Ouabain reduces net acid secretion and increases pHi by inhibiting NH+4 uptake on rat tIMCD Na+-K+-ATPase

Susan M. Wall

Division of Renal Diseases and Hypertension, University of Texas Medical School at Houston, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In the rat terminal inner medullary collecting duct (tIMCD), Na+ pump inhibition reduces transepithelial net acid secretion (JtAMM) [JH = total CO2 absorption (JtCO2) + total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHi and reduction in JH that follow ouabain addition do not occur in the absence of NH+4 nor when NH+4 is substituted with another weak base. The purpose of this study was to explore the mechanism of the NH+4-dependent reduction in JtCO2 and increase in pHi that follow ouabain addition. We hypothesized that NH+4 enters the tIMCD cell through the Na+-K+-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+ and NH+4 compete for a common binding site on the Na+ pump, increasing extracellular K+ should limit NH+4 (and hence net H+) uptake by the Na+ pump. Upon increasing extracellular K+ concentration from 3 to 12 mM, the NH+4-dependent, ouabain-induced increase in pHi and reduction in JtCO2 were attenuated. In the presence but not in the absence of NH+4, reducing Na+ pump activity by limiting Na+ entry reduced JtCO2 and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of HCO<SUP>−</SUP><SUB>3</SUB>/CO2 and was not reproduced with BaCl2 or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na+-K+-2Cl- cotransporter or a HCO<SUP>−</SUP><SUB>3</SUB> transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell, NH+4 uptake is mediated by the Na+-K+-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+ loading.

ammonia; sodium-potassium-chloride cotransport; potassium channels; acidification

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE TERMINAL inner medullary collecting duct (tIMCD) is the final nephron site of urinary acidification within the mammalian kidney. Ammonium (NH+4) secretion in the tIMCD occurs, in part, through active proton transport in parallel with the passive diffusion of NH3 (15). However, our laboratory has demonstrated an important role of direct NH+4 transport in this segment. In cultured IMCD cells, NH+4 and K+ compete for a common binding site on the Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) (34, 37). In native IMCD cells in suspension, both ions support ouabain-sensitive ATP hydrolysis (34). Thus both NH+4 and K+ are transported directly by the Na+ pump.

To test the significance of Na+ pump-mediated NH+4 uptake on proton secretion, the effect of ouabain on transepithelial net acid secretion was examined in rat tIMCD tubules perfused in vitro. Since deoxycorticosterone pivalate (DOCP) increases Na+ pump activity in the rat tIMCD (30), DOCP-treated rats were studied. In the absence of NH4Cl, total CO2 absorption (JtCO2) was low and not affected by ouabain addition to the bath (30). Baseline JtCO2 was higher in the presence of NH+4 than in its absence (30). Moreover, in the presence of NH4Cl, JtCO2 was significantly inhibited upon ouabain addition to the bath (30). It was reasoned that NH+4 enters the tIMCD cell on the basolateral membrane through an Na+-K+-ATPase-dependent pathway with release of protons in the cytosol. NH+4 serves as a source of NH3 and H+, which are secreted across the apical membrane. If such a model were true, then blockade of NH+4 uptake through Na+ pump inhibition should decrease net proton entry and alkalinize the cell. To test this hypothesis, the effect of ouabain on intracellular pH (pHi) was examined. Resting pHi was lower in the presence than in the absence of NH4Cl (30). Moreover, in the presence of NH4Cl, addition of ouabain to the bath alkalinized the cell (30). Ouabain-induced alkalinization was not observed in the absence of NH4Cl or when NH+4 was substituted with another weak base (30). Thus NH+4 and the Na+-K+-ATPase are important determinants of both net acid secretion and resting pHi.

However, the mechanism for the increase in pHi and the reduction in JtCO2 observed following ouabain addition could be due to blockade of Na+-K+-ATPase-mediated NH+4 uptake or through changes in ion gradients generated by the Na+ pump. The Na+ pump could generate ion gradients that affect other NH+4/OH-/H+/HCO<SUP>−</SUP><SUB>3</SUB> transporters independent of direct Na+-K+-ATPase-mediated NH+4 uptake. The purpose of this study was to further characterize the NH+4-dependent increase in pHi and reduction in JtCO2 that follow ouabain addition and to determine whether these observations can be explained by a transport mechanism other than Na+-K+-ATPase-mediated NH+4 uptake.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparation. tIMCD tubules were dissected from pathogen-free male Sprague-Dawley rats weighing 65-120 g (Rm. 205G; Harlan, Indianapolis, IN). All animals were housed in microisolator cages and fed a low-Na+, 0.8% K+ diet (Ziegler Brothers, Gardners, PA) (36). Rats were injected with 5 mg DOCP (CIBA-Geigy Animal Health, Greensboro, NC) by intramuscular injection 5-7 days prior to death. DOCP was employed to increase Na+-K+-ATPase activity, as described previously (30). Animals were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation to induce a rapid diuresis. This furosemide-induced diuresis reduces the inner medullary axial solute concentration gradient (36) and attenuates changes in the extracellular osmolality of the tubule.

Unless otherwise stated, all experiments were performed in bicarbonate-buffered solutions (Table 1), gassed with 95% air-5% CO2 before use. The measured osmolalities of all solutions are listed (Table 1).

