Pathways for HCOminus 3 exit across the basolateral membrane in rat thick limbs

Françoise Leviel, Dominique Eladari, Anne Blanchard, Jean-Stéphane Poumarat, Michel Paillard, and René-Alexandre Podevin

Laboratoire de Physiologie et Endocrinologie Cellulaire Rénale, Faculté de Médecine Broussais-Hôtel Dieu, Institut National de la Santé et de la Recherche Médicale, Unité 356, 75270 Paris, France


    ABSTRACT
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ABSTRACT
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METHODS
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We studied the pathways for HCO-3 transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward HCO-3 gradient in the presence of an inside-positive potential stimulated the rate of 22Na uptake minimally and did not induce a 22Na overshoot, arguing against the presence of electrogenic Na+-HCO-3 cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of 86Rb+ (a K+ analog) with or without HCO-3. Conversely, applying an outward K+ gradient caused a modest intracellular pH (pHi) decrease of ~0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by HCO-3, indicating the absence of appreciable K+-HCO-3 cotransport. On the other hand, imposing an inward Cl- gradient in the presence of HCO-3 caused a marked pHi decrease of ~1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a HCO-3/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked HCO-3 transport could not be demonstrated. These and other data suggest that basolateral Cl-/HCO-3 exchange could be the major pathway for HCO-3 transport in the MTAL.

medullary thick ascending limb; bicarbonate; lactate; membrane vesicles


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INTRODUCTION
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THE CELLS OF THE MEDULLARY thick ascending limb (MTAL) are significantly engaged in absorption of filtered HCO-3. In the rat and mouse MTALs, the first step of this HCO-3 absorption is believed to be mediated virtually completely by apical membrane Na+/H+ exchangers (NHEs) (reviewed in Ref. 11), presumably via operation of the NHE3 (1, 3) and/or the NHE2 (9, 29) isoforms.

In contrast, little is known about the specific pathways implicated in exit of HCO-3 across the basolateral membrane of these cells. Indeed, conflicting results regarding the presence or absence of basolateral Na+-HCO-3 cotransport have been obtained in whole tubule experiments by measuring intracellular pH (pHi). Kikeri et al. (19) proposed that an electrogenic Na+-nHCO-3 cotransporter mediates base exit in suspensions of mouse MTAL (s-MTAL). However, a subsequent study (20) failed to obtain similar results in rat s-MTAL. In the latter study, it was concluded that those cells possess an electroneutral Na+-independent K+-HCO-3 cotransporter that is inhibited by DIDS. There is also conflicting evidence for a basolateral Cl-/HCO-3 exchanger in whole tubule experiments. Kikeri et al. (19) and Leviel et al. (20) failed to detect Cl-/base exchange in rat and mouse MTAL suspensions by measuring pHi. More recently, basolateral Na+-independent and Cl--dependent HCO-3 transport that was inhibited by DIDS has been reported in isolated, perfused mouse MTAL by monitoring pHi in response to changes in peritubular fluid composition (28). However, in view of the multiple pathways for HCO-3 and H+ transport in the MTAL, it is difficult to establish, with certainty, direct coupling between fluxes of ions on a given plasma membrane by measuring pHi.

We recently demonstrated the presence in basolateral membrane vesicles (BLMV) purified from rat MTAL of a Na+-independent, stilbene-sensitive Cl-/HCO-3 exchanger activated by an internal pH-sensitive modifier site (10). It was the purpose of these studies to reexamine directly, using these plasma membrane vesicles, the other possible basolateral membrane HCO-3 transport pathways. We were unable to detect substantial coupling of either Na+, K+, or lactate flux to HCO-3 gradients, indicating that Na+-HCO-3 and K+-HCO-3 cotransporters and a lactate/HCO-3 antiporter postulated to reside in the basolateral membrane of the MTAL are not important for net HCO-3 absorption. These results indicate that the main physiological basolateral HCO-3 extrusion pathway in rat MTAL may be a Na+-independent Cl-/HCO-3 exchanger.


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Preparation of MTAL tubules. Male Sprague-Dawley rats (250-300 g) were anesthetized with pentobarbital (50 mg/kg intraperitoneal), and the kidneys were removed quickly, decapsulated, and sliced sagittally. Slices were transferred in fresh iced (0-4°C) Hanks' modified medium containing (in mM) 115 NaCl, 0.4 MgSO4, 0.5 MgCl2, 0.4 KH2PO4, 0.3 Na2HPO4, 25 NaHCO3, 10 HEPES, 4 KCl, 1.2 CaCl2, 5 glucose, and 5 L-leucine, as well as 1 mg/ml bovine serum albumin, pH 7.40 (bubbled with 95% O2-5% CO2). Under stereomicroscopic control, the inner stripe of the outer medulla, recognized by its reddish color, was carefully separated from each slice by removing completely the outer part of the outer medulla as well as the inner medulla. The resulting tissue was subjected to collagenase treatment as previously described (3, 5). In the final suspensions, most of the tubules (>95%) proved to be MTAL in origin, based on immunofluorescent staining with Tamm-Horsfall protein (3), a specific marker for the thick ascending limb. Moreover, we were unable to detect significant activity of maltase, a marker of the proximal tubule brush-border membrane, in homogenates from s-MTAL, whereas its specific activity in homogenates from the immediately adjacent outer stripe of the outer medulla averaged 160 ± 7 nmol · mg protein-1 · min-1 (n = 5 in each group), excluding significant contamination of the starting material with tubule segments from the outer stripe of the outer medulla.

Isolation of plasma membranes. Typically, the preparation began with 15-20 mg protein of MTAL tubules obtained from the kidneys of 10 rats. Luminal membrane vesicles (LMV) and BLMV were prepared from purified rat MTAL tubules as recently described in detail (3, 5). We have recently demonstrated that the BLMV and LMV preparations have a right-side-out orientation (5). Compared with homogenate, the basolateral marker activity of Na+-K+-ATPase was enriched ~9-fold in the BLMV and only 0.5-fold in the LMV. In contrast, the apical marker activity of gamma -glutamyltransferase was enriched >10-fold in the LMV and ~1.5-fold in BLMV. Utilizing 22Na+ uptake determinations, we have also recently evaluated the purity of the membrane vesicle preparations in functional terms. In these studies, we detected bumetanide-sensitive Na+-K+-2Cl- cotransport activity only in LMV, and not in BLMV (24).

