Basolateral membrane Clminus -, Na+-, and K+-coupled base transport mechanisms in rat MTALH

Soline Bourgeois, Sandrine Massé, Michel Paillard, and Pascal Houillier

Université Pierre et Marie Curie, Institut National de la Santé et de la Recherche Médicale Unité 356, Institut Fédératif de Recherche 58, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, 75015 Paris, France


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INTRODUCTION
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Mechanisms involved in basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl- induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and blunted in the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The alkalinization elicited by removing peritubular Cl- persisted in the bilateral absence of Na+, together with a voltage clamp. When studied in Cl--free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Removing peritubular Na+ elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport and inhibition of a basolateral Na+/H+ exchange. Increasing bath K+ induced an intracellular alkalinization that was inhibited in the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. At 2 mM, peritubular Ba2+, which inhibits the K+-Cl- cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K+-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba2+-sensitive K+-Cl- cotransport that may operate as a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport mechanisms are functional under physiological conditions: electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, DIDS- and EIPA-insensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, and Ba2+-sensitive electroneutral K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport.

medullary thick ascending limb; bicarbonate; in vitro microperfusion; intracellular pH


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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TUBULAR REABSORPTION OF FILTERED bicarbonate (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) is a major function of the kidney. Under normal conditions, all the filtered HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is actively reabsorbed by the renal tubule, thereby preventing renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loss and metabolic acidosis. The main site of renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is the proximal tubule, where the mechanisms of apical and basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport are reasonably well known (for review see Ref. 1). The second most important tubular segment for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reclamation is the thick ascending limb of the loop of Henle (TALH), which reabsorbs ~15% of the filtered HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (13). In addition, in the medulla, the reabsorbed HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is delivered to the interstitium, characterized by a low blood flow; therefore, the reabsorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by the medullary TALH (MTALH) is a critical determinant of interstitial pH that controls secretion of protons by the neighboring medullary collecting duct (15).

The apical step of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entrance into the MTALH cell relies on proton secretion. Two different mechanisms of proton secretion have been described in these cells: an electroneutral Na+/H+ exchange and an electrogenic, ATP-dependent proton pump (11). However, only the former seems to be physiologically significant under the various conditions tested. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is almost completely inhibited in the absence of luminal Na+ and/or in the presence of luminal amiloride (16). Finally, the expression and activity of the apical Na+/H+ exchanger is enhanced during metabolic acidosis and depressed during Cl--depleted metabolic alkalosis (26, 27), conditions under which MTALH HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is increased or decreased, respectively (12, 14).

In contrast to the apical mechanisms involved in proton secretion, the mechanisms involved in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit from the MTALH cells under physiological conditions are unsettled. However, several HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit mechanisms have been successively suggested. Previous experiments examining HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit mechanisms have been mainly conducted with MTALH suspensions. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na+-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>)n and Ba2+-insensitive electroneutral K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransports have been described in mouse and rat MTALH suspensions, respectively, and no evidence for the presence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange has been found in mouse and rat MTALH suspensions (23, 28). However, these data have recently been challenged by the results of a study using purified basolateral membrane vesicles from rat MTALH cells (29). A weak Na+-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>)n cotransport activity and no electroneutral K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity were observed in this preparation. In contrast, an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity was apparent under isosmotic conditions and in the absence of arginine vasopressin in basolateral membrane vesicles from rat MTALH cells (10). In addition, two distinct isoforms of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers were reported in this preparation (10). These results are in keeping with those recently obtained by Sun (37) in microperfused mouse MTALH, demonstrating a basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity under baseline conditions.

Because a growing body of evidence indicates that tubule suspensions do not appear to be suitable for identification of membrane transporters (10, 29, 32) and because a transporter itself may be altered and/or a regulatory factor may be lost during the preparation of membrane vesicles, the present study used microperfused MTALH to identify the mechanisms physiologically involved in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport across the basolateral membrane of intact rat MTALH cells. Here, we demonstrate the presence and the functionality, under isosmotic conditions, of an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, and an electroneutral Ba2+-sensitive K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport.


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Tubule isolation and perfusion. Pathogen-free male Sprague-Dawley rats (60-75 g body wt; Iffa Credo) had free access to standard rat chow and distilled water until the time of experiments. Animals were injected intraperitoneally with 2 mg of furosemide 10 min before they were anesthetized with pentobarbital sodium (50 mg/kg body wt). Both kidneys were cooled in situ with control bath solution for 1 min and then removed and cut into thin coronal slices for tubule dissection. MTALH were dissected from the inner stripe of the outer medulla at 4°C in the control solution of the respective experiment. The isolated tubule was transferred to the bath chamber on the stage of an inverted microscope (Axiovert 100, Carl Zeiss), mounted on concentric pipettes, and perfused in vitro. To prevent motion of the tubule during intracellular pH (pHi) measurement, the average tubule length exposed to bath fluid was limited to 300-350 µm.

Solution composition. The composition of the various solutions is given in Table 1. In nominally Na+-free solutions, Na+ was isosmotically replaced with N-methyl-D-glucamine (NMDG+). Cl--free solutions contained gluconate as a replacement for Cl-. We compensated for Ca2+ chelated by Cl- substitutes by increasing total Ca2+ concentration from 2 to 7.5 mM in Cl--free solutions. The solutions containing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were continuously gassed with 95% O2-5% CO2 at 37°C, and the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions were continuously gassed with 100% O2 passed through a 3 N KOH CO2 trap. Before each experiment, Na+, K+, and Cl- concentrations, osmolality, and pH were measured in bulk solutions.

                              
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Table 1.   Composition of solutions

Alanine and HEPES were obtained from Research Organics (Cleveland, OH), DIDS from Acros Organics, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester from Molecular Probes (Eugene, OR). 5-Nitro-2-(3-phenylpropylamino)benzoate (NPPB), [(dihydroindenyl)oxy]alkanoic acid (DIOA), and all other chemicals were obtained from Sigma (Saint-Quentin Fallavier, France).

pHi measurement. MTALH cells were loaded with the fluorescent probe BCECF, prepared as a 20 mM stock in DMSO, by exposing the cells for ~20 min at room temperature to the control bath solution containing 20 µM BCECF. Loading was continued until the fluorescence intensity at 440-nm excitation was at least one order of magnitude higher than background fluorescence. The loading solution was then washed out by initiation of bath flow, and the tubule was equilibrated with dye-free control bath solution for 5-10 min. The bath solution was delivered at 20 ml/min and warmed to 37 ± 0.5°C by a water jacket immediately upstream to the chamber. The perfusion rate was adjusted by hydrostatic pressure to ~20 nl/min to prevent axial changes in composition of the luminal fluid.