                              
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Table 1.   Solution compositions

Coronal slices were cut from the kidneys and placed into a dissection dish containing the chilled experimental solution (11°C). IMCDs were dissected from the middle third of the inner medulla as described previously (36). Tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C. Experiments were performed with identical solutions in the perfusate and bath. In some experiments, ouabain (2.5 or 5 mM) or bumetanide (100 µM) was added to the bath fluid only. To maintain the desired CO2 concentration, the perfusate was passed through jacketed concentric tubing through which 95% air-5% CO2 was blown in a countercurrent direction around the perfusate line. To maintain pH in HCO<SUP>−</SUP><SUB>3</SUB>-containing solutions, the bath fluid was constantly bubbled with 95% air-5% CO2. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, bath fluid was bubbled with 100% O2. Bath pH was measured continuously during all experiments as described previously (36). Bumetanide was prepared as a 100 mM stock in 500 mM tris(hydroxymethyl)aminomethane (Tris). Ouabain was dissolved directly into the bath solution.

Measurement of bicarbonate flux. Tubule fluid samples were collected under oil in calibrated constriction pipettes. Flow rate was determined as described previously (36). Total CO2 (tCO2) concentration was measured in the collected fluid (CL) and perfusate (Co) using a continuous flow fluorometer (30). The CO2 reagent was purchased as a kit (no. 132-A; Sigma, St. Louis, MO) and diluted to 50% strength with water. Using this method, bicarbonate (total CO2, tCO2) concentration differences of less than 1 mM can be detected using a pipette of 8 nl (30). Bicarbonate flux, JtCO2, was calculated according to the equation
<IT>J</IT><SUB>b</SUB> = (C<SUB>o</SUB> − C<SUB><IT>L</IT></SUB>)V<SUB><IT>L</IT></SUB>/<IT>L</IT>
where Co and CL are the perfusate and collected fluid tCO2 concentration. VL is the flow rate (in nl/min), and L is the tubule length (in mm). This equation assumes zero net fluid transport in the absence of an imposed osmolality gradient (36). To eliminate detection of tCO2 flux which represents artifact generated by loss of CO2 in the collection pipette, Co was estimated by measuring tCO2 concentration in the collected fluid at very fast flow rates (>50 nl · mm-1 · min-1). Thus CO2 loss was matched in Co and CL measurements, allowing the loss terms to cancel (36). Amiloride at a 1 µM concentration did not affect the fluorescence signal of the CO2 reagent (n = 3, data not shown).

Measurement of pHi. pHi was measured using the esterified form of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (30). The detailed methodology for measurement of pHi in tubules perfused in vitro has been described previously (30).

Tubules were cannulated on concentric glass pipettes and then perfused for 20 min at 37°C. The bath solution was then changed to include 5 µM of the acetoxymethyl ester of BCECF (BCECF-AM). Tubules were perfused with BCECF present in the bath solution for 20 min. BCECF was then removed from the bath. Measurements of pHi were performed at least 10 min following removal of BCECF from the bath solution.

The excitation light source was a 75-W xenon short-arc lamp (Photoscan II; Nikon, Melville, NY) (30). The excitation light hit a rotating chopper disc (30), allowing light to pass alternately through 440- and 495-nm band-pass filters (Omega Optical, Brattleboro, VT) at a frequency of 60 Hz. The excitation light was reflected by a dichroic mirror with 50% reflectance at 515 nm (Omega Optical) and passed through the ×40 objective to strike the tubule (30). The emitted light was collected by the objective and passed through the dichroic mirror and long-pass filter with transmission >535 nm (Omega Optical) (30). The transmitted light was detected with a photomultiplier tube (Photoscan II, Nikon) (30). This detected signal was sampled at 20 points/s (30).

In most experiments, pHi was measured in three periods. In period 1 no inhibitor was present. In period 2, ouabain was present in the bath. In period 3 ouabain was removed from the bath fluid. Each period was begun with a rapid bath exchange, performed by introduction of new solution (preheated and pregassed) from a separate closed reservoir at a rate of >30 ml/min. At the same time, the new solution was introduced to the bath exchange reservoir. Bath fluid was exchanged continuously at 0.5 ml/min. Thus bath fluid could be exchanged completely in less than 10 s (30). Unless otherwise stated, fluorescence was measured for 90 s, beginning 4 min after the bath was exchanged. The fluorescence recorded over each time period was fit to a line digitally by the method of least squares. The fluorescence for that period was taken to be the fluorescence value given by that line at the midpoint of the recording.

A two-point standard curve was constructed for each tubule by perfusing the lumen and peritubular space at 37°C, with a high-K+ containing solution, pH 6.9-7.4, buffered with HEPES-Tris (30). The full composition of this calibration solution is given in Table 1 (solution 8). In addition, the bath contained 14 µM nigericin. After 10 min of exposure to nigericin, fluorescence was recorded. Dark current values were obtained by taking readings in the absence of transmitted light. Dark current values were subtracted from the unknown and the standard curve values.

Transepithelial potential difference. To measure transepithelial potential difference (VT), the solution in the perfusion pipette was connected to an electrometer (model KS-700; World Precision Instruments, New Haven, CT) through an agar bridge saturated with 0.16 M NaCl and a calomel cell as described previously (30). The reference was an agar bridge from the bath to a calomel cell. VT was recorded 1 h after warming the tubule and then 20-30 min after the addition of amiloride to the perfusate.