Basolateral membranes were isolated from the superficial rat renal cortex by differential and Percoll gradient centrifugations, as described previously (8). Compared with the starting homogenate, the purification of these BLMVs was 10- to 15-fold, on the basis of enrichment in Na+-K+-ATPase.

Transport assays were performed after overnight storage of the vesicles at -85°C.

Isotopic flux measurements. 22Na+, 86Rb+, and [14C]lactate uptakes into the membrane vesicles were assayed at ambient temperature (20-25°C) by a rapid filtration technique. Media were gassed for at least 120 min with either humidified 100% N2 or 5% CO2-95% N2 as specified in the legends to Figs. 1-9. For each experiment, pH and total CO2 of incubation solutions were measured. For highly buffered media that were used in the radioactive experiments, it was found that total CO2 and pH were stable for at least 30 min after stopping equilibration with the appropriate gas. Total CO2 values measured with a CO2 analyzer (Ciba-Corning, Halstead, UK) were inferior to 0.1 and 0.5 mM in nominally HCO-3-free solutions at pH 6.0 and 7.8, respectively. The vesicles were preequilibrated at room temperature for 2 h to load with desired constituents. For each experiment, the specific conditions are given in the legends to Figs. 1-9. In general, a 10-µl aliquot of plasma membrane vesicles (10-40 µg protein) was added to 100-300 µl of appropriate reaction medium containing either 22Na+, 86Rb+, or [14C]lactate (~1 µCi/ml). Incubation periods of 9 s were timed with a metronome and used to estimate initial rates. The reaction was stopped with 1.5-ml ice-cold solution containing 20 mM Tris-HEPES, pH 7.4, and the desired potassium gluconate concentration to maintain constant osmolarity. This suspension was rapidly filtered on the center of a 0.45-µm prewetted Millipore cellulose filter (HAWP) and washed with an additional 16 ml ice-cold stop solution. In all experiments, vesicle uptake was corrected for nonspecific isotopic binding to the filter. The filters were dissolved in 3 ml of scintillant (Filter-count, Packard), and radioactivity was determined using a beta scintillation counter.

Carboxyfluorescein fluorescence measurements. The fluorescent dye carboxyfluorescein (CF) was chosen for pHi measurements because of its negligible leakage rate and because of the availability of an anti-CF antibody (kindly provided by Dr. M. L. Zeidel), which permits complete quenching of extravesicular CF. This method is also advantageous because, in contrast to the widely used acridine orange method, the rate of change in fluorescence was constant over a wide range of membrane protein concentrations and unaffected by the presence of amiloride and DIDS. For these experiments, BLMV and LMV were prepared exactly as described previously (3, 5), except for the presence of 0.5 mM CF in the homogenization buffer. Both types of vesicles were washed by centrifugation at 400,000 g for 60 min at 0°C in a large excess of an appropriate preincubation buffer (indicated in text) to remove extravesicular CF. The pellets thus obtained were resuspended (~1 mg/ml) in the preincubation buffer by passage through a 27-gauge needle and equilibrated for 2 h at room temperature until used. The vesicles and the media were bubbled with either 5% CO2-95% N2 or 100% N2 prior to the fluorescence measurements and gassed at the surface of the cuvette during the fluorescence measurements. Fluorescence of the labeled vesicle suspensions was measured at 20-25°C in a Jobin-Yvon spectrofluorometer. All experiments were performed in the presence of an excess anti-CF antibody to eliminate completely extravesicular fluorescence. Excitation and emission wavelengths were 490 and 525 nm, respectively, and 10-nm slits were used. Usually, pHi measurements were performed by adding 20 µl (corresponding to ~20 µg membrane protein) of either BLMV or LMV to a cuvette containing 2 ml of the indicated stirred medium. Calibration of pHi vs. fluorescence was carried out for each experiment by adding 20 µl of labeled vesicles in a cuvette containing 2 ml of the indicated stirred medium. For experiments using K+-loaded vesicles, calibration was based on the nigericin-high-K+ technique of Thomas et al. (30). In brief, vesicles were subjected, in the presence of nigericin (2 µM), to stepwise decrements in extravesicular pH (pHo) (over a pHo range of 7.35-6.4) with concentrated gluconic acid, resulting in quenching of entrapped CF. Disruption of vesicles with 0.4% Triton X-100 resulted in almost complete quenching of CF. Assuming that the ionophore sets H+i/H+o = K+i/K+o (where "o" indicates extravesicular and "i" indicates intravesicular), we estimated an apparent pHi value from the linear correlation between relative fluorescence and pH. A typical experiment is shown in Fig. 1. Increasing threefold the vesicle membrane protein concentration did not affect the calibration of the dye.


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Fig. 1.   Typical experiment: relationship between relative fluorescence of entrapped carboxyfluorescein (CF) and extravesicular pH (pHo). Either 15 µg () or 45 µg (open circle ) of basolateral membrane vesicles (BLMV) were subjected to sequential decreases in pHo with use of concentrated gluconic acid in presence of nigericin (see METHODS). Relative fluorescence is plotted as a function of pH. Slopes are 0.53 and 0.52 in experiments using 15 and 45 µg of vesicle membrane protein, respectively. Regression lines were calculated by the method of least squares.

In experiments designed to evaluate Cl-/HCO-3 exchange activity, a comparable linear correlation between relative fluorescence and pH was obtained using the protonophore FCCP (4 µM), which sets H+i = H+o (data not shown). dpHi/dt represents the initial rate of change in pHi calculated in the first 9 s after addition of the vesicles. The intravesicular buffer capacity was determined in vesicles preequilibrated in the presence and absence of CO2/HCO-3 using the technique of rapid sodium acetate addition (21) where 25 mM tetramethylammonium (TMA) acetate replaced TMA gluconate. The acetic acid in the TMA acetate solution rapidly enters the vesicles, leading to immediate vesicle acidification with no significant pHi recovery for at least 20 s.