Intracellular dye was excited alternately at 490 and 440 nm with a 75-W xenon lamp and a computer-controlled chopper assembly. Emitted light was collected through a dichroic mirror, passed through a 530-nm filter, and focused onto a charge-coupled device camera (model ICCD 2525F, Videoscope International) connected to a computer. The measured light intensities were digitized with eight-bit precision (256 gray level scale) for further analysis. For each tubule, a region of interest was drawn, and the mean gray level for each excitation wavelength was calculated with Starwise Fluo software (Imstar, Paris, France). Background fluorescence was subtracted from fluorescence intensity at each excitation wavelength to obtain intensities of intracellular fluorescence. The ratio of fluorescence at 490 nm to that at 440 nm was used as an indicator of pHi.

Intracellular dye was calibrated at the end of each experiment using the high-K+-nigericin technique. Tubules were perfused and bathed with a HEPES-buffered, 95 mM K+ solution containing 10 µM nigericin, a K+/H+ exchanger. Four different calibration solutions, titrated to pH 6.5, 6.9, 7.3, or 7.5, were used.

Determination of intrinsic buffering capacity. The intrinsic buffering capacity (beta i) of MTALH cells was determined using a method similar to that used by Watts and Good (40), except a weak acid, instead of a weak base, was used to change pHi and Ba2+ was omitted from the solutions. To exclude HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2 as a buffering component and block Na+-dependent pHi regulatory mechanisms, Na+-free, HEPES-buffered solutions containing 1 mM amiloride were used in the luminal perfusate and basolateral bath. Addition of 20 mM acetate to the bath induced a decrease in pHi. The dissociation constant of acetic acid (pKa; 4.74) was used to calculate the intracellular acetate concentration when cell acidification reached a plateau. The beta i was calculated as the ratio of the change in intracellular acetate concentration to the change in pHi; beta i ranged from 32.8 to 48.5 mM/pH unit over the range of initial pHi observed in the experiments.

Calculation of base flux values. Base flux (Jbase) values (in pmol · min-1 · mm-1) were calculated using the following equation: Jbase = dpHi/dt × beta total × V, where dpHi/dt is the initial rate of change in pHi (in pH units/min), beta total is the buffering capacity (in mM · pH unit-1 · l-1), and V is the cell volume (in nl) per 1 mm of tubule length.

Values for dpHi/dt were determined by a computer-assisted fitting of the pHi vs. time data to a linear regression line over the first 15-30 s. In the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, beta total is equal to beta i; in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, beta total is equal to beta i + 2.3 × [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i, where [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i is intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, calculated as follows: [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i = 0.03PCO2 × 10<SUP>(pH<SUB>i</SUB>−6.10)</SUP>, with the assumption that intracellular PCO2 equals extracellular PCO2 (37 mmHg). V was calculated by determining the volume of the tubule [(radius of tubule)2 × Pi  × length] and subtracting the volume of the lumen [(radius of lumen)2 × Pi  × length] and was equal to 0.31 ± 0.01 nl/mm. A negative value of Jbase indicates a net base efflux, and a positive value indicates a net base influx.

Measurement of transepithelial voltage. The transepithelial voltage (Vte) was measured with a DP-301 differential electrometer (Warner) by use of a Ag-AgCl electrode connected to the perfusion pipette via a 0.15 M NaCl or sodium gluconate agar bridge, as appropriate. A 0.15 M NaCl or sodium gluconate agar bridge also connected the peritubular bath to an Ag-AgCl electrode.

Statistics and data analysis. Values are means ± SE. Statistical significance was assessed by paired or impaired Student's t-test, as appropriate. P < 0.05 was considered significant.


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A functional electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is present in the basolateral membrane of MTALH cells. Because a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger electroneutrally exchanges Cl- that enters the cell for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that leaves the cell, the presence of a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity in the basolateral membrane of the MTALH cells is expected to yield the following results (31, 37). 1) The isosmotic removal of bath Cl- should induce a reversible intracellular alkalinization. 2) This effect should be blunted in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer, as well as in the basolateral presence of an anion-exchange inhibitor such as DIDS. 3) The exchange should operate independently of the presence of Na+ and changes in the membrane voltage.

As shown in Fig. 1A and Table 2, in the bilateral presence of Cl- and in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (extracellular pH 7.40), pHi was stable at ~7.08 (segment a-b, Fig. 1A). The isosmotic removal of basolateral Cl- (solution A to solution B) induced a prompt and significant intracellular alkalinization (segment b-c, Fig. 1A; Table 2). Conversely, readdition of bath Cl- (solution B to solution A) elicited a rapid fall in pHi, which returned to baseline values (segment c-d, Fig. 1A; Table 2).


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Fig. 1.   A: effect of basolateral Cl- removal on intracellular pH (pHi). Cells were initially perfused and bathed with a standard solution buffered with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (external pH 7.40) and containing 121 mM Cl- (segment a-b). Removing Cl- from the bath (replaced with gluconate) caused a large and fast pHi increase (segment b-c). Returning Cl- to the bath elicited a prompt decrease of pHi toward control values (segment c-d; n = 5 tubules). B: effect of basolateral Cl- removal on pHi in the presence of 0.15 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the bath. Cells were initially perfused and bathed with a standard solution buffered with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (external pH 7.40) also containing 0.15 mM DIDS in the bath. In the bilateral presence of 121 mM Cl-, pHi values were steadily increasing (segment a-b): the feature was clearly different from that observed in the absence of basolateral DIDS. Removing Cl- from the bath (replaced with gluconate) in the presence of DIDS caused small changes in pHi (segment b-c; n = 6 tubules). C: effect of basolateral Cl- removal in the bilateral absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and the presence of 0.1 mM acetazolamide to inhibit endogenous HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production on pHi. In the bilateral presence of 144 mM Cl-, pHi values were nearly constant (segment a-b). Removing peritubular Cl- (segment b-c) and then returning bath Cl- (segment c-d) caused only small changes in pHi (n = 6 tubules). D: effect of peritubular Cl- addition in the presence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, voltage clamp, and Na+ depletion on pHi. Tubules were initially perfused and bathed in Na+-free solutions containing 120 mM K+ and 10 µM valinomycin. Voltage clamp resulted in intracellular alkalinization (segment a-b). Isosmotic addition of Cl- to the bath (Cl- replacing gluconate) elicited a decrease in pHi (segment b-c) that was reversed by removal of peritubular Cl- (segment c-d).