Statistical analysis. For each tubule wherein JtCO2 was measured, two to four measurements were averaged to obtain a single value for each experimental condition. For pHi measurements, a single measurement was made for each condition. Mean values were used in the statistical analysis. Statistical significance was determined by a paired or unpaired two-tailed Student's t-test, as appropriate, with P < 0.05 indicating statistical significance. Data are displayed as means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of limiting Na+ entry on JtCO2 and resting pHi. It was hypothesized that across the basolateral membrane of the rat tIMCD, NH+4 uptake is mediated by the Na+-K+-ATPase. NH+4 thus provides a source of H+ and NH3 for luminal secretion and the titration of other luminal buffers (30). In the presence of NH4Cl, the reduction in JtCO2 and the increase in pHi observed following ouabain addition (30) can be explained by inhibition of Na+ pump-mediated NH+4 uptake with reduced net H+ entry and increased pHi. However, Na+ pump inhibition affects other H+/OH-/HCO<SUP>−</SUP><SUB>3</SUB>/NH+4 pathways. Ouabain-induced changes in activity of these other transporters could also explain the above observation. The purpose of this study was to explore further the mechanism responsible for the NH+4-dependent, ouabain-induced reduction in JtCO2 and increase in pHi observed previously (30).

On the apical membrane of the tIMCD, Na+ enters the cell through amiloride-sensitive Na+ channels (17, 28) and exits across the basolateral membrane through the Na+ pump (17, 19). Increased intracellular Na+ (Na+i) facilitates Na+ pump-mediated NH+4 or K+ uptake. If NH+4-dependent, ouabain-induced changes in pHi and JtCO2 reflect Na+ pump-mediated NH+4 transport, then reducing Na+i by limiting Na+ entry should reduce JtCO2 and increase pHi.

To determine the effect of limiting Na+i entry on Na+ pump-mediated NH+4 uptake, JtCO2 was measured in the presence and the absence of the Na+ channel inhibitor, amiloride. In the presence of 3 mM KCl and 6 mM NH4Cl in the bath and perfusate (solution 1), baseline JtCO2 was 3.7 ± 0.7 and then fell to 2.0 ± 0.9 pmol · mm-1 · min-1 upon the addition of 1 µM amiloride to the perfusate (n = 5, P < 0.05; Fig. 1 and Table 2). This reduction in JtCO2 was not observed in time controls [Fig. 1, Table 2; n = 3, P = not significant (NS)] and was not observed in the absence of NH4Cl (solution 3, Table 2; n = 3, P = NS). To test whether the amiloride-induced reduction in JtCO2 results from a membrane potential-induced change in paracellular transport, VT was measured in the presence and absence of 1 µM amiloride (solution 1). Baseline VT was 0.0 ± 0.2 and +0.1 ± 0.1 mV (n = 3) upon the addition of 1 µM amiloride to the perfusate (P = NS). Thus the reduction in JtCO2 observed with amiloride addition is NH+4 dependent and is not mediated by a nonspecific effect of amiloride on VT that drives changes in paracellular transport.


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Fig. 1.   Effect of luminal amiloride on total CO2 absorption (JtCO2) with NH4Cl present in bath and perfusate. A: effect of 1 µM amiloride on JtCO2. During control period, mean flux was 3.7 ± 0.7 pmol · mm-1 · min-1. After addition of 1 µM amiloride to the luminal perfusate, flux declined to 2.0 ± 0.9 (n = 5, P < 0.05). B: experiment in A was repeated, but with a mock perfusate exchange, i.e., amiloride was not introduced into luminal perfusate. Baseline JtCO2 was 3.4 ± 1.1 and 3.6 ± 0.8 pmol · mm-1 · min-1 after perfusate exchange (n = 3, P = NS).

                              
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Table 2.   Total CO2 transport

The effect of luminal amiloride on resting pHi was tested. It was reasoned that if limiting apical Na+ entry limits Na+ pump-mediated NH+4 uptake, then in the presence of NH4Cl luminal amiloride should decrease net H+ uptake and alkalinize the cell. To test this hypothesis, the effect of amiloride on pHi was tested (Table 3). In the presence of NH4Cl (solution 1), following the addition of amiloride to the perfusate, a small increase in pHi was observed. This amiloride-induced alkalinization was not observed in the absence of NH4Cl (solution 3). However, these amiloride-induced changes in pHi were very small. Therefore, the effect of limiting Na+ availability on pHi was explored further.

                              
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Table 3.   Effect of amiloride on pHi

Effect of limiting Na+ entry on NH+4-dependent, ouabain-induced changes in pHi. If Na+ pump-mediated NH+4 entry supplies the cell cytosol with H+ and NH+4, then Na+ pump blockade should limit NH+4 uptake, attenuating net H+ entry and alkalinizing the cell. The effect of ouabain on resting pHi was therefore explored. Results were compared when the experiment was repeated with substitution of ouabain for its vehicle (solution 1). In the presence of 3 mM KCl, 6 mM NH4Cl, and 25 mM NaHCO3/5% CO2 in the bath and perfusate (solution 1), resting pHi averaged 7.18 ± 0.02 (n = 16). As shown in Fig. 2 (solution 1), with ouabain addition to the bath, a prompt and sustained increase in pHi was observed. Since pHi remained elevated, compared with control (vehicle), for at least 5.5 min following the addition of ouabain to the bath, pHi was measured 4 min after the addition of ouabain to the peritubular bath. These results confirm our previous observations that ouabain addition to the bath results in increased pHi when perfused in the presence of NH4Cl (30). The effect of ion substitution on ouabain-induced alkalinization was studied when each tubule was used as its own control.