Statistics. All data are represented as means ± SE. Comparisons among groups were carried out by ANOVA. Statistical significance was defined as P < 0.05.

Materials. From Amersham, we obtained L-[U-14C]lactic acid, carrier-free 22NaCl, and 86RbCl. DIDS was obtained from Research Organics (Cleveland, OH). All other reagents were from Sigma Chemical (St. Louis, MO).


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Na+-dependent HCO-3 transport in BLMVs purified from s-MTAL and cortical tubules. If an electrogenic Na+-nHCO-3 cotransporter is present in MTAL BLMVs, then imposing an inwardly directed HCO-3 gradient in the presence of an inside-positive membrane potential should lead to a marked stimulation of 22Na+ uptake. As can be seen in Fig. 2A, however, 22Na+ uptake at 9 s was stimulated only minimally (36%) by an inwardly directed HCO-3 gradient (pHi 6.0/pHo 7.8, HCO-3) and an inside-positive potential compared with the same conditions but in the nominal absence of HCO-3 (64 ± 3.1 vs. 47 ± 2.3 pmol · mg protein-1 · 9 s-1, P = 0.02). In the presence of inwardly directed HCO-3 gradients, 22Na+ uptake was significantly inhibited by amiloride, an inhibitor of the Na+/H+ exchangers, and at a lesser degree by DIDS, an established anion transport inhibitor (control, 64 ± 3.1; vs. 32 ± 4 and 48 ± 6 pmol/mg protein for amiloride and DIDS, respectively). These data suggest that, in addition to an Na+/H+ exchanger, these membranes might also contain a HCO-3-dependent and DIDS-sensitive component of Na+ uptake. Because, however, inward gradients of HCO-3 stimulated the rate of 22Na uptake only marginally in these membranes, we performed control experiments using BLMVs isolated from rat renal cortex, which contain a robust Na+-nHCO-3 cotransporter (13). As shown in Fig. 2B, imposing a HCO-3 and a pH gradient, under the conditions of Fig. 2A, in these membrane vesicles, stimulated 22Na+ uptake 326% compared with the same experimental conditions but in the nominal absence of HCO-3, a stimulation nearly 10 times greater than that we detected in the BLMV prepared simultaneously. Another major difference between the two types of BLMVs was the differential sensitivity of 22Na+ uptake by basolateral membranes purified from the renal cortex to amiloride and DIDS. The HCO-3 gradient-driven 22Na+ influx was inhibited 67% by DIDS (434 ± 58 vs. 143 ± 12 pmol/mg protein, P < 0.001), whereas amiloride had no significant effect. It should be noted that, in the above-described experiments, the transmembrane Na+ gradient was unfavorable for operation of the Na+/H+ exchangers due to preincubation of the membrane vesicles with 1 mM disodium DIDS.


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Fig. 2.   Comparison of effect of HCO-3 and inhibitors on 22Na+ uptake by BLMV isolated simultaneously from suspensions of medullary thick ascending limbs (s-MTAL; A) and cortical tubules (B). Both types of membrane vesicles were preincubated for 2 h with a pH 6.0 medium consisting of (in mM) 100 mannitol, 3 EGTA, 140 tetramethylammonium (TMA) gluconate, and 100 MES-Tris and gassed either with 100% N2 (no HCO-3) or with 95% N2-5% CO2 (HCO-3) and, as indicated, in presence or absence of either 1 mM amiloride (AMIL) or 1 mM disodium DIDS. Sodium gluconate was added as appropriate to maintain the Na+ concentration constant at 2 mM. All vesicles were pretreated for 2 h with valinomycin (10 µg/mg protein). Sodium (final concentration of 22Na+ was 0.18 mM) uptake at 9 s was then assayed by diluting vesicles 1:11 into pH 7.8 buffer consisting of (in mM) 100 mannitol, 3 EGTA, 85 TMA gluconate, and 100 Tris-HEPES, that contained either 55 potassium gluconate gassed with 100% N2 (no HCO-3) or 55 KHCO3 gassed with 95% N2-5% CO2 (HCO-3). Values are means ± SE of 6 determinations from 2 different basolateral membrane preparations isolated from either s-MTAL or superficial renal cortex. * P < 0.05 and ** P < 0.005 vs. respective control values. $ P < 0.05 and $$ P < 0.0005 vs. respective values obtained in presence of HCO-3 (ANOVA).