                              
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Table 2.   Changes in pHi caused by bath Cl- removal or addition

In the presence of 0.15 mM bath DIDS and in the basolateral presence of Cl-, pHi was not stable but steadily increasing (segment a-b, Fig. 1B), at variance with data obtained in the absence of peritubular DIDS (Fig. 1A). This steadily increasing pHi was probably accounted for by a DIDS-induced inhibition of basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit pathway(s) under basal conditions. Furthermore, the effect of basolateral Cl- removal (solution A to solution B) was significantly blunted when tubules were studied in the presence of 0.15 mM basolateral DIDS: the initial rate of alkalinization was reduced by 63% (P < 0.01), and the rise in pHi after removal of basolateral Cl- was diminished by 46% (P < 0.05; segment b-c, Fig. 1B; Table 2). The isosmotic readdition of bath Cl- induced only a transient and nonsignificant decrease in pHi (segment c-d, Fig. 1B).

When studied in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (solution C, external pH 7.40) and in the bilateral presence of 0.1 mM acetazolamide to inhibit endogenous HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production, pHi measured in the bilateral presence of Cl- was significantly higher than pHi measured in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (segment a-b, Fig. 1C; Table 2). In addition, the basolateral replacement of Cl- with gluconate (solution C to solution D) did not induce any significant change in pHi (segment b-c, Fig. 1C; Table 2), indicating that the increase in pHi elicited by basolateral Cl- removal was dependent on the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

Finally, we studied the effect of basolateral Cl- addition in the bilateral absence of Na+ (isosmotically replaced with NMDG+) together with a voltage clamp obtained with 10 µM valinomycin in high (120 mM)-K+-containing Ringer solution (solution F); this maneuver has previously been shown to efficiently depolarize the plasma membrane when used with intact tubules (4). First, pHi values were higher (~7.45; segment a-b, Fig. 1D) under these conditions (absence of extracellular Na+ and Cl-, high extracellular K+, and valinomycin) than under control conditions (cf. segment a-b, Fig. 1, A and D). A high pHi has previously been noted by others in cortical or medullary TALH, studied under similar conditions (24, 37). This probably indicates that, in these cells, a base exit mechanism is inhibited by the absence of external Cl-, the high external K+ concentration, and/or the membrane depolarization induced by valinomycin and K+. The addition of basolateral Cl- (solution F to solution E) induced a prompt and significant intracellular acidification (segment b-c, Fig. 1D). The subsequent removal of bath Cl- elicited another increase in pHi to initial values (segment c-d, Fig. 1D), consistent with the presence of an electroneutral and Na+-independent Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (Fig. 1D, Table 2).

Taken together, these data demonstrate the presence of an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity under basal conditions in the basolateral membrane of the rat MTALH cells.

Because of the presence of an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity in the basolateral membrane of MTALH cells, all the following experiments have been carried out in the bilateral absence of Cl-.

A Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit pathway is present in MTALH cells. Two Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux mechanisms have been described in MTALH suspensions: an Na+-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>)n cotransport in mouse MTALH cells (which is DIDS sensitive), but not in rat MTALH cells (23), and a DIDS-sensitive and Ba2+-insensitive K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in rat MTALH (28). Moreover, in a recent study using purified basolateral membrane vesicles from rat MTALH, besides the presence of a robust Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and a weak Cl--independent Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, no conclusive evidence for a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport could be obtained. To clarify this issue, we wondered whether a basolateral Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux was present in the rat microperfused MTALH.

A Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit pathway in the basolateral membrane is expected to affect pHi in the following way. 1) In the bilateral absence of Cl-, a decrease in bath pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration at constant PCO2 should increase HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux and, therefore, induce a fall in pHi. 2) The outwardly directed base efflux induced by a given decrease in bath pH should be greater in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> than in its absence. 3) Because many HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport mechanisms previously described in the kidney are stilbene sensitive, DIDS should reduce the effect of bath HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> decrease on pHi. 4) The inhibitory effect of DIDS should be absent in a nominally CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free Ringer solution.

As shown in Fig. 2, when tubules were studied in a Cl--free Ringer solution (Cl- replaced with gluconate), a decrease in bath HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration from 23 mM (external pH 7.40) to 3 mM (external pH 6.60) at constant PCO2 (solution B to solution G) elicited a prompt intracellular acidification (segment b-c, Fig. 2) with an initial rate of change in pHi of -0.22 ± 0.04 pH unit/min. The initial base efflux induced by the decrement in peritubular pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration was calculated to be -4.2 ± 0.8 pmol · min-1 · mm-1 (see MATERIALS AND METHODS). In paired experiments conducted in the same tubules in the presence of 0.15 mM peritubular DIDS, the magnitude and the initial rate of decrease in pHi after the decrement in bath HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration were significantly reduced (segment e-f, Fig. 2). The initial rate of base efflux in the presence of peritubular DIDS was reduced and calculated to be -2.1 ± 0.4 pmol · min-1 · mm-1 (P < 0.05 vs. without DIDS). Also, before the decrease in peritubular pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, pHi values were rather stable in the absence of DIDS (segment a-b, Fig. 2) but steadily increased in the presence of 0.15 mM bath DIDS (segment d-e, Fig. 2); this indicates that, in the absence of external Cl-, peritubular DIDS inhibited a base efflux pathway under basal conditions.


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Fig. 2.   Effect on pHi of lowering bath pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the nominal absence of Cl- by decreasing basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration at fixed PCO2 in the absence and then in the presence of 0.15 mM DIDS in the bath. Tubules were initially perfused and bathed with a Cl--free solution (Cl- replaced with gluconate) buffered with CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, external pH 7.40; segment a-b). Lowering basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration from 23 to 3 mM at constant PCO2 caused a larger pHi decrease in the absence (segment b-c) than in the presence of 0.15 mM DIDS in the bath (segment e-f). Both effects on pHi were reversed by returning basolateral pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration to normal (segments c-d and f-g). Reported values of pHi were measured at the end of each experimental period (n = 5 tubules); dpHi/dt, rate of change of pHi. *P < 0.05, **P < 0.02, and ***P < 0.01 vs. the same period without DIDS.

