq of HCO3- per Na+ (9, 10). Recent studies have indicated that the actual ionic mechanism involves the cotransport of Na+, HCO3-, and CO32- in a 1:1:1 ratio (11). In addition to reabsorption of HCO3- in proximal tubule, NBC also plays an important role in cell pH regulation in several tissues, including brain, liver, heart, and lung (12-17).

Functional studies support the presence of more than one NBC isoform as judged by direction and stoichiometry of the cotransporter. In kidney, NBC activity leads to cell acidification, whereas in other tissues (such as liver and heart) its function leads to cell alkalinization (5-8, 13, 14). Furthermore, NBC has a stoichiometry of 3 eq of HCO3- per Na+ ion in the kidney (9, 10) but shows a stoichiometry of 2 eq of HCO3- per Na+ in other tissues (12).

We recently cloned and functionally expressed a human kidney Na+:HCO3- cotransporter (18). In addition to the kidney, the Na+:HCO3- cotransporter (called here NBC-1) is highly expressed in pancreas, with detectable levels in brain (18). The human NBC-1 (18) shows 80% homology to the amphibian Na+:HCO3- cotransporter (19). Both the human NBC-1 (18) and the amphibian NBC (19) mediate Na+-dependent HCO3- cotransport in a DIDS-sensitive manner. The purpose of the current study was to examine the functional properties of the human kidney NBC. Accordingly, cultured HEK-293 cells were transiently transfected with the NBC-1 cDNA and studied.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture Procedures-- HEK-293 cells were cultured in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin-G and supplemented with 10% fetal bovine serum. Cultured cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day.

Transient Transfection-- Cultured HEK-293 cells were plated on coverslips and transfected at 60% of confluence with 8 µg of the full-length human NBC-1 cDNA construct (in the cloning/expression vector pCMV.SPORT1) by calcium phosphate-DNA coprecipitation (20). Cells were assayed 44-52 h after transfection.

Intracellular pH Measurement-- Changes in intracellular pH (pHi) were monitored using the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) as described (21-23). HEK-293 cells were grown to confluence on coverslips and incubated in the presence of 5 µM BCECF in a Na+-free solution consisting of 115 mM tetramethylammonium-Cl and 25 mM KHCO3, pH 7.4 (solution A, Table I). pHi was measured in a thermostatically controlled holding chamber (37 °C) in a Delta Scan dual excitation spectrofluorometer (Photon Technology International, South Brunswick, NJ). The monolayer was then perfused with the appropriate solutions (Table I). The fluorescence ratio at excitation wavelengths of 500 and 450 nm was utilized to determine intracellular pH values in the experimental groups by comparison with the calibration curve that was generated by KCl/nigericin technique. The fluorescence emission was recorded at 525 nm. The Na+:HCO3- cotransporter activity was determined as the initial rate of the DIDS-sensitive, Na+-dependent pHi recovery (dpHi/dt, pH/min) in a HCO3--containing solution following an acid load induced by NH3/NH4+ withdrawal. The experiments were performed in the presence of 1 mM amiloride to block the Na+/H+ exchanger activity. The experiments were repeated in the absence of HCO3- and CO2 and bubbled with O2 to determine whether NBC can mediate Na+:OH- (hydroxyl) cotransport. The dpHi/dt was calculated by fitting to a linear equation the first 2 min of the time course of intracellular pH recovery. Correlation coefficients for these linear fits averaged 0.984 ± 0.004

Materials-- Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. BCECF-AM was from Molecular Probes Inc. Nigericin, DIDS, amiloride, and other chemicals were purchased from Sigma.

Statistics-- Results are expressed as means ± S.E. Statistical significance between experimental groups was determined by Student's t test or by one-way analysis of variance.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Na+ and HCO3- Dependence of the Cloned Human Kidney NBC (Influx Mode)-- In the first series of experiments, cells were incubated and loaded with BCECF in Na+-free solution (solution A, Table I), exposed to NH4+ for 10 min (solution B, Table I), and acid-loaded by switching to an NH4+-free solution (solution A, Table I). The base-line pHi in sodium-free solution was 7.11 ± 0.02. In the presence of 1 mM amiloride (to block Na+/H+ exchange), switching to a Na+ (115 mM) and HCO3--containing solution (solution C, Table I) resulted in rapid pHi recovery from acidosis in transfected cells (Fig. 1A), which was completely inhibited by 300 µM DIDS. The rate of pHi recovery was 0.190 ± 0.013 pH/min in transfected cells (n = 5). This recovery was HCO3--dependent, as shown by lack of significant pHi recovery in the absence of HCO3- (solution D, Table I) (Fig. 1B). In HCO3--free solution, pHi recovery was 0.015 ± 0.003 pH/min in transfected cells (p < 0.001 versus HCO3--containing solution, n = 5). Nontransfected cells showed little Na+-dependent HCO3- cotransport (Fig. 1C) with pHi recovery of 0.017 ± 0.002 pH/min (p < 0.001 versus transfected cells, n = 5).