Since, in absolute terms, the DIDS-sensitive component of 22Na+ uptake detected in BLMV in the presence of added HCO-3/CO2 is negligible compared with that observed in cortical BLMVs (16 vs. 290 pmol · mg-1 · 9 s-1), we wanted to see whether a pH and a HCO-3 transmembrane gradient in the presence of an inside-positive potential would result in uphill transport of 22Na+. The results of these experiments are illustrated in Fig. 3. Uptake of 22Na+ in the presence of a transmembrane pH gradient (pHi 6.0/pHo 7.8) slowly approached equilibrium. Imposing the same inside-acid pH gradient in the presence of an inwardly directed HCO-3 gradient caused no significant additional uptake of 22Na+ at the P < 0.05 level, for incubation periods of 0.5, 1, and 3 min, and failed to induce an overshoot of this cation. Thus these data argue against the existence of direct coupling between the flux of Na+ and the flux of HCO-3 (i.e., no Na+-HCO-3 cotransport) in the basolateral membranes of the rat MTAL. The possibility exists, however, that a Na+-HCO-3 cotransporter is actually present in these vesicles but that, unlike the cortical Na+-HCO-3 cotransporter, it could have been inhibited at pH 6.0 inside. This hypothesis was tested in two ways. In the first approach, we investigated the time course of 22Na+ uptake under conditions comparable to those illustrated in Fig. 3 but at pH 7.2 inside. As can be seen in Fig. 3 (bottom), imposing a HCO-3 gradient with the same pH gradient caused no appreciable additional uptake of 22Na+ at the P < 0.05 level, for incubation periods of 0.5 and 1 min, and did not induce a 22Na+ overshoot. In the second approach, we evaluated whether, at the relatively alkaline pHi of 7.37, imposing outwardly directed Na+ gradients would drive net HCO-3 efflux and thereby cause intravesicular acidification. This possibility was evaluated by using the pH-sensitive fluorescent dye CF. In these experiments, the membrane potential was clamped at 0 mV by using valinomycin and equimolar internal and external K+ concentrations. As can be seen in Fig. 4, imposing an outward Na+ gradient of 100 mM:3 mM modestly decreased pHi at an initial rate of 0.52 ± 0.07 pH units/min, which was not significantly different at the P < 0.05 level than in the absence of a Na+ gradient (0.25 ± 0.11 pH units/min). Addition of 1 mM DIDS was without effect on the initial rate of acidification measured in the presence of an outward Na+ gradient (control, 0.52 ± 0.07 pH units/min; DIDS, 0.35 ± 0.08 pH units/min; P > 0.05, n = 8 in each group). Taken together with the results in Figs. 2 and 3, these findings argue against the existence of a HCO-3-dependent, DIDS-sensitive component of Na+ transport in the basolateral membranes of the rat MTAL. Of note, because the BLMV contains the NHE1 isoform (3), one would have expected transient uphill 22Na transport via operation of this exchanger in the experiments conducted in the presence of outward H+ gradients (pHi = 6.0, pHo = 7.8). Failure to detect an overshoot in these experiments may be attributed to the presence of 55 mM external K+, used to create an interior-positive potential, insofar as it has been demonstrated that K+o competitively inhibits H+i-activated 22Na+ influx by NHE1 expressed in Na+/H+ exchanger-deficient Chinese hamster ovary cells (22).


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Fig. 3.   Time course of effect of H+ and HCO-3 gradients on Na+ uptake by BLMV at two internal pH levels (pHi). At pHi 6.0/pHo 7.8, BLMV were preincubated for 2 h in pH 6.0 media and incubated in pH 7.8 media as in Fig. 2. At pHi 7.2/pHo 8.2, BLMV were preincubated for 2 h with a pH 7.2 medium consisting of (in mM) 100 mannitol, 3 EGTA, and 100 MES-Tris and either 140 TMA gluconate gassed with 100% N2 or 126 TMA gluconate and 14 TMA HCO3 gassed with 95% N2-5% CO2. Uptake of 0.1 mM 22Na+ was then assayed by diluting vesicles 1:11 into pH 8.2 buffer consisting of (in mM) 100 mannitol, 3 EGTA, and 100 Tris-HEPES that contained either 140 potassium gluconate gassed with 100% N2 or 140 K HCO3 gassed with 95% N2-5% CO2. All vesicles were pretreated for 2 h with valinomycin (10 µg/mg protein). Values are means ± SE of 6 determinations from 2 different preparations.



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Fig. 4.   Effect of outwardly directed Na+ gradients and DIDS on the rates of intravesicular acidification in BLMV. BLMV previously loaded with CF were equilibrated for 2 h in a medium consisting of (in mM) 50 mannitol, 3 EGTA, 100 sodium gluconate, 100 potassium gluconate, 20 Tris-HEPES, pH 7.37, 20.5 TMA HCO3, and 0.2 mg/ml of carbonic anhydrase and gassed with 95% N2-5% CO2. At time 0, vesicles were diluted 1:101 in either the same medium or in media in which sodium gluconate was replaced by 100 mM TMA gluconate. All media were gassed with 95% N2-5% CO2 and contained 0.1 mM ethylisopropylamiloride. DIDS (1 mM) was added as the disodium salt, and sodium gluconate was added as appropriate to maintain the Na+ concentration constant. All vesicles were pretreated for 2 h with valinomycin (10 µg/mg protein). Values are means ± SE of 8 determinations from 2 different preparations of BLMV.

Effects of H+ and HCO-3 gradients on 86Rb+ uptake and intravesicular acidification. To determine whether a K+-coupled HCO-3 transporter was present in basolateral membrane from the rat MTAL, we first tested whether a transmembrane HCO-3 gradient would be capable of driving 86Rb+ uptake, used as an analog of K+. As shown in Fig. 5, an outward H+ gradient (pHi = 6.0, pHo = 7.8) in CO2/HCO-3-buffered solutions stimulated 86Rb+ uptake by 56 ± 19 and 72 ± 12%, compared with uptake values observed under non-pH gradient conditions at pH 6.0 and 7.8, respectively. However, imposing the same inside-acid pH gradient in the absence of added CO2/HCO-3 stimulated 86Rb+ uptake to the same degree (67 ± 16 and 54 ± 11% vs. non-pH gradient conditions at pH 6.0 and 7.8, respectively), indicating that the H+-activated 86Rb influx was on a HCO-3-independent pathway. Figure 5 also shows that this H+-activated 86Rb influx was markedly abolished (~70%) by Ba2+ (a blocker of K+ channels), whether measured in the presence of HCO-3 or in its absence, suggesting that this movement of 86Rb+ occurred mainly via K+ channels. These results, therefore, argue against the existence of direct coupling between the flux of Rb+ and the flux of HCO-3 (i.e., no K+-HCO-3 cotransport) in these membranes. Since this hypothetical basolateral transporter has been described using s-MTAL (6, 20), and since this approach cannot distinguish between transport events occurring at the basolateral or at the apical surface of this epithelium, we next searched for its possible existence in the luminal membrane. As illustrated in Fig. 5, right, however, imposing an inside-acid pH gradient in the presence or absence of CO2/HCO-3 modestly stimulated 86Rb+ uptake compared with that measured under non-pH gradient conditions at pH 6.0. Moreover, the stimulatory effects of outward H+ gradients on 86Rb+ uptake become barely detectable compared with those measured in the absence of a pH gradient at pH 7.8 instead of at pH 6.0. For example, in HCO-3-buffered solutions, 86Rb+ influx in the presence of a pH gradient tended to be greater than under non-pH gradient conditions at pH 7.8 (36 ± 3.5 vs. 28 ± 1.8), but this difference was not significant at the P < 0.05 level. This may be attributed to an effect of pHi per se, insofar as it has been demonstrated that intracellular acidification inhibits the apical K+ conductance of the thick ascending limb (7, 18, 34).