In separate experiments, the same maneuvers were performed in the nominal absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and in the bilateral presence of 0.1 mM acetazolamide (Fig. 3). Under these conditions, a decrease in basolateral pH from 7.4 to 6.6 (solution D to solution H) elicited a significant intracellular acidification (segment b-c, Fig. 3) that appeared to be completely DIDS resistant (segment e-f compared with segment b-c, Fig. 3); the decrement in pHi and the rate of change in pHi were the same in the absence and presence of 0.15 mM DIDS. The average base efflux elicited by the decrease in bath pH was calculated to be -2.6 ± 0.3 pmol · min-1 · mm-1 (P < 0.05 vs. Jbase measured in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>).


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Fig. 3.   Effect on pHi of lowering basolateral pH in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in Cl--free solutions. The study was performed in the absence and then in the presence of 0.15 mM DIDS in the bath. Tubules were perfused and bathed with a CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>- and Cl--free solution (HEPES-Tris solution, external pH 7.40; segment a-b). Lowering basolateral pH from 7.40 to 6.60 in the absence of bath DIDS caused a pHi decrease (segment b-c) that was unchanged in the presence of 0.15 mM DIDS in the bath (segment e-f). Both effects on pHi were reversed by returning basolateral pH to normal (segments c-d and f-g). Reported values of pHi were measured at the end of each experimental period.

In summary, in the bilateral absence of Cl-, a decrease in bath pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration caused a reversible intracellular acidification and a net base efflux; both values were significantly decreased in the presence of bath DIDS or in the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Taken together, these data demonstrate a Cl--independent, DIDS-sensitive HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway in the basolateral membrane of MTALH cells.

DIDS-insensitive, EIPA-insensitive Na+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport activity is present in the basolateral membrane of MTALH cells. In the presence of an Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in the basolateral membrane of MTALH cells, the following changes in pHi should be observed. The isosmotic removal of bath Na+ (Na+ replaced with NMDG+) induces an outwardly directed Na+ efflux that drives HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> out of the cell, resulting in a decrease in pHi; however, the analysis is complicated by the presence of Na+/H+ exchange activity in this basolateral membrane that acidifies the cell as a consequence of peritubular Na+ removal. Nevertheless, if an Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport is present in addition to an Na+/H+ exchange, removal of peritubular Na+ should induce a higher base efflux in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> than in its absence (HEPES-Tris condition).

The results are shown in Fig. 4. In the presence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pHi remained stable in the peritubular presence of 140 mM Na+ (segment a-b, Fig. 4A). Replacing peritubular Na+ with NMDG+ (solution B to solution I) elicited a prompt intracellular acidification (from 6.92 ± 0.04 to 6.41 ± 0.05 pH units; segment b-c, Fig. 4A), indicating a net base efflux. The initial base efflux was calculated to be -6.32 ± 0.56 pmol · min-1 · mm-1; finally, returning bath Na+ concentration to the control value induced a cellular realkalinization (segment c-d, Fig. 4A). When the same maneuvers were performed in paired experiments in the peritubular presence of 30 µM EIPA, the changes in pHi induced by the removal/readdition of bath Na+ were significantly decreased, but not suppressed, indicating an EIPA-sensitive component in the Na+-dependent net base efflux (Fig. 4A).


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Fig. 4.   Effect on pHi of basolateral Na+ removal (replaced by N-methyl-D-glucamine+) in Cl--free solutions in the absence and then in the presence of 30 µM ethylisopropylamiloride (EIPA) in the bath in the presence (A) and absence (B) of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. A: tubules were initially perfused with a Cl-- and Na+-free solution containing 23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (1 mM amiloride was also added in the luminal fluid) and bathed with a Cl--free solution containing 23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 140 mM Na+ (external pH 7.40; segment a-b). In the absence of EIPA, basolateral Na+ removal induced a reversible decrease in pHi (segment b-c) that was partially blunted in the presence of 30 µM EIPA in the bath (segment f-g). Both effects on pHi were reversed by returning basolateral Na+ concentration to normal (segments c-d and g-h; n = 7 tubules). B: tubules were initially perfused with a Cl-- and Na+-free solution (also containing 1 mM amiloride) and bathed with a Cl--free solution containing 140 mM Na+ in the bilateral absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and in the presence of 0.1 mM acetazolamide (external pH 7.40; segment a-b). In the absence of EIPA, basolateral Na+ removal induced a reversible pHi decrease (segment b-c) that was dramatically reduced in the presence of 30 µM EIPA in the bath (segment f-g). Both effects on pHi were reversed by returning basolateral Na+ concentration to normal (segments c-d and g-h; n = 6 tubules). Reported values of pHi were measured at the end of each experimental period. **P < 0.01 vs. the same period in the absence of EIPA; ***P < 0.001 vs. the same period in the absence of EIPA.

The same experimental maneuvers were then performed in the complete absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 4B). Removal of peritubular Na+ (solution D to solution J) induced a significant intracellular acidification (segment b-c, Fig. 4B) that was reversed by returning peritubular Na+ concentration to its initial value (segment c-d, Fig. 4B). The initial rates of change in pHi after peritubular Na+ removal were almost identical in the presence and absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>; however, because the intracellular buffering power was significantly higher in the presence than in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (58.0 ± 0.55 vs. 34.9 ± 3.61 mmol · pH unit-1 · l-1, P < 0.001), the Na+-driven base efflux was accordingly lower under CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions (-3.42 ± 0.38 pmol · min-1 · mm-1, P < 0.01). Finally, the effect of peritubular EIPA was studied in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. The magnitude and the rate of changes in pHi were significantly decreased in the presence of 30 µM peritubular EIPA (segment a-b-c-d compared with segment e-f-g-h, Fig. 4B).

Taken together, these results indicated the presence in the basolateral membrane of an EIPA-sensitive Na+/H+ exchange activity and an additional Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity, the latter being demonstrated by the higher Na+-driven base efflux measured in the presence than in the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In this regard, it is noteworthy that the sum of the EIPA-resistant Na+-driven base efflux measured in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions (-2.92 ± 0.21 pmol · min-1 · mm-1) and the Na+-driven base efflux in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions (-3.42 ± 0.38 pmol · min-1 · mm-1) was identical to the Na+-driven base efflux measured in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions (-6.32 ± 0.56 pmol · min-1 · mm-1). In addition, because the Na+-driven base efflux was always higher in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing than in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions, irrespective of the absence or presence of EIPA, the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity was EIPA resistant, whereas the Na+/H+ exchange activity was EIPA sensitive.