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Fig. 2.   Effect of ouabain on resting intracellular pH (pHi) with NH4Cl present in bath and perfusate. Tubules were perfused with symmetrical solutions containing 3 mM KCl + 6 mM NH4Cl (solution 1). Bath was exchanged with the introduction of 2.5 mM ouabain and pHi measured every 45 s. Results are the mean of 3 tubules studied. In separate tubules, the experiment was repeated, but with a mock bath exchange and without introduction of ouabain. Ouabain vehicle was solution 1. Results are the mean of 3 experiments.

The mechanism responsible for the NH+4-dependent, amiloride-induced reduction ion in JtCO2 was studied further. In principal cells, amiloride attenuates but does not fully inhibit the increase in Na+i that follows Na+ pump inhibition (25). These results imply that inhibiting Na+ uptake through the apical Na+ channel attenuates but does not fully inhibit activity of the Na+ pump. These results might explain the greater increase in pHi and the greater decline in JtCO2 observed following the addition of ouabain to the bath than that observed following the addition of amiloride to the perfusate.

In principal cells, limiting Na+ entry from both the bath and perfusate by removal of extracellular Na+ fully inhibits the Na+ pump (25). Therefore removal of Na+ from the bath and perfusate should fully inhibit ouabain-induced alkalinization in the tIMCD, if mediated by NH+4 uptake on the Na+-K+-ATPase. The effect of removing Na+ from the bath and perfusate on NH+4-dependent, ouabain-induced alkalinization was therefore studied. With Na+ present in the bath and perfusate (solution 1; Fig. 3 and Table 4), pHi increased 0.07 ± 0.01 pH units (P < 0.05) following ouabain addition and returned to baseline upon ouabain withdrawal, as reported previously (30). In the absence of extracellular Na+ (solution 2), pHi did not increase following ouabain addition to the bath (Fig. 3).


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Fig. 3.   Effect of limiting Na+ entry on NH+4-dependent, ouabain-induced alkalinization. Tubules were perfused and bathed with NH4Cl present in bath and perfusate (solution 1). Baseline pHi was measured. Bath solution was exchanged to include ouabain (2.5 mM), and pHi was measured 4 min after the exchange. Bath was again exchanged, removing ouabain, and pHi was measured 4 min later (recovery). In the same tubules, perfusate and bath was exchanged, removing extracellular Na+ (solution 2). Ouabain-induced alkalinization was measured as above. Dotted line indicates that the experiment was performed in the reversed order, i.e., ouabain-induced alkalinization was measured first in absence and then in presence of extracellular Na+.

In the tIMCD, upon the removal of extracellular Na+, a prompt and sustained reduction in Na+i is observed (35). In the present experiment, when Na+ was removed from the bath and perfusate (solution 2; Fig. 3 and Table 4), pHi decreased (n = 3). 1 A reduction in Na+ pump activity under Na+-free conditions could be due to reduced Na+i or the reduced pHi (37). The pHi dependency of the Na+ pump in mouse tIMCD has been reported by our laboratory (37). With decreasing pHi, a reduction in ouabain-sensitive Rb+ uptake was observed. However, in the present study, the decline in pHi upon Na+ removal was modest (7.22 ± 0.02 to 6.98 ± 0.03, n = 3). Over this pHi range, ouabain-sensitive Rb+ uptake is reduced by only ~15% (37). Therefore, the absence of ouabain-induced alkalinization that follows extracellular Na+ removal cannot be explained fully by a pHi-induced change in Na+ pump activity. Thus limiting Na+ uptake by the tIMCD attenuates NH+4-dependent, ouabain-induced alkalinization.

Effect of extracellular K+ on ouabain-induced alkalinization. Our laboratory has demonstrated that NH+4 and K+ are competitive inhibitors for a common extracellular binding site of the Na+-K+-ATPase (34, 37). Thus increased extracellular K+ concentration attenuates NH+4 uptake by the tIMCD cell (34, 37). Using the model of Kurtz and Balaban (18) and employing kinetic values measured in our laboratory (34), we were able to estimate NH+4 uptake through the Na+-K+-ATPase. The model predicts NH+4 uptake through the Na+ pump to be increased two- to threefold when the extracellular K+ concentration is reduced from 12 to 3 mM. We reasoned that if the model were true, then in the presence of NH4Cl, increasing extracellular K+ concentration should attenuate ouabain-induced alkalinization as well as total and ouabain-sensitive JtCO2. To test this hypothesis, NH+4-dependent, ouabain-induced alkalinization was measured at two extracellular K+ concentrations (3 and 12 mM). In the presence of NH4Cl, pHi was 7.10 ± 0.06 at a K+ concentration of 3 mM (solution 1, Table 4) and 7.16 ± 0.05 (n = 4) at a K+ concentration of 12 mM (solution 4, Table 4). These results compare with JtCO2 flux rates of 3.4 ± 0.4 (n = 11) and 2.5 ± 0.3 pmol · mm-1 · min-1 (n = 7) when the extracellular K+ concentrations were 3 and 12 mM, respectively (Table 2). Thus, on average, JtCO2 was increased, and resting pHi was reduced, at lower extracellular K+ concentrations. These differences, however, did not reach statistical significance.