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Fig. 5.   Effect of HCO-3 gradient and Ba2+ on 86Rb+ uptake by BLMVs (left) and luminal membrane vesicles (LMV, right) isolated simultaneously. At pHi 6.0/pHo 6.0, membrane vesicles were preincubated for 2 h with a pH 6.0 medium consisting of (in mM) 50 mannitol, 200 Tris-MES, and 92.5 N-methyl-D-glucamine (NMG) gluconate, gassed with either 95% N2-5% CO2 (HCO-3) or 100% N2 (no HCO-3). Uptake of 0.1 mM 86Rb+ at 9 s was then assayed in presence of respective gassed media of the same composition. At pHi 7.8/pHo 7.8, membrane vesicles were preincubated for 2 h with a pH 7.8 medium consisting of (in mM) 50 mannitol and 200 Tris-HEPES that contained either 37.5 NMG gluconate and 55 NMG-HCO3 gassed with 95% N2-5% CO2 (HCO-3) or 92.5 NMG gluconate gassed with 100% N2 (no HCO-3). Uptake of 86Rb+ was then assayed in presence of respective gassed media of the same composition. At pHi 6.0/pHo 7.8, vesicles were preincubated in pH 6.0 media gassed with either 100% N2 or 95% N2-5% CO2, as indicated above. Uptake of 86Rb+ was assayed in a pH 7.8 medium consisting of (in mM) 50 mannitol and 200 Tris-HEPES that contained either 37.5 NMG gluconate and 55 NMG HCO3 gassed with 95% N2-5% CO2 (HCO-3) or 92.5 NMG gluconate gassed with 100% N2 (no HCO-3). BaCl2 (3 mM) or TMA Cl (6 mM), used as a control, was added to both the preincubation and incubation media. Open bars, results from control vesicles; solid bars, results from vesicles treated with Ba2+. Each datum is the mean ± SE for 6 experiments performed on 2 different BLMV and LMV preparations.

Failure to detect significant K+-HCO-3 cotransport activity in the foregoing 86Rb experiments may be caused by the absence of intravesicular K+, since recent s-MTAL studies (6) have suggested that operation of this transport system required high internal K+ concentrations. To evaluate this possibility, BLMV and LMV were equilibrated with high (120 mM) K+ concentrations and pHi was determined, using the pH-sensitive fluorescent dye CF. As can be seen in Fig. 6, imposing an outward K+ gradient of 120 mM:1.18 mM in the absence of CO2/HCO-3 decreased pHi in BLMV at an initial rate of 0.37 ± 0.08 pH units/min, compared with no K+ gradient, which was not significantly different than in the presence of CO2/HCO-3 (0.38 ± 0.04 pH units/min; P = 0.8, n = 8 in each group). As can be seen in Fig. 6, right, qualitatively similar results were obtained in studies using LMV. Imposing an outward K+ gradient caused intravesicular acidification, which was not significantly different regardless of whether measured in the presence of CO2/HCO-3 or measured in its absence (0.35 ± 0.001 vs. 0.22 ± 0.08 pH units/min; n = 8, P = 0.2).


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Fig. 6.   Effect of outwardly directed K+ gradients on rates of intravesicular acidification in BLMV (left) and LMV (right). For K+i = K+o, both types of membrane vesicles previously loaded with CF were equilibrated for 2 h in a medium consisting of (in mM) 75 mannitol, 3 EGTA, and 120 potassium gluconate that contained either 22 Tris-HEPES, pH 7.0, 8.7 NMG HCO3, and 0.2 mg/ml of carbonic anhydrase gassed with 95% N2-5% CO2 or 56 Tris-HEPES, pH 7.0, gassed with 100% N2. pHi was then monitored by adding 20 µl of vesicles in 2 ml of respective gassed media of the same composition. For K+i > K+o, membrane vesicles previously loaded with CF were preincubated in pH 7.0 media gassed with either 100% N2 or 95% N2-5% CO2, as indicated above. They were then diluted 1:101 in respective gassed media consisting of (in mM) 75 mannitol, 3 EGTA, and 120 TMA gluconate that contained either 22 Tris-HEPES, pH 7.0, and 8.7 NMG HCO3 gassed with 95% N2-5% CO2 or 56 Tris-HEPES, pH 7.0, gassed with 100% N2. Values are means ± SE of 8 determinations from 2 different preparations of BLMV and LMV.

Since in s-MTAL studies the hypothetical K+-HCO-3 cotransporter has been detected at alkaline pHi values (7.3-7.8), it is possible that the failure to detect K+-HCO-3 cotransport could have been due to the relatively low pHi values (6.0 and 6.95) employed in the foregoing isotopic and fluorescent experiments. To evaluate this possibility, the effect of HCO-3 on the rate of intravesicular acidification was determined at an internal pH value of 7.37. In experiments not illustrated, it was found that imposing an outward K+ gradient of 120 mM:1.18 mM in the absence of CO2/HCO-3 decreased pHi in BLMV at an initial rate of 0.19 ± 0.04 pH units/min, compared with no K+ gradient, which was not significantly different than in the presence of CO2/HCO-3 (0.17 ± 0.05 pH units/min; P = 0.8, n = 8 in each group). Thus these findings further argue against the presence of K+-HCO-3 cotransport in the BLMV isolated from rat MTAL.