In a second set of experiments, we studied the sensitivity to DIDS of the Na+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport across the basolateral membrane of the rat MTALH cells (Fig. 5). Under control conditions, pHi was stable in the presence of 140 mM bath Na+ (segment a-b, Fig. 5). Removing bath Na+ (solution B to solution I) induced a fall in pHi (from 6.98 ± 0.04 to 6.63 ± 0.04 pH units; segment b-c, Fig. 5) that was reversed after bath Na+ concentration was returned to the control value (segment c-d, Fig. 5). Paired experiments have been conducted in the presence of 0.15 mM (n = 3) or 0.5 mM (n = 3) DIDS in the bath. First, in the bilateral presence of Na+, the presence of DIDS in the bath led the pHi to steadily increase with time, at variance with our observation in the absence of DIDS (segment d-e compared with segment a-b, Fig. 5). As previously noted, this favors a Cl--independent, DIDS-sensitive base exit mechanism in the basolateral membrane of the MTALH cells under basal conditions. Second, removing peritubular Na+ in the presence of DIDS elicited a fall in pHi (segment e-f, Fig. 5) that was indistinguishable from the cell acidification observed in the absence of DIDS (segment b-c, Fig. 5). Indeed, the magnitude and the rate of cell acidification were the same in the presence and absence of DIDS. Third, the magnitudes of pHi increase after readdition of Na+ to the bath were the same in the presence and absence of DIDS (segment f-g compared with segment c-d, Fig. 5). Finally, the rates of change in pHi after bath Na+ readdition in the presence and absence of bath DIDS were not readily comparable, because pHi measured at the end of the 0 Na+ period significantly differed when DIDS was present and when it was absent (point f compared with point c, Fig. 5). Also, the rates of changes in pHi were not readily comparable between Fig. 4 and 5, because luminal Na+ was present in experiments in Fig. 5 and absent in experiments in Fig. 4.


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Fig. 5.   Effect on pHi of basolateral Na+ removal (replaced by N-methyl-D-glucamine+) in Cl--free solutions in the absence and then in the presence of 0.15 mM (n = 3 tubules) or 0.5 mM (n = 3 tubules) DIDS in the bath. Tubules were initially perfused and bathed with a Cl--free solution containing 23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 140 mM Na+ (external pH 7.40; segment a-b). In the absence of DIDS, basolateral Na+ removal induced a reversible pHi decrease (segment b-c) that was unchanged in the presence of 0.15 or 0.5 mM DIDS in the bath (segment e-f). Both effects on pHi were reversed by returning basolateral Na+ concentration to normal (segments c-d and f-g). Reported values of pHi were measured at the end of each experimental period (n = 6 tubules). **P < 0.001 vs. the same period in the absence of DIDS.

These results indicated that the EIPA-resistant Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in the basolateral membrane of MTALH cells was also DIDS resistant.

A Ba2+-sensitive electroneutral K+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport is present in the basolateral membrane of MTALH cells. The presence of a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport is expected to affect pHi in the following way. Increasing peritubular K+ concentration reduces the outwardly directed K+ efflux; therefore, if K+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux are electroneutrally coupled, increasing bath K+ concentration should reduce HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux and elicit a rise in pHi. However, because of the presence of basolateral K+ channel(s), increasing peritubular K+ concentration may induce an acute depolarization of the basolateral membrane, thereby affecting any electrogenic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit mechanism located in the same membrane. That is, an increase in pHi elicited by a rise in peritubular K+ concentration does not allow a distinction between an electrogenic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport mechanism and an electroneutral K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport.

However, in the case of the thick ascending limb (TAL), the mechanism through which an increase in peritubular K+ concentration induces a basolateral membrane depolarization is not quite so simple. Greger and Schlatter (19) proposed that a high peritubular K+ concentration may directly inhibit an electroneutral K+-Cl- cotransport, leading to a rise in cytosolic Cl- activity and a secondary basolateral membrane depolarization. In addition, in their experiments, peritubular Ba2+ was able to abolish the effect of peritubular K+ through a direct inhibition of the K+-Cl- cotransport activity. More recently, Di Stefano et al. (9) confirmed that peritubular Ba2+ does not act on a basolateral K+ channel but on the electroneutral K+-Cl- cotransport. Therefore, to precisely determine the nature of a putative K+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, we tested the effect of the changes in the peritubular concentrations of K+ and Ba2+, alone or in combination, on pHi. In addition, we measured the acute effect of peritubular Ba2+ addition on Vte, taken as an index of basolateral membrane voltage. We reasoned that, if K+ and Ba2+ are able to inhibit the same K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, both would be expected to induce a rise in pHi; in addition, the effect of the increase in peritubular K+ concentration on pHi would be attenuated in the presence of Ba2+ (absence of an additive effect); finally, if K+ and Ba2+ inhibit an electroneutral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, the addition of peritubular Ba2+ would not change Vte.

In the bilateral presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, increasing bath K+ concentration from 4 to 40 mM (solution K to solution L) provoked a sharp and reversible intracellular alkalinization (from 6.99 ± 0.03 to 7.28 ± 0.05 pH units; segment b-c, Fig. 6). This alkalinization rapidly reversed (segment c-d, Fig. 6) when bath K+ concentration was returned to 4 mM (solution L to solution K).


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Fig. 6.   Effect on pHi of basolateral K+ concentration increase in the absence and then in the presence of 2 mM Ba2+ in the bath. Tubules were initially perfused and bathed with a Cl--free solution containing 4 mM K+ and 23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (segment a-b). Basolateral increase in K+ concentration from 4 to 40 mM caused a large and rapid rise in pHi (segment b-c). When the same maneuver was performed in the presence of 2 mM Ba2+ in the bath, the rate of change in pHi was dramatically inhibited (segment f-g). Both effects on pHi were reversed by returning basolateral K+ concentration to normal (segments c-d and g-h). Reported values of pHi were measured at the end of each experimental period (n = 7 tubules). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the same period without Ba2+.

When bath K+ concentration was changed in paired experiments on the same tubules in the presence of 2 mM Ba2+ in the bath (solutions M and N), the magnitude and rate of change in pHi were clearly inhibited (Fig. 6, right). The rate of change in pHi was inhibited by 55 ± 7% when bath K+ was changed from 4 to 40 mM (segment f-g compared with segment c-d, Fig. 6) and by 75 ± 6% when bath K+ was decreased from 40 to 4 mM (segment g-h compared with segment c-d, Fig. 6).