                              
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Table I
Composition of experimental solutions
Concentrations are in mM. Solutions (A, B, C, E, F) were bubbled with 5% CO2, 95% O2; solution D was gassed with 100% O2. The pH was adjusted to 7.40 with tris(hydroxymethyl)aminomethane. For Cl--free solution, NH4Cl was replaced with NH4SCN and other Cl--containing chemicals were replaced with gluconate salts.


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Fig. 1.   Na+ and HCO3- dependence of the cloned human kidney NBC (influx mode). Transfected cells were acid loaded by NH4+ withdrawal and monitored for pHi recovery. A, in Na+ and HCO3--containing solution, in the presence of 300 µM DIDS (n = 4) or its vehicle (n = 5); B, in HCO3--free Na+-containing solution (n = 4); C, nontransfected cells were exposed to Na+ and HCO3--containing solution (n = 5). Amiloride, 1 mM, was present to block Na+/H+ exchange.

Na+ and HCO3- Dependence of NBC (Efflux Mode)-- NBC activity in transfected cells was also measured in the efflux mode. As shown in Fig. 2, switching the cells from a Na+ and HCO3--containing medium (solution C, Table I) to a Na+-free solution (solution A, Table I) resulted in rapid cell acidification,2 with Delta pH of 0.27 pH unit (n = 3). This cell acidification was reversible, with pHi returning to base line upon switching back to the Na+-containing solution (solution A, Table I) (Fig. 2A), with Delta pH of 0.31 pH unit (n = 3). The pHi recovery back to base line was completely inhibited in the presence of 300 µM DIDS (Fig. 2A) (n = 3). Neither cell acidification nor pHi recovery were observed in the absence of HCO3- (Fig. 2B), indicating the dependence of the transporter on Na+ and HCO3-. Taken together with Fig. 1, these studies indicate that the cloned cDNA encodes a Na+:HCO3- cotransporter.


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Fig. 2.   Na+ and HCO3- dependence of the cloned NBC (efflux mode). A, transfected cells incubated in Na+ and HCO3--containing solution were exposed to a Na+-free solution (resulting in rapid cell acidification). Thereafter, cells were switched back to the Na+-containing solution (resulting in pHi recovery back to normal) (n = 3), in the presence of 300 µM DIDS or its vehicle (n = 3). Amiloride, 1 mM, was present to block Na+/H+ exchange. B, transfected cells were subjected to the same maneuver as above in the absence of HCO3- in the environment.

Interaction of Lithium (Li+) with the Cloned NBC-- The ability of NBC to mediate Li+:HCO3- cotransport was next tested. As shown in Fig. 3A, exposing the cells to a Li+-containing solution (solution E, Table I) caused significant recovery from intracellular acidosis. Switching from the Li+-containing solution to the Na+-containing solution (solution A, Table I) further increased the rate of pHi recovery (Fig. 3A). At comparable acidic pHi (nadir pH of 6.282 ± 0.039 and 6.32 ± 0.027 for Li+ and Na+ experiments, respectively, p > 0.05, n = 4) the rate of pHi recovery caused by Na+ was 4-fold higher then Li+ (Fig. 3, A and B) (pHi recovery was 0.185 ± 0.003 pH/min in the presence of Na+ and 0.045 ± 0.003 in the presence of Li+, p < 0.001, n = 4 for each group). The Li+-dependent HCO3- cotransport was completely inhibited in the presence of 300 µM DIDS (Fig. 3C) and was not detected in nontransfected cells (Fig. 3D). These results indicate that Li+ can substitute for Na+ on NBC, with Li+ showing lower rates in mediating HCO3- transport. This is in contrast to the proximal tubule luminal NHE, where Li+ has much stronger affinity than Na+ (1).