Further studies were done to determine whether the pH-sensitive fluorescent dye CF method is sufficiently sensitive to detect operation of ion-coupled processes in plasma membrane vesicles. To this end, we evaluated the activity of the BLMV Cl-/HCO-3 antiporter, a robust transport system, sensitive to inhibition by DIDS, which has been recently detected in these membranes using radioisotopic methods (10). As illustrated in Fig. 7, when an inward Cl- gradient was imposed in the presence of CO2/HCO-3, the change in pHi occurred at a ~440% faster initial rate than in vesicles subjected to an outwardly directed K+ gradient (1.68 ± 0.21 vs. 0.38 ± 0.004 pH units/min). As expected, this Cl- gradient-stimulated intravesicular acidification was markedly inhibited (60%) by 1 mM DIDS. Accordingly, the failure to detect significant coupling between the flux of either Na+ or K+ with HCO-3 in Figs. 4 and 6 cannot be attributed to an insufficient sensitivity of the CF assays.


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Fig. 7.   Effect of Cl- gradients and DIDS on rates of BLMV acidification. BLMV previously loaded with CF were equilibrated for 2 h in a medium consisting of (in mM) 50 mannitol, 3 EGTA, 120 TMA gluconate, 20 Tris-HEPES, pH 7.3, 16 TMA HCO3, and 0.2 mg/ml of carbonic anhydrase and gassed with 95% N2-5% CO2. At time 0, vesicles were diluted 1:101 in either the same medium or in medium in which TMA gluconate was replaced by 120 mM TMA Cl. All media were gassed with 95% N2-5% CO2 and contained 0.1 mM amiloride. DIDS (1 mM) was added as the disodium salt, and sodium gluconate was added as appropriate to maintain the Na+ concentration constant. Values are means ± SE of 8 determinations from 2 different preparations of BLMV.

However, this interpretation is only correct if the buffer capacity is the same in the presence and absence of added CO2/HCO-3. To evaluate this, we compared the intravesicular buffer capacity of vesicles incubated in HCO-3-containing solutions to those incubated in Tris-HEPES-buffered solutions, using the technique of TMA acetate addition. Note that, in the experiments illustrated in Fig. 6, the concentration of Tris-HEPES was reduced 2.5 times in preincubation and incubation media containing CO2/HCO-3. The buffer capacity of BLMV incubated in the absence of CO2/HCO-3 was 14.6 ± 1.0 mmol · l-1 · pH unit-1, which was not different from that obtained in vesicles incubated in the presence of CO2/HCO-3, 17 ± 2 mmol · l-1 · pH unit-1 (n = 4 in each group, P > 0.05). In LMV, the buffer capacity averaged 12 ± 3 and 9 ± 1 mmol · l-1 · pH unit-1 (n = 4 in each group, P > 0.05) in the absence and presence of CO2/HCO-3, respectively. Thus the lack of effect of HCO-3 in the rate of intravesicular acidification was not due to a higher buffer capacity in these vesicles.

Lactate/HCO-3 exchange in BLMV. Since outwardly directed HCO-3 gradients have been reported to stimulate lactate uptake into BLMVs purified from the dog MTAL (32), we investigated for the presence of this possible mode of HCO-3 transport in the rat BLMV. Figure 8 shows the effects of pH and/or HCO-3 gradients on time-dependent [14C]lactate uptake. In these experiments, the membrane potential was clamped at 0 mV by using valinomycin and equimolar internal and external K+ concentrations. Imposing an inside-alkaline pH gradient (pHi 7.8, pHo 5.5) caused the transient accumulation of lactate to a level approximately eightfold higher than equilibrium. However, the presence of outwardly directed HCO-3 gradients of 55 mM:2.62 mM with the same pH gradient caused no further stimulation of lactate uptake, arguing against the existence of direct coupling between the flux of lactate and the flux of HCO-3 (i.e., no lactate/HCO-3 antiport) in the basolateral membrane of the rat MTAL.


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Fig. 8.   Effect of H+ and HCO-3 gradient on [14C]lactate uptake by BLMV. Vesicles were preincubated for 2 h in presence of HCO-3 with a pH 7.8 medium consisting of (in mM) 100 mannitol, 3 EGTA, 100 Tris-HEPES, 100 potassium gluconate, and 55 NMG HCO3 gassed with 95% N2-5% CO2 or in absence of HCO-3 (110 mannitol, 3 EGTA, 200 Tris-HEPES, and 100 potassium gluconate gassed with 100% N2). Lactate (0.1 mM [14C]lactate) uptake was then assayed by dilution of vesicles 1:21 into pH 5.5 buffer containing either (in mM) 100 mannitol, 3 EGTA, 100 Tris-MES, 100 potassium gluconate, and 55 NMG gluconate gassed with 95% N2-5% CO2 (open circle ) or 100 mannitol, 3 EGTA, 100 potassium gluconate, and 200 Tris-MES gassed with 100% N2 (). All vesicles were pretreated for 2 h with valinomycin (10 µg/mg protein). Each datum is the mean ± SE for 6 experiments performed on 2 different preparations. When SE value is not shown, it was smaller than the symbols.

However, it is possible that, at the alkaline pHi of 7.8, OH- might compete at an internal transport site with HCO-3, and thus mask an effect of this anion. To test this possibility, we compared, on the same preparations of BLMV, the effect of HCO-3 on the uptake of [14C]lactate at internal pH values of 7.8 and 6.8. The presence of HCO-3 did not stimulate the initial rate of [14C]lactate uptake at either pHi value (Fig. 9). Moreover, decreasing pHi from 7.8 to 6.8 inhibited by ~300% [14C]lactate uptake, rendering unlikely the possibility that the lack of stimulation by HCO-3 at pHi 7.8 is secondary to saturation of an internal transport site by the 0.6 µM OH- anion present in the intravesicular medium. Figure 9 also shows that furosemide, an inhibitor of lactate/OH- exchange (32), inhibited to a similar degree pH gradient-stimulated uptake of lactate regardless of the presence of HCO-3. Thus carrier-mediated H+-lactate cotransport (or lactate/OH- exchange), rather than lactate/HCO-3 exchange or passive nonionic diffusion of lactic acid, is the likely mechanism underlying pH gradient-activated lactate uptake in the basolateral membrane of the rat MTAL.