To evaluate the mechanism of NH+4 uptake in the tIMCD further, NH+4-dependent, ouabain-induced changes in JtCO2 and resting pHi were measured when the extracellular K+ concentration was either 3 or 12 mM. In the presence of 3 mM KCl + 6 mM NH4Cl (solution 1), resting pHi rose 0.07 ± 0.01 pH units (n = 4, P < 0.05) with ouabain addition and fell to baseline with ouabain withdrawal (Fig. 4; Table 4). However, at a K+ concentration of 12 mM no change in pHi was detected following the addition or withdrawal of ouabain (n = 4, P = NS; Fig. 4 and Table 4).

                              
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Table 4.   Effect of Ouabain on pHi


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Fig. 4.   Effect of increased extracellular K+ concentration on ouabain-induced alkalinization. Ouabain-induced alkalinization was measured in presence of 3 mM KCl + 6 mM NH4Cl (solution 1), as outlined in Fig. 3. Ouabain concentration was 5 mM. Perfusate and bath were then exchanged to solution containing 12 mM KCl + 6 mM NH4Cl (solution 4), and ouabain-induced alkalinization was again measured. In 2 tubules, the experiment was performed in the reverse order, i.e., ouabain-induced alkalinization was first measured with 12 mM KCl + 6 mM NH4Cl present. Results are means ± SE of 4 experiments.

We have demonstrated previously that in DOCP-treated rats, in the presence of 3 mM NH4Cl + 6 mM NH4Cl in the bath and perfusate (solution 1), baseline JtCO2 was 3.8 ± 0.5 and then 1.6 ± 0.3 pmol · mm-1 · min-1 (n = 7) following the addition of ouabain to the bath (30). These previously published data are given in Fig. 5A. In the present study, we asked whether ouabain-sensitive JtCO2 could be detected when NH+4 concentration was held constant but extracellular K+ was increased to 12 mM (solution 4). As shown in Table 2 and Fig. 5B, at a K+ concentration of 12 mM a reduction in JtCO2 could not be detected with the addition of ouabain to the bath. These results support the hypothesis that K+ and NH+4 compete for a common binding site on the Na+-K+-ATPase. Upon increasing extracellular K+ concentration, Na+ pump-mediated NH+4 uptake is attenuated, which results in a decrease in the ouabain-sensitive component of JtCO2 and resting pHi.


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Fig. 5.   Effect of ouabain on JtCO2 when the extracellular K+ concentration is increased. A: effect of ouabain on JtCO2 in presence of 3 mM KCl + 6 mM NH4Cl. During control period, mean flux was 3.8 ± 0.5 pmol · mm-1 · min-1. After addition of 2.5 mM ouabain to the bath, flux declined to 1.6 ± 0.3 pmol · mm-1 · min-1 (n = 7, P < 0.05). These data were taken from Ref. 30. B: experiment in A was repeated, but in presence of 12 mM KCl + 6 mM NH4Cl (solution 4). Baseline JtCO2 was 2.9 ± 0.4 and 2.9 ± 0.3 pmol · mm-1 · min-1 following the addition of 5 mM ouabain to bath (n = 4, P = NS).

Role of Ba2+-sensitive K+ channels. Increasing extracellular K+ concentration could increase pHi by limiting Na+ pump-mediated NH+4 entry. An alternative hypothesis, however, is that increasing extracellular K+ depolarizes the cell, which alters the activity of other H+/OH- transporters. If this hypothesis were true, then NH+4-dependent, ouabain-induced alkalinization results from membrane depolarization, which in turn alters electrogenic H+/OH- transport rather than Na+-K+-ATPase-mediated NH+4 uptake. At a concentration of 1 mM, BaCl2 induces a rapid and sustained cellular depolarization through K+ channel blockade in rat IMCD tubules perfused in vitro (26). Thus the effect of BaCl2 on pHi was explored to test the effect of changes in membrane potential on pHi, independent of direct Na+ pump blockade or changes in extracellular K+ concentration. Resting pHi was examined in the presence and the absence of Ba2+ (solution 5). Following the addition of 1 mM Ba2+ to the peritubular bath (n = 5), pHi was similar to that observed in time controls (n = 3, solution 5, Ba2+ absent; Fig. 6 a nd Table 5). Thus Ba2+ does not induce cellular alkalinization commensurate with that observed upon ouabain addition (solution 5; Fig. 6B and Table 5).2 Moreover, NH+4-dependent, ouabain-induced alkalinization was not abolished when 1 mM BaCl2 was present in the bath solution (Table 5).


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Fig. 6.   Effect of Ba2+ on resting pHi. A: tubules were perfused in presence of 3 mM KCl + 6 mM NH4Cl but in absence of SO<SUP>2−</SUP><SUB>4</SUB> and PO<SUP>2−</SUP><SUB>4</SUB> (solution 5) (see footnote 2.). Then, baseline pHi was measured (left). Bath solution was exchanged with the introduction of 1 mM BaCl2. pHi was measured 4 min later. In a time control series (right), the experiment above was repeated, but NaCl was substituted for BaCl2 isosmotically (n = 3). B: ouabain-induced alkalinization was measured under the same conditions as above (solution 5), but in absence of Ba2+. Baseline pHi was measured. pHi was again measured 4 min after ouabain addition (2.5 mM) and then 4 min following ouabain withdrawal.