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Fig. 3.   Interaction of lithium (Li+) with the cloned NBC. A, transfected cells were acidified and then exposed to a Li+ or Na+-containing solution (n = 4). B, the rate of pHi recovery caused by Li+ or Na+ at the same acidic pHi (n = 4 for each). C, cells were exposed to a Li+-containing solution in the absence or presence of 300 µM DIDS (n = 4). D, nontransfected cells were acidified and then exposed to a Li+-containing solution (n = 4). HCO3- was present during the duration of the experiment. Amiloride, 1 mM, was present to block Li+/H+ exchange.

Inhibition of the Cloned NBC by Harmaline-- The purpose of the next series of experiments was to examine the interaction of harmaline with NBC. As indicated in Fig. 4, the presence of 0.2 mM harmaline completely blocked the Na+-dependent pHi recovery in HCO3--containing media (the experiments were performed in the presence of 1 mM amiloride to block Na+/H+ exchange). The inhibition by harmaline was reversible as shown by recovery from cell acidosis upon switching to a harmaline-free solution (Fig. 4). pHi recovery from acidosis was 0.184 ± 0.005 pH/min in the absence of harmaline and 0.007 ± 0.002 in the presence of harmaline (p < 0.001, n = 4 for each group). pHi recovery upon removal of harmaline was 0.178 ± 0.006 pH/min (n = 4).


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Fig. 4.   Inhibition of the cloned NBC by harmaline. Transfected cells were acidified and then exposed to a Na+-containing solution in presence of 200 µM harmaline or its vehicle (n = 4). HCO3- was present during the duration of the experiment. Amiloride, 1 mM, was present to block Na+/H+ exchange.

Effect of External pH on the Cloned NBC-- NBC in rabbit kidney cortex is absolutely dependent on HCO3- (24) and does not demonstrate any affinity for OH- whereas in colon it can also accept OH- (25). To examine the interaction of human kidney NBC with OH-, cells were transfected and assayed for pHi recovery from an acid load in the presence of varying pHo and the absence of HCO3-. As shown in Fig. 5A, in the absence of HCO3- and at pHo 7.4, transfected cells showed little recovery from an acid load, indicating that at normal pH, NBC-1 has very low affinity for OH- (pHi recovery from acidosis was 0.020 ± 0.004 pH/min in the absence of HCO3- (p < 0.001 versus HCO3--containing solution, n = 5 for each group). Increasing the inward OH- gradient by increasing the external pH to 7.8 caused significant Na+-dependent pHi recovery from an acid load (0.085 ± 0.005, p < 0.001 versus pHo 7.4, n = 5) that was abolished in the presence of DIDS (Fig. 5B).


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Fig. 5.   Effect of external pH on the cloned NBC. Cells were acidified and pHi recovery caused by Na+ addition was monitored at varying pHo and in the absence of HCO3-. A, Na+-dependent pHi recovery at pHo 7.40 (n = 5). B, Na+-dependent pHi recovery at pHo 7.80 in the presence of 300 µM DIDS (n = 4) or its vehicle (n = 5). Nontransfected cells were exposed to Na+ at pHo 7.80 in the absence (C) (n = 4) or presence of 300 µM DIDS (D) (n = 3). Fig. 5E shows the Na+-dependent and Na+-independent pHi recovery at pHo 7.8 in transfected cells.

These results are consistent with the operation of NBC on Na+:OH- cotransport mode. These results further indicate that the human kidney NBC predominantly carries HCO3- at physiologic pH (Fig. 5A versus Fig. 1A), but can accept OH- under certain conditions. Nontransfected cells demonstrated little pHi recovery at pHo 7.4, but showed moderate DIDS-sensitive pHi recovery from acidosis at pHo 7.8 (Fig. 5, C and D), with the pHi recovery at 0.023 ± 0.003 pH/min (n = 4).

To examine the Na+-dependent pHi recovery at pHo 7.8 further, transfected cells were acidified and then switched to a Na+-free solution at pHo 7.8. The rate of Na+-independent pHi recovery at pHo 7.8 was 0.027 ± 0.004 (Fig. 5D). These experiments were performed in Cl--free solution (Table I); however, the possibility that Na+-independent pHi recovery (Fig. 5E) is mediated via either Cl-:OH- exchange (or OH- conductance) cannot be excluded, as this process was inhibited by DIDS. When cells were switched to a Na+-containing solution at pHo 7.8 (Fig. 5E), the rate of pHi recovery increased to 0.11 ± 0.007 pH/min (p < 0.009 versus Na+-free pHo 7.8 solution, n = 5 for each group), consistent with Na+:OH- cotransport.