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Fig. 9.   Effect of internal HCO-3 and furosemide on [14C]lactate uptake by BLMV at two pHi values. At pHi 7.8/pHo 5.5, BLMV were preincubated for 2 h in pH 7.8 media and incubated in pH 5.5 media as in Fig. 8. The 9-s uptake of 0.1 mM [14C]lactate was measured in presence or absence of 1 mM furosemide (FURO). At pHi 6.8/pHo 5.5, vesicles were preincubated for 2 h in presence of HCO-3 with a pH 6.8 medium consisting of (in mM) 100 mannitol, 3 EGTA, 100 Tris-HEPES, 100 potassium gluconate, 50 NMG gluconate, and 5.5 NMG HCO3 gassed with 95% N2-5% CO2 or in absence of HCO-3 (100 mannitol, 3 EGTA, 200 Tris-HEPES, and 100 potassium gluconate gassed with 100% N2). Lactate (0.1 mM [14C]lactate) uptake was then assayed at 9 s in pH 5.5 media in presence or absence of 1 mM furosemide. All vesicles were pretreated for 2 h with valinomycin (10 µg/mg protein). Each datum is the mean ± SE for 6 experiments performed on 2 different preparations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The specific mechanism(s) implicated in the final step of HCO-3 absorption by the MTAL remains largely unknown. In the present study, we used BLMVs purified from rat MTAL to characterize directly the transport pathway(s) by which HCO-3 exits across the basolateral membrane of this nephron segment.

In this study, we found no evidence compatible with electrogenic Na+-HCO-3 cotransport across basolateral membrane in the MTAL: 1) an inwardly directed HCO-3 gradient in the presence of an inside-positive membrane potential increased only marginally the initial rate of 22Na+ uptake (17 pmol · mg-1 · 9 s-1), nearly 20 times less of the increment (330 pmol · mg-1 · 9 s-1) we detected using cortical BLMVs as a positive control assay (Fig. 2); 2) an inward HCO-3 gradient in the presence of an inside-positive membrane potential failed to induce a 22Na+ overshoot in BLMV at markedly different pHi values (Fig. 3); 3) studies using the pH-sensitive dye CF revealed that imposing outwardly directed Na+ gradients induced only marginal intravesicular acidification in BLMV that was unaffected by DIDS (Fig. 4). These findings, therefore, argue against the presence of basolateral membrane electrogenic Na+-nHCO-3 cotransport in the rat MTAL. This conclusion is consistent with recent immunohistochemical (27) and in situ hybridization (25) studies which have shown that the electrogenic Na+-HCO-3 cotransporter from rat kidney, termed rNBC, localizes to the straight portion of the rat proximal tubule, whereas rNBC mRNA expression and staining by polyclonal antibodies to this transporter were not detected in any other tubule segment. It must be noted, however, that Kikeri et al. (19) provided evidence for a Na+-nHCO-3 cotransporter in mouse s-MTAL by measuring pHi; whether this is due to species difference remains to be determined.

In this study, we found that a K+-HCO-3 cotransporter postulated to reside in the rat MTAL (6, 20), presumably at the basolateral membrane (20), cannot be detected by two complementary approaches. In studies using the pH-sensitive dye CF, we found that imposing outwardly directed K+ gradients induced only marginal intravesicular acidification in both BLMV and LMV and at identical rates, regardless of whether measured in the absence of HCO-3 or measured in its presence (Fig. 6). These negative results cannot be accounted for by a higher buffer capacity in the presence of CO2/HCO-3, which was identical in the presence and absence of HCO-3. Thus these fluorescence dye measurements of pHi demonstrate that K+ gradient-induced acidification has no requirement for CO2/HCO-3. Conversely, we have found that imposing an inside-acid pH gradient in BLMV and in LMV induced identical stimulation of the rates of 86Rb+ uptake in the presence and absence of CO2/HCO-3 (Fig. 5), further arguing against K+-HCO-3 cotransport across both types of plasma membranes in the MTAL.

Our results showing directly an absence of coupling between the flux of K+ and the flux of HCO-3 are in apparent contradiction with indirect studies evaluating pHi changes in response to application of K+ gradients in rat s-MTAL (6, 20). In studies by Leviel and colleagues (20), dilution of CO2-loaded tubules into CO2/HCO-3-free buffer resulted in a near-immediate pHi increase from ~7.2 to ~7.8, due to CO2 exit, followed by pHi recovery. This pHi recovery was significantly slowed by DIDS, as well as by reducing [K+]i or increasing [K+]o, results consistent with the presence of a K+-HCO-3 cotransporter in this nephron segment. However, the thick ascending limb possesses multiple K+- and Cl--conductive pathways (14-17, 23, 26, 33). In this regard, we have recently demonstrated that generation of an inside-positive membrane potential markedly stimulated uptake of 36Cl- in BLMV and LMV, indicating significant Cl--conductive pathways in these plasma membranes (10). Accordingly, outward K+ gradients would be expected to cause a large hyperpolarization, which would activate a conductive pathway for Cl- transport leading to a decrease in [Cl-]i. The resulting increase in the size of the inward Cl- gradient would then acidify the cells via operation of Cl-/HCO-3 exchangers, which have been recently shown to be expressed, under isotonic conditions and in the absence of arginine vasopressin, in the mouse (28) and rat MTAL (10). Importantly, we have recently shown (10) that the BLMV Cl-/HCO-3 exchanger is markedly activated at alkaline pHi values, i.e., under the experimental conditions (pHi 7.3-7.8) that have been used to detect K+-HCO-3 cotransport in rat s-MTAL (6, 20).