The K+ channel present on the basolateral membrane of the IMCD recycles K+ (26), taken up by the Na+-K+-ATPase (26). It is possible that this K+ channel mediates NH+4 efflux as well (39). Thus ouabain addition could alter K+ or NH+4 transport through a Ba2+-sensitive pathway such as K+ channels. However, ouabain-induced alkalinization was observed in the presence of Ba2+ (Table 5). Therefore, NH+4 transport through Ba2+-sensitive pathways cannot fully explain NH+4-dependent, ouabain-induced alkalinization.

Role of HCO<SUP>−</SUP><SUB>3</SUB> transport. Another explanation for the observed NH+4-dependent reduction in JtCO2 and increase in pHi observed following ouabain addition is through changes in HCO<SUP>−</SUP><SUB>3</SUB> transport. The Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchanger and Na+-HCO<SUP>−</SUP><SUB>3</SUB> symporter are located on the basolateral membrane of the rat tIMCD (12, 33). In other cell types such as the proximal tubule, NH3 uptake "back-titrates" intracellular protons and thus increases pHi (29). The NH3-induced increase in pHi could stimulate HCO<SUP>−</SUP><SUB>3</SUB> exit through either of the above HCO<SUP>−</SUP><SUB>3</SUB> pathways. Moreover, in other cells the Na+ pump modulates both Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange and Na+-HCO<SUP>−</SUP><SUB>3</SUB> symport activity (27, 31). Thus the increase in pHi and the reduction in JtCO2, which follow ouabain addition, could be explained by changes in either Na+-HCO<SUP>−</SUP><SUB>3</SUB> or Cl-/HCO<SUP>−</SUP><SUB>3</SUB>-mediated HCO<SUP>−</SUP><SUB>3</SUB> efflux.

To resolve this question, NH+4-dependent, ouabain-induced alkalinization was tested in the absence of HCO<SUP>−</SUP><SUB>3</SUB>/CO2. To do so, bicarbonate-free, HEPES-buffered solutions (solutions 6 and 7) were employed, which were bubbled with ultrapure O2 (<3 ppm CO2). In the presence of 3 mM KCl + 6 mM NH4Cl in the bath and perfusate (solution 6), pHi rose 0.08 ± 0.01 pH units (n = 5) with ouabain addition and fell to baseline with ouabain withdrawal (Fig. 7; Table 4). This ouabain-induced alkalinization, however, was not observed in the absence of NH4Cl (solution 7; Fig. 8 and Table 4). Thus ouabain-induced alkalinization was independent of HCO<SUP>−</SUP><SUB>3</SUB>/CO2 in the extracellular media but was completely dependent on the presence of NH4Cl.


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Fig. 7.   Effect of ouabain on resting pHi in absence of HCO<SUP>−</SUP><SUB>3</SUB>/CO2. Tubules were perfused and bathed in presence of 3 mM KCl + 6 mM NH4Cl but in absence of HCO<SUP>−</SUP><SUB>3</SUB>/CO2 (solution 6). Baseline pHi was measured. Resting pHi was then measured following addition and then withdrawal of ouabain (2.5 mM) to peritubular bath. Bath was then exchanged to include 0.1 mM ethoxzolamide, a carbonic anhydrase inhibitor. Resting pHi was again measured in presence and absence of ouabain (2.5 mM) in peritubular bath. Results from 5 separate tubules are displayed.

                              
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Table 5.   Effect of Ba2+ on pHi


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Fig. 8.   Effect of ouabain on resting pHi in absence of both HCO<SUP>−</SUP><SUB>3</SUB>/CO2 and NH4Cl. Tubules were perfused and bathed in presence of 3 mM KCl, but without NH4Cl (solution 7). Baseline pHi was measured. pHi was again measured following addition and then withdrawal of ouabain (2.5 mM) in peritubular bath. Results obtained from 3 separate tubules are displayed.

Considerable CO2 can be generated through cellular metabolism, even in the nominal absence of HCO<SUP>−</SUP><SUB>3</SUB>/CO2 (3). In other cell types, the hydration of CO2, catalyzed by carbonic anhydrase, can produce cellular HCO<SUP>−</SUP><SUB>3</SUB> and hence substantial Na+-HCO<SUP>−</SUP><SUB>3</SUB> or Cl-/HCO<SUP>−</SUP><SUB>3</SUB>-mediated HCO<SUP>−</SUP><SUB>3</SUB> transport (3). Therefore the role of HCO<SUP>−</SUP><SUB>3</SUB> transport in ouabain-induced alkalinization was further explored. To do so, NH+4-dependent, ouabain-induced alkalinization was measured when 0.1 mM ethoxzolamide, a membrane permeant inhibitor of carbonic anhydrase, was added to the bath (solution 6). As shown (Fig. 7; Table 4), carbonic anhydrase inhibition did not abolish ouabain-induced alkalinization. Thus changes in HCO<SUP>−</SUP><SUB>3</SUB> transport cannot explain NH+4dependent, ouabain-induced alkalinization.