The effect of addition of 2 mM peritubular Ba2+ on pHi (solution K to solution S) is shown in Fig. 7. Peritubular Ba2+ acutely alkalinizes the cell (from 7.13 ± 0.03 to 7.21 ± 0.04 pH units, P < 0.05).


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Fig. 7.   Effect on pHi of 2 mM Ba2+ addition in the bath in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions in the nominal absence of Cl-. Tubules were initially perfused and bathed with a Cl--free solution containing 4 mM K+ and 23 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Peritubular addition of 2 mM Ba2+ induced a significant cellular alkalinization. Reported values of pHi were measured at the end of each experimental period (n = 6 tubules).

The effect of 2 mM peritubular Ba2+ on Vte is presented in Fig. 8. In the presence of Cl-, the peritubular addition of 2 mM Ba2+ (solution A to solution Q) induced a quick and significant increase in Vte (from 9.6 ± 1.3 to 15.0 ± 2.3 mV, P < 0.05). In contrast, Ba2+ was unable to induce a change in Vte in the bilateral absence of Cl- (from 2.0 ± 0.73 to 2.3 ± 0.73 mV; solutions B and R).


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Fig. 8.   Effect on transepithelial voltage of 2 mM Ba2+ addition in the bath in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions in the presence () and nominal absence of Cl- (replaced with gluconate; open circle ). Ba2+ induced an increase in Vte in the presence but not in the absence of Cl-.

Finally, when the effect of the same changes in bath K+ concentration was studied in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions and in the absence of Ba2+ (solutions O and P), the changes in pHi were blunted compared with those observed in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (segment a-b-c-d, Fig. 6 compared with Fig. 9). The rate and the magnitude of cellular alkalinization were inhibited by 55 and 59%, respectively, in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the solutions compared with values obtained in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solutions. Furthermore, the basolateral presence of 2 mM Ba2+ (solutions S and T) did not influence the magnitude or the rate of change in pHi when it was studied in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (segment a-b-c-d compared with segment e-f-g-h, Fig. 9). Thus increasing peritubular K+ concentration in HEPES-buffered solutions inhibits a Ba2+-insensitive proton influx (or OH- efflux).


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Fig. 9.   Effect on pHi of basolateral K+ concentration increase in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. The study was performed in the absence and then in the presence of 2 mM peritubular Ba2+. Tubules were initially perfused and bathed with a CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution containing 4 mM K+ (HEPES-Tris solution; segment a-b). Basolateral K+ concentration increase from 4 to 40 mM caused a small pHi increase (segment b-c) that was totally unchanged in the presence of 2 mM peritubular Ba2+ (segment f-g). Both effects on pHi were reversed by returning basolateral K+ concentration to normal (segments c-d and g-h).

Taken together, these results are consistent with the presence of a Ba2+-sensitive, K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit pathway in the basolateral membrane of rat MTALH cells that accounts for the major component of Cl--independent base efflux. Moreover, this pathway appears to be electroneutral, because addition of Ba2+ inhibited the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit but did not change Vte. Our results are also consistent with the presence of a Ba2+-resistant K+/H+ exchange (or K+-OH- cotransport) in the same membrane that accounts for a minor proportion of the Cl--independent basolateral base efflux, in agreement with previous studies from our laboratory using basolateral membrane vesicles from rat MTALH (29).

Pharmacological characterization of the electroneutral K+-coupled HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> exit pathway. The Ba2+ sensitivity of the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit is reminiscent of that described for the electroneutral K+-Cl- cotransport (3, 9, 19, 30).

To address this issue, we tested the effect of several drugs previously reported to be efficient inhibitors of the K+-Cl- cotransport on the activity of the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit pathway. We compared the initial rates of change in pHi induced by increasing peritubular K+ concentration from 4 to 40 mM in the absence and then in the peritubular presence of one of the following drugs: furosemide (2 mM), DIOA (0.1 mM), and NPPB (0.1 mM). Finally, because the Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway was sensitive to DIDS (Fig. 2), we also tested the effect of this latter drug on the K+-induced cellular alkalinization. As shown in Fig. 10 and Table 3, bath furosemide, bath DIOA, bath NPPB, and bath DIDS induced a significant inhibition in the initial rate of change in pHi, elicited by increasing basolateral K+ concentration from 4 to 40 mM.


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Fig. 10.   Effect of furosemide (Furo), [(dihydroindenyl)oxy]alkanoic acid (DIOA), 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), and DIDS on dpHi/dt induced by an increase in peritubular K+ concentration. Cells were initially alkalinized by increasing basolateral K+ concentration from 4 to 40 mM in Cl--free, CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions. dpHi/dt was reported as 100% and corresponded to control. The same experiment was performed in the same tubule in the presence of 2 mM furosemide, 0.1 mM DIOA, 0.1 mM NPPB, or 0.15 mM DIDS in the bath. In all cases, this maneuver induced a significant inhibition in dpHi/dt.


                              
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Table 3.   Effects of peritubular furosemide, DIOA, NPPB, and DIDS on cellular alkalinization induced by an increase in peritubular K+ concentration


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show that cells of the rat MTALH possess at least three distinct mechanisms for transporting HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across the basolateral membrane: an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange; a DIDS-resistant, EIPA-resistant Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport; and a Ba2+-sensitive, K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway. In addition, they exclude the presence of a DIDS-sensitive Na+-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>)n. Finally, they provide evidence for a basolateral Ba2+-resistant K+/H+ exchange (or K+-OH- cotransport) with a minor activity.

Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. The major evidence for a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is summarized in Fig. 1 and Table 2: removal of bath Cl- leads to an intracellular alkalinization that is dependent on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and sensitive to DIDS; in addition, Cl--coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport across the basolateral membrane of MTALH cells is independent of the presence of Na+ and changes in basolateral membrane voltage. Such a basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity is demonstrable under isosmotic conditions and in the absence of arginine vasopressin.

These results do not confirm the absence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange described in rat MTALH suspensions (28). In contrast, they are in complete agreement with the results recently obtained in our laboratory using purified basolateral membrane vesicles from rat MTALH cells. Eladari et al. (10) clearly demonstrated an electroneutral, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-driven Cl- movement in purified basolateral membrane vesicles from rat MTALH cells. In addition, they showed that two distinct isoforms of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger are present in this membrane: AE1-related polypeptide and AE2. Our results extend these data by demonstrating that the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is physiologically present in intact rat MTALH cells and, therefore, is likely involved in the active, transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption measured under isosmotic conditions.

Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway. In the proximal tubule, the basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit relies on the parallel activity of distinct transporters (1, 2), and it may be considered that the same may occur in other tubular segments. In addition, at least two other basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport mechanisms have been suggested to be present in the murine MTALH cell suspensions (23, 28). Therefore, it was mandatory to assess whether any of these transporters is present in intact rat MTALH cells. The first step was to demonstrate that a Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway is present in our preparation. This was achieved by studying the effect on pHi of lowering peritubular pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration at constant PCO2 in Cl--free solutions. This maneuver is expected to enhance an outwardly directed HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux and to elicit an intracellular acidification, provided a Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway is present in these cells. Such a decrease in peritubular pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration induces a significant fall in pHi, consistent with an enhancement of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux. In addition, the cellular acidification elicited by decreasing bath HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration is significantly blunted in the peritubular presence of DIDS, a drug that inhibits many HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport mechanisms. Furthermore, when the effect of the same decrease in peritubular pH (from 7.40 to 6.60) is studied in the nominal absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> together with acetazolamide to inhibit endogenous HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> production, the initial rate of change in pHi is the same as in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solution, despite a lower buffering power. Indeed, beta total was 60 ± 1 mM · pH unit-1 · l-1 in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solutions vs. 40 ± 2 mM · pH unit-1 · l-1 in HEPES-buffered solutions (P < 0.001). The net base efflux that occurs after a given decrease in peritubular pH is higher in the presence than in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Finally, when studied in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions, peritubular DIDS significantly decreases net base efflux to the level measured in a CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free, HEPES-buffered solution. In addition, DIDS has no effect on the changes in pHi elicited by a decrease in bath pH in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. In summary, these data demonstrate the presence of a Cl--independent, DIDS-sensitive mechanism that specifically drives HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> out of the MTALH cells. The cellular acidification induced by the decrease in bath pH in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free Ringer solution is, at least in part, ascribable to the inhibition of basolateral Na+/H+ exchange activity (see below).

Na+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport activity. Our evidence for the presence of a DIDS-insensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport is summarized in Figs. 4 and 5. Removal of peritubular Na+ induces a cellular acidification, indicating a net base efflux; in addition, under basal conditions, the Na+-driven base efflux is significantly larger when measured in the presence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> than in its absence (-6.32 ± 0.56 vs. -3.42 ± 0.38 pmol · min-1 · mm-1; Fig. 4). These data provide direct evidence for the coexistence in the basolateral membrane of the MTALH cells of an Na+/H+ exchange activity that operates in the absence as well as in the presence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and an additional Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity that operates only in the presence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

EIPA and DIDS were used to further precisely determine the pharmacological sensitivities of these Na+-coupled transporters. Clearly, the Na+/H+ exchange activity is EIPA sensitive (Fig. 4B). In contrast, the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity is EIPA insensitive: indeed, the EIPA-induced decrease in Na+-driven base efflux is not significantly different in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (from -6.32 ± 0.56 to -2.92 ± 0.21 pmol · min-1 · mm-1) and in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (from -3.43 ± 0.38 to -1.18 ± 0.15 pmol · min-1 · mm-1). In addition, the algebraic sum of the Na+-driven base efflux in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and EIPA (i.e., Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity) and the Na+-driven base efflux in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and EIPA (i.e., Na+/H+ exchange activity) is identical to the Na+-dependent base efflux in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and in the absence of EIPA (Na+/H+ exchange plus Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activities). In addition, the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity is also insensitive to DIDS. Indeed, the intracellular acidification elicited by peritubular Na+ removal in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is the same with and without DIDS in the bath. In the tubular segments where the DIDS-sensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport NBC1 has been demonstrated, that is, the convoluted proximal tubule and the S3 proximal tubule, basolateral DIDS or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid significantly inhibits the magnitude and the rate of change in pHi induced by the isosmotic bath Na+ removal (25, 31). We have obtained strikingly different results, since neither the magnitude nor the rate of change in pHi after peritubular Na+ removal is influenced by bath DIDS, even if DIDS is present at a concentration as high as 0.5 mM.

Our results are in keeping with the recent demonstration of the electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport NBCn1 in the basolateral membrane of the rat MTALH cells (38). This cotransport has been shown to be weakly and variably inhibited by 500 µM DIDS and to be insensitive to EIPA (8), as is our basolateral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity, which is resistant to EIPA and DIDS. Our results are also in accordance with the fact that neither the mRNA for the rat electrogenic, DIDS-sensitive Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC1 nor the protein itself has been detected in a significant amount in this segment (5, 35, 36). In addition, a recent study from our laboratory has demonstrated a small but significant Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in purified basolateral membrane vesicles from rat MTALH cells (29).

Our results differ from those obtained by Krapf (24) in the rat microperfused cortical TAL. By monitoring pHi, he observed that removing Na+ from the bath elicited an intracellular acidification that was independent of Cl-, inhibited by bath 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, electrogenic, and much more marked in the presence than in the absence of external CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Therefore, it can be proposed that the pathways for basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit differ between the cortical and the medullary part of the rat TAL. However, neither the electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBC1 nor the electroneutral Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter NBCn1 of rat kidney has been detected in cortical TAL cells (35, 38).

Ba2+-sensitive electroneutral K+-HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> cotransport. Our evidence for the presence of a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport essentially derives from the results obtained during changes in peritubular K+ and/or Ba2+ concentrations and from the effect of peritubular Ba2+ on Vte. In the absence of Ba2+, the increase in basolateral K+ concentration elicits a very significant intracellular alkalinization. Such an effect of bath K+ concentration may be the consequence of the inhibition of an electroneutral K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in the basolateral membrane or, alternatively, the consequence of the K+-induced depolarization of the basolateral membrane that subsequently inhibits any electrogenic basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux mechanism. Indeed, the increase in bath K+ concentration causes a membrane depolarization in cortical TALH (9, 19), in agreement with the presence of a K+ channel (9, 22, 33). The analysis of our data and those previously obtained by others allows us to propose that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> may exit the cell, together with K+, by substitution to Cl- in the basolateral K+-Cl- cotransport. In fact, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit is independently inhibited by an increase in peritubular K+ concentration and by addition of 2 mM peritubular Ba2+ to the bath. In addition, the effect of high peritubular K+ concentration is significantly inhibited in the presence of Ba2+: the lack of additive effect of K+ and Ba2+ strongly suggests that both act on the same HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit mechanism.