Further studies by Blanchard and colleagues (6), using rat s-MTAL, have shown that increasing [K+]i from 60 to 120 mM markedly stimulated the initial rate of intracellular acidification under conditions in which K+-conductive pathways were assumed to be completely blocked by 0.1 mM verapamil and 10 mM Ba2+. These findings were taken as evidence of an important modifier role for internal K+ as an allosteric activator of a putative K+-HCO-3 cotransporter. The validity of this conclusion, however, is uncertain since the adequacy of the voltage clamp was not verified. Furthermore, the possibility exists that the degree of blockade of K+-conductive pathways by Ba2+ could be [K+]i dependent. Evidence to support this contention was provided in studies using K+ channels from rabbit muscle incorporated into planar bilayers (31) and MTAL of mouse (16), which showed that the effectiveness of Ba2+ block can be mitigated by increasing K+ concentrations. If at high [K+]i Ba2+ did indeed become less effective at inhibiting K+-conductive pathways, then it is possible that most of the [K+]i-induced acidification may have been due to negative shifts in the membrane potential.

In our studies using 86Rb+, we have obtained evidence compatible with some mode of coupling between K+ and H+ fluxes in the BLMV (Fig. 5). The marked inhibition (80-90%) of BLMV 86Rb+ uptake by Ba2+ under non-pH gradient conditions, at pH 6.0 and 7.8, suggested that most of these movements of 86Rb+ were mediated by K+ channels. Nevertheless, we found that ~30% of the pH gradient-activated uptake of 86Rb persisted in the presence of Ba2+. This could be due to the fact that Ba2+ inhibition is voltage dependent, insofar as Greger and coworkers (14) demonstrated that the Ba2+ inhibition of K+ channels was reduced by hyperpolarization in the thick ascending limb of Henle's loop. Because, however, these membranes exhibited low permeabilities to H+ (24), an alternative possibility is that the pH-stimulated Ba2+-insensitive 86Rb+ uptake could reflect carrier-mediated basolateral K+/H+ exchange of very low magnitude. Graber and Pastoriza-Munoz (12) have already reported the presence of a Ba2+-insensitive K+/H+ exchanger in the OK opossum kidney cell, presumably at the basolateral membrane. Two studies report the presence of a K+/H+ exchanger in the rat MTAL, which has been located, albeit indirectly, on the apical membrane (2, 4), which disagrees with our findings. Indeed, the experiments reported in Fig. 5 demonstrate conclusively that an inside-acid pH gradient caused only marginal increments in the rates of 86Rb uptake into LMV (8-14 pmol · mg-1 · 9 s-1), about six to nine times less of the corresponding increments (72-82 pmol · mg-1 · 9 s-1) we detected using BLMV. It is possible that the marginal H+i-activated 86Rb+ uptake observed here in LMV represents contamination by basolateral membrane. The difference between our results and those of Attmane-Elakeb et al. (4) can be readily explained by the fact that their luminal fractions were heavily contaminated with basolateral membranes, as judged by a threefold to approximately fivefold enrichment in Na+-K+-ATPase. Of note, failure to detect appreciable H+-activated 86Rb+ uptake in LMV cannot be attributed to a general defect in the transport capacities of these membrane vesicles. Indeed, we recently demonstrated robust bumetanide-sensitive Na+-K+-2Cl- cotransport activity in these fractions, and not in the BLMV (24), as well as uphill 36Cl- transport via operation of a Na+-independent, DIDS-sensitive Cl-/HCO-3 exchanger (10).

The present studies confirm the observations of Vinay et al. (32) that outwardly directed OH- gradients can induce uphill lactate uptake into BLMVs purified from the MTAL. The experiments reported in Figs. 8 and 9, however, demonstrate that the combination of a pH gradient and an outward HCO-3 gradient did not provide additional stimulation of lactate uptake at markedly different pHi values, strongly suggesting that there is not direct coupling between the flux of lactate and the flux of HCO-3 (i.e., no lactate/HCO-3 exchange). Vinay et al. (32) reported HCO-3 gradient stimulation of lactate uptake, in the absence of a pH gradient, and concluded that it was due to lactate/HCO-3 exchange, because the stimulated uptake persisted unchanged in the presence of a pH clamp with K+i = K+o and nigericin. In this study, however, the adequacy of the pH clamp was not demonstrated. In the experiments of Vinay et al. (32), imposing an outward HCO-3 gradient in media not gassed with CO2 may have led to a near-instantaneous pHi increase due to CO2 exit, raising the possibility that most of the HCO-3 gradient-induced lactate uptake may have been via nonionic diffusion of lactic acid and/or by lactate-H+ cotransport or lactate/OH- exchange. Since we have found that the pH gradient-stimulated uptake of L-lactate was sensitive to inhibition by furosemide, carrier-mediated H+-lactate cotransport is the likely mechanism underlying pH gradient-activated lactate uptake in the BLMV. This view is supported by immunocytochemical studies showing that the proton-linked monocarboxylate transporter isoform MCT2 is expressed in the basolateral membrane of the rat MTAL (D. Eladari and R. Chambrey, unpublished observations).

Finally, the observations that inward HCO-3 gradients did not affect significantly 86Rb+ uptake (Fig. 5) and that K+ gradient-induced acidification was not significantly affected by the presence of HCO-3 (Fig. 6) suggest that basolateral HCO-3 conductance could not play an important role in net transcellular HCO-3 absorption.

In conclusion, we found no evidence for the presence in BLMVs purified from rat MTAL of appreciable Na+-, K+-, or lactate-linked HCO-3 transport. It appears from this and other recent studies (10, 28) that basolateral Cl-/HCO-3 exchange may be the major transport pathway by which this nephron segment absorbs HCO-3.


    ACKNOWLEDGEMENTS

We thank F. Pezy for excellent technical assistance.


    FOOTNOTES

Portions of this work were presented at the Annual Meeting of the American Society of Nephrology, Philadelphia, PA, and have been published in abstract form (J. Am. Soc. Nephrol. 9: 8, 1998).

R.-A. Podevin is an Established Investigator of the Institut National de la Santé et de la Recherche Médicale.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R.-A. Podevin, Institut Biomédical des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France (E-mail: podevin{at}ccr.jussieu.fr).

Received 9 July 1998; accepted in final form 18 February 1999.


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