Effect of Na+-K+-2Cl- inhibition on JtCO2 and resting pHi. Our laboratory has shown that NH+4 and K+ compete for a common binding site on the Na+-K+-2Cl- cotransporter in mouse tIMCD cells (37). Furthermore, both ions are transported through this carrier (37). These results are in keeping with a recent report of high levels of Na+-K+-2Cl- cotransport expression in the mouse tIMCD (14). In many cell types, increased Na+i reduces K+ (or NH+4) uptake through the Na+-K+-2Cl- cotransporter (27, 40). Thus the reduction in JtCO2 and the increase in resting pHi observed with ouabain addition could occur from reduced Na+-K+-2Cl- cotransport-mediated NH+4 uptake. If this hypothesis were true, then addition of an Na+-K+-2Cl- cotransport inhibitor, such as bumetanide, should inhibit JtCO2 the same or more than that observed with ouabain addition. Bumetanide at a concentration of 100 µM fully inhibits the Na+-K+-2Cl- cotransporter in the rat tIMCD (11). Thus bumetanide in a 100 µM concentration was employed, and its effects on JtCO2 and resting pHi were tested (Table 2; Fig. 9). Our laboratory reported previously that in tIMCD tubules from DOCP-treated rats perfused in vitro in the presence of 3 mM KCl + 6 mM NH4Cl (solution 1), JtCO2 fell from 3.8 ± 0.5 to 1.6 ± 0.3 pmol · mm-1 · min-1 upon the addition of ouabain to the bath (n = 7, P < 0.05) (30). We asked ether the reduction in JtCO2 observed following ouabain addition could be reproduced with bumetanide. Under these conditions (solution 1), baseline JtCO2 was 3.1 ± 0.4 and 2.8 ± 0.5 pmol · mm-1 · min-1 with the application of 100 µM bumetanide to the bath (n = 3, P = NS). Thus, although ouabain addition to the bath reduced JtCO2, the addition of bumetanide did not.


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Fig. 9.   Effect of bumetanide on JtCO2. Tubules were perfused and bathed in presence of 3 mM KCl + 6 mM NH4Cl (solution 1). During control period, mean flux was 3.1 ± 0.4 pmol · mm-1 · min-1. After addition of 100 µM bumetanide to peritubular bath, flux was 2.8 ± 0.5 pmol · mm-1 · min-1 (n = 3, P = NS).

As an independent assessment of the effect of bumetanide on NH+4 transport, pHi was measured following the addition of bumetanide to the bath. Under conditions identical to those above (solution 1), pHi did not increase following bumetanide addition to the bath (Table 6). We conclude that the effect of ouabain on JtCO2 and pHi cannot be explained by NH+4 transport on the Na+-K+-2Cl- cotransporter. These results are also consistent with immunolocalization studies that indicate low levels of expression of Na+-K+-2Cl- cotransport protein (BSC-2) in the rat tIMCD (9), which contrasts with the high levels of expression reported in the tIMCD of the mouse (14).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Our laboratory has demonstrated previously that net acid secretion in the tIMCD is greater in the presence of NH4Cl than in its absence (30). Furthermore, in the presence but not in the absence of NH4Cl, ouabain addition reduces net acid secretion and increases pHi. These observations cannot be explained by differences in buffering of the luminal fluid or the cytosol which might occur in the presence and absence of NH4Cl3 (30). The present study demonstrates that the NH+4- dependent, ouabain-induced alkalinization and reduction in JtCO2 occur through inhibition of NH+4 uptake by the Na+-K+-ATPase. We conclude that Na+-K+-ATPase-mediated NH+4 uptake is an important determinant of pHi and net acid secretion in the rat tIMCD.

In mouse tIMCD cells in culture, a K+/NH+4 exchanger has been reported (1). Since the driving force for K+ exit/NH+4 uptake, or K+/NH+4 exchange (Fig. 10) (1), is decreased with Na+ pump inhibition, NH+4- dependent, ouabain-induced alkalinization might be explained by changes in K+/NH+4 exchange. This question cannot be tested directly, since no specific inhibitors of K+/NH+4 exchange are available (1, 38). However, it is unlikely that ouabain-induced changes in pHi and JtCO2, as observed in this study, are mediated by changes in K+/NH+4 exchange activity. Inhibition of the Na+ pump decreases the driving force for K+/NH+4 exchange by reducing intracellular K+ (K+i) concentration. In many cells, including the proximal tubule (22, 41), following the addition of ouabain, Na+i increases, and hence K+i decreases, over a time period of at least 20 min. However, as shown in Fig. 2, the alkalinization observed following ouabain addition is complete within 2 min. Thus pHi changes that follow ouabain addition do not parallel expected changes in K+i. Moreover, we have shown that both NH+4 and K+ support equivalent rates of ouabain-sensitive ATP hydrolysis in permeabilized, native rat IMCD cells (34). Thus both cations are transported directly by the Na+-K+-ATPase. Since these experiments were performed in permeabilized cells, inhibition of NH+4 transport following ouabain addition occurred independent of changes in intracellular ion composition.

In the rat tIMCD, two K+-ATPases have been identified (16). One of these K+-ATPases, like the gastric H+-K+-ATPase, is sensitive to low concentrations of Sch-28080 but insensitive to ouabain. The other K+-ATPase is insensitive to Sch-28080 but sensitive to ouabain and thus resembles the "colonic" H+-K+-ATPase isoform. NH+4 transport by the H+-K+-ATPase has been described (8). Therefore, it is possible that the ouabain-sensitive "colonic" H+-K+-ATPase transports NH+4 and therefore mediates the NH+4-dependent, ouabain-induced increase in pHi and decrease in JtCO2 observed in the present study. However, activity of the colonic H+-K+-ATPase is insensitive to changes in Na+ availability (4) and therefore cannot explain the observations of the current study. We have reported Sch-28080-sensitive bicarbonate absorption in the rat tIMCD (38). The transporter mediating Sch-28080-sensitive bicarbonate absorption, however, is distinct from the transport mechanism that mediates the NH+4-dependent, ouabain-sensitive changes in pHi and JtCO2 described herein (38).