Interaction of potassium (K+) with the cloned NBC. A K+-dependent HCO3- cotransport has been described in the kidney (26) and squid giant axons (27). The purpose of the next series of experiments was to determine whether K+ can substitute for Na+ on NBC. As shown in Fig. 6, exposing the cells to a Na+-free, K+-containing solution (solution F, Table I) causes minimal recovery from intracellular acidosis, whereas exposing the cells to a Na+-containing solution (solution C, Table I) resulted in rapid recovery from cell acidosis (Fig. 6), indicating that NBC-1 does not function in K+:HCO3- cotransport mode. In HCO3--containing solution, pHi recovery from acidosis was 0.210 ± 0.006 pH/min in the presence of Na+ and 0.015 ± 0.008 in the presence of K+ (p < 0.001, n = 4 for each group).


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Fig. 6.   Interaction of K+ with the cloned NBC. A, transfected cells were acidified and then exposed to a K+ (solution F, Table I) or Na+-containing solution (solution C, Table I) (n = 4). B, the rate of pHi recovery caused by Na+ or K+ addition in the same monolayer (n = 4). HCO3- was present during the duration of the experiment. Amiloride, 1 mM, was present to block possible K+/H+ exchange.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The results of current experiments indicate that the cloned human kidney Na+:HCO3- cotransporter (NBC-1) accepts Na+ and HCO3- and is inhibited by DIDS (Figs. 1 and 2). The results further indicate that Li+ can substitute for Na+ on NBC-1, with Li+ showing decreased capacity to mediate HCO3--dependent pHi recovery compared with Na+ (Fig. 3). NBC-1 is inhibited by DIDS and harmaline (Fig. 4) and can accept OH- in place of HCO3-, with HCO3- showing much higher affinity than OH- (Fig. 5). NBC does not function in K+:HCO3- cotransport mode (Fig. 6).

We recently cloned the human kidney NBC-1 (18). Based on the deduced amino acid sequence, the cDNA encodes a protein with a molecular mass of ~116 kDa. Northern blot analysis reveals that NBC-1 encodes a 7.6-kilobase mRNA that is highly expressed in kidney and pancreas, with lower levels of expression in the brain (18). NBC-1 mRNA was not detected in the liver, lung, and heart (18) despite functional studies indicating the presence of Na+:HCO3- cotransport in these tissues. These results strongly suggest that the Na+:HCO3- cotransport in these latter tissues might be another isoform from this family. The human kidney NBC cDNA is highly homologous with the amphibian kidney NBC and shares striking similarities at both structural and functional levels with that cotransporter (19).

We have cloned rat NBC-1 and examined its mRNA distribution in different tissues as well as various nephron segments (28). Rat NBC-1 is highly expressed in kidney and brain but shows low levels of expression in stomach and colon (28). Nephron segment distribution studies revealed that NBC-1 is predominantly expressed in proximal tubules (28).

The results in Fig. 1 demonstrate that the Na+:HCO3- cotransport does not mediate complete pHi recovery from acidosis to baseline values in the presence of amiloride. Whereas in the absence of amiloride, intracellular pHi returns to base-line values (data not shown), further studies are needed to determine whether NHE activity is indeed essential for complete pHi recovery from acidosis.

The results of Fig. 3 demonstrate the interaction of Li+ with NBC and indicate that the human kidney NBC can accept Li+ in place of Na+, with Li+:HCO3- cotransport demonstrating lower transport rate than Na+:HCO3- cotransport (Fig. 3). This is very similar to the NBC in basolateral membranes of rabbit kidney proximal tubule (11, 29) and is opposite to the luminal NHE, which has much higher affinity for Li+ than Na+ (1).

The human kidney NBC was completely inhibited in the presence of harmaline (Fig. 4) and DIDS (Fig. 1), indicating that it has an inhibitory profile similar to the mammalian NBC in basolateral membranes of rat or rabbit kidney proximal tubule (11, 30). Harmaline is known to inhibit the rabbit kidney proximal tubule NBC by binding to the Na+ site (11), whereas DIDS binds to the HCO3- site (5-8). This latter conclusion is based on the fact that DIDS inhibits both Na+-dependent as well as Na+-independent HCO3- transporters.