The target of Ba2+ in the basolateral membrane of the TAL has been identified by Greger and Schlatter (19) and Di Stefano et al. (9). In electrophysiological studies, they showed that Ba2+ does not inhibit a basolateral K+ channel but, rather, the electroneutral basolateral K+-Cl- cotransport. Accordingly, in the presence of Cl-, Ba2+ leads to a depolarization of the basolateral membrane because of the induced increase in intracellular Cl- activity (3, 9, 19). The effect of Ba2+ on the membrane voltage disappears in the absence of Cl- (3) and when transepithelial Cl- reabsorption is prevented by luminal furosemide (9). Our results are in perfect accordance. In fact, 2 mM peritubular Ba2+ induces a rise in Vte (very likely via a decrease in the basolateral membrane voltage) in the presence but not in the absence of extracellular Cl- (Fig. 8). In conclusion, our results demonstrate that, in the absence of Cl-, K+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exits across the basolateral membrane of the MTALH cell are Ba2+ sensitive and electroneutrally coupled.

The Ba2+ sensitivity of this cotransport is reminiscent of that reported for the electroneutral K+-Cl- cotransport (9, 19, 30). This leads us to propose that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> could be efficiently transported by a basolateral K+-Cl- cotransport, with a substitution of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> for Cl-. Therefore, we have tested the effect of several known inhibitors of the K+-Cl- cotransport on the K+-induced changes in pHi. As shown in Fig. 9, peritubular furosemide, DIOA, or NPPB induced a significant inhibition of the initial rate of change in pHi after the increase in peritubular K+ concentration from 4 to 40 mM. These data strengthen our hypothesis that a basolateral K+-Cl- cotransport could operate as a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. Finally, because the Cl--independent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux is DIDS dependent (Fig. 2), we tested the effect of DIDS under the same conditions; DIDS also appeared to be an efficient inhibitor of the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, which is also consistent with the involvement of a member of the K+-Cl- family in the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit, since the KCC1 and KCC4 isoforms have been shown to be inhibited by DIDS (30). However, the definitive demonstration that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> can be efficiently transported by a member of the KCC family requires further experimentation.

The present results suggesting a Ba2+- and DIDS-sensitive K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in rat MTALH basolateral membrane differ from those obtained previously with suspensions or basolateral membrane vesicles from rat MTALH. An electroneutral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux that is K+, but not Cl- or Na+, dependent and DIDS, but not Ba2+, sensitive has been previously suggested in MTALH suspensions (28). Because a DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is clearly present in rat MTALH, as evidenced in basolateral membrane vesicles (10) and basolateral membrane of MTALH perfused in vitro (present study), the absence of Cl--dependent efflux of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in rat MTALH suspensions is very likely explained by methodological limitations of tubule suspension preparations (10). In addition, the K+ dependence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux observed in MTALH suspensions was tested in Cl--containing solutions, a condition in which indirect effects of K+ concentration changes on the DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange cannot be excluded (29). Moreover, the lack of conclusive evidence for a K+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in purified basolateral membrane vesicles from rat MTALH might be related to the presence of K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport in an inactive form, in agreement with the failure to find evidence for K+-Cl- cotransport activity in purified basolateral membrane of renal cortex vesicles (7, 17), except when homogenizing solutions used for vesicles preparation were hypotonic to stimulate the K+-Cl- cotransport activity (39).

In summary, at least three distinct HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport pathways are present in the basolateral membrane of MTALH cells: a DIDS-sensitive electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, a DIDS- and EIPA-resistant Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, and a Ba2+-sensitive electroneutral K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport. The physiological significance of the coexistence of these three distinct pathways is unknown. Under physiological conditions, it is likely that the Na+-coupled pathway, which possesses the same pharmacological profile as NBCn1, is probably electroneutral and operates as a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> loader (38) and is therefore not directly involved in the active HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by the rat MTALH cell but, rather, in the control of pHi. The Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and the K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport are expected to drive HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> out of the cell and, thus, to be involved in the active, transcellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption that occurs in the MTALH. The reason for the requirement of two distinct HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathways in the basolateral membrane of MTALH cells is not obvious, however. In addition, the respective contribution of each pathway to overall active HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is unknown, and this question is probably difficult to address. In fact, we have observed that removing bath Cl- inhibits an electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (Fig. 1) and reduces the net transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by 57%, suggesting that the electroneutral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange process is of major importance in mediating HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux (21). However, removing bath Cl- acutely alkalinizes the cell, thereby favoring HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit along the other pathway. Therefore, it is likely that the estimation of the contribution of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in overall active HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is underestimated by bath Cl- removal. On the other hand, blocking the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway with DIDS, furosemide, or NPPB will also provide false estimates of the role of this latter pathway, because blockers of the K+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> efflux pathway are also efficient inhibitors of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (6).

As previously suggested in squid giant axon (20), we may propose that the availability of two acid loaders gives the MTALH cell the possibility of lowering pHi with an accompanying accumulation of Cl- or a depletion of K+. Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is expected to lead to cell swelling (because transported HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is not fully active osmotically), and K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport is expected to lead to cell shrinkage. The availability of two acid loaders with opposite effects on cell volume would allow MTALH to regulate cell pH independently of cell volume. In addition, in the particular case of the MTALH cell that also reabsorbs a significant amount of NaCl, it may be considered that an increase in the apical entry of NaCl leads to an increase in intracellular Cl- concentration (34) and cell volume that, in turn, reduces the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, activates the K+-Cl-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) cotransport (because the KCC cotransporters are stimulated by cell swelling), and, finally, allows the maintenance of cell volume and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption, independently of the change in NaCl reabsorption.


    ACKNOWLEDGEMENTS

We thank C. Nicolas for excellent secretarial assistance.


    FOOTNOTES

This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Université Pierre et Marie Curie.

Address for reprint requests and other correspondence: P. Houillier, Dépt. de Physiologie, Hôpital Européen Georges Pompidou, 20-40 rue Leblanc, 75015 Paris, France (E-mail: pascal.houillier{at}egp.ap-hop-paris.fr).

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.

10.1152/ajprenal.00220.2000

Received 24 July 2001; accepted in final form 8 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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