Our results do not support significant K+ channel-mediated NH+4 efflux on the basolateral membrane of the rat tIMCD. However, they cannot exclude channel-mediated NH+4 efflux on the apical membrane. ATP-sensitive K+ channels sensitive to intracellular but not extracellular Ba2+ have been localized to the apical membrane of the mouse IMCD (24). Similarly, a nonspecific, amiloride-sensitive cation channel on the apical membrane has been reported (20). Both of these channels transport NH+4. Thus depolarization of the cell with ouabain addition could facilitate NH+4 efflux across the apical membrane mediated by these channels. The result would be increased NH+4 secretion into the luminal fluid upon ouabain addition. However, in a previous study (30), when ouabain was applied to the peritubular bath, total ammonium secretion fell from -0.3 ± 0.1 to -0.1 ± 0.1 pmol · mm-1 · min-1. Thus ouabain does not increase channel-mediated NH+4 efflux across the apical membrane. Thus changes in K+ channel-mediated NH+4 transport cannot explain the observations of the present study.

In epithelia that utilize carbonic acid as the main proton source, net luminal proton secretion occurs when proton secretion across the apical membrane is accompanied by bicarbonate (base) secretion across the basolateral membrane (31). However, cytosolic carbonic anhydrase activity is less abundant in the tIMCD than in other nephron segments (32, 33). Therefore, the regulation of basolateral HCO<SUP>−</SUP><SUB>3</SUB> exit and apical proton secretion in parallel may be less important in the tIMCD than in other nephron segments. Our results show that NH+4, rather than carbonic acid, provides the primary source of protons in the tIMCD.

                              
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Table 6.   Effect of bumetanide on pHi


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Fig. 10.   Proposed NH+4 pathway in rat terminal inner medullary collecting duct (tIMCD). NH+4 is taken up on basolateral membrane by the Na+-K+-ATPase. Intracellular NH+4 provides a source of H+ and NH3 for the cell. H+ is secreted across the apical membrane through the H+-K+-ATPase. NH3 leaves the cell by passive diffusion across the apical membrane, or it recycles across the basolateral membrane. Other NH+4/OH-/H+/HCO<SUP>−</SUP><SUB>3</SUB> transport pathways reported in tIMCD are shown (1, 11-13, 20, 24, 26, 28, 30, 35, 38).

In the tIMCD, NH+4 is an important H+ source. Our data show that NH+4 is taken up across the basolateral membrane by the Na+ pump, providing a source of H+ and NH3. Protons are then secreted across the apical membrane. However, since the pKa for NH3/NH+4 is 9.03 (15), in the range of physiological pHi, only ~1% of NH+4 releases a proton (39). Thus NH+4 uptake alone should not result in measurable changes in pHi. However, rapid entry of NH+4 coupled with NH3 exit across the basolateral membrane should provide significant net proton uptake with a substantial fall in pHi (30, 39). Across the basolateral membrane, NH+4 uptake with NH3 efflux would provide an NH3 shuttle, with net proton uptake. Across the apical membrane, NH3 secretion in parallel with H+ secretion by the H+-K+-ATPase (or H+-ATPase) (38) would trap NH+4 and facilitate net acid secretion (Fig. 10).

Amiloride administration decreases K+ excretion and impairs urinary acidification (5, 7). These defects in H+ and K+ secretion generate a hyperkalemic, hyperchloremic metabolic acidosis (7). In the cortical collecting duct (21) and turtle bladder (2), amiloride administration reduces the lumen-negative potential difference and decreases proton secretion. The acidification defect that follows amiloride administration is felt to occur from elimination of this lumen-negative potential difference, which decreases the driving force for H+ secretion. In the turtle bladder, if the lumen-negative potential difference is restored, then the defect in proton secretion that follows amiloride administration is reversed, despite the ongoing presence of amiloride (2). Thus the amiloride-induced reduction in proton secretion has been attributed to a "voltage defect." In the rat tIMCD, micropuncture studies have demonstrated a reduction in net acid secretion with luminal amiloride (5). In bicarbonate-loaded rats, DuBose and Caflisch (5) observed that the papillary urine-to-blood PCO2 gradient, an index of proton secretion, was reduced with amiloride administration. The present study demonstrates a reduction in JtCO2 upon addition of amiloride to the perfusate that cannot be explained by changes in VT. The present study, therefore, provides an explanation for this impaired proton secretion in the tIMCD. Amiloride reduces Na+i availability, which attenuates Na+ pump-mediated NH+4 uptake and therefore reduces net proton secretion.4 Luminal acidification in the tIMCD is thus dependent on apical Na+ entry.

Hyperkalemia is frequently associated with metabolic acidosis (6). Hyperkalemia reduces ammonium production by the proximal tubule and reduces ammonium absorption by the thick ascending limb, which together attenuate net acid excretion (6). In rats receiving dietary K+ loading, DuBose