The anion exchanger AE1 (band-3) in red blood cells can mediate the transport of Na+ and HCO3- via ion pair (Na+:CO32-) (31-33). According to this mechanism, the positive charge of the Na+ ion is being shielded by a CO32- (31), allowing the cotransport to be mediated as an anion via AE1. In support of the ion pair functioning as an anion, we find that cation inhibitors do not inhibit the transport of Na+ via band-3 (31). NBC was cloned based on homology to anion exchanger isoforms (18), raising the possibility that the Na+:CO32- ion pair may be the actual substrate for transport on this protein. The results of the experiments in Figs. 3 and 4, however, argue against Na+:CO32- ion pair as the substrate. The first argument against ion pair formation concerns the relative affinities of Na+ and Li+. The association constant of Li+ for ion pair formation with CO32- is 4-fold greater than of Na+ (32), consistent with Li+ having higher affinity than Na+ for transport via red cell band-3 (33). The results of Fig. 3 indicate that the ability of Li+ to mediate HCO3- transport via NBC is significantly lower than Na+. This difference is opposite to that for ion pair formation with CO32-. The other argument against ion pair formation is inhibition of NBC by alkaloid harmaline (Fig. 4). Harmaline is an organic cation known to compete at the Na+ site of several Na+-coupled transport systems in kidney proximal tubules such as the Na+/H+ exchanger and the Na+/glucose cotransporter. Although we have not studied the kinetics of NBC inhibition by harmaline, we suggest that based on similarities with the renal basolateral Na+:HCO3- cotransporter in rabbit and rat kidney (11, 30), harmaline inhibits the cloned human NBC by interaction with the Na+ site. These results, in conjunction with the results of Li+ studies, strongly argue that Na+ directly interacts at a distinct site on the NBC rather than via ion pair formation with CO32-.

The human kidney NBC accepts OH- in place of HCO3-, with OH- demonstrating much lower Na+-dependent transport rates than HCO3- (Fig. 5). These results are in agreement with functional studies in rabbit colon, indicating lower affinity of NBC for OH- (25). The rabbit kidney NBC on the other hand shows absolute dependence on HCO3- and does not demonstrate any affinity for OH- (24). It is worth mentioning that functional studies in rabbit kidney cortex were performed at physiologic pH (pHo 7.4) (24). As such, the affinity of rabbit kidney cotransporter for OH- at external pH values similar to current experiments (i.e. pHo 7.8) remains unknown.

Kidney NBC functions in an outwardly directed mode under physiologic conditions (5-8), resulting in trans-vectorial transport of HCO3- from lumen to blood. As such, the physiologic role of the Na+:OH- cotransport mode remains speculative, as carbonic anhydrase activity in the kidney proximal tubule cells couples the CO2 with OH- to generate HCO3-, thereby diminishing the concentration of intracellular OH-. We propose that the physiologic significance of Na+:OH- cotransport mode may be in the colon, where secretion of acid into the lumen via NHE-3 may not lead to the reabsorption of HCO3-, as the luminal [HCO3-] is negligible (34), but rather results in generation of intracellular OH-. The Na+:OH- cotransporter will then exit the cell across the basolateral membrane, thereby regulating cell pH.

In conclusion, based on functional properties (Figs. 1-6) and nephron segment distribution studies (28), we propose that NBC-1 is the kidney proximal tubule Na+:HCO3- cotransporter.

    ACKNOWLEDGEMENT

The technical assistance of Holli Shumaker is greatly appreciated.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grants DK 46789 and DK 52821 and by funds from Dialysis Clinic Inc. (to M. S.).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.

To whom correspondence should be addressed: University of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585. Tel.: 513-558-5462; Fax: 513-558-4309; E-mail: manoocher.soleimani{at}uc.edu.

1 The abbreviations used are: NBC, Na+:HCO3- cotransporter; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein.

2 For the Na+:HCO3- cotransporter to work in the efflux mode, the cells were loaded with a high sodium-containing solution (140 mM) for 30 min as compared with regular solution (Table I, solution A), which has 115 mM sodium. Although the difference between the sodium concentrations in the two solutions is only 25 mM, we observe that NBC can function in the efflux mode only at higher Na+ concentration, indicating that incubation with high Na+ solution increases intracellular Na+ and therefore allows for the Na+:HCO3- cotransporter to function in the efflux mode.

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Abstract
Introduction
Procedures
Results
Discussion
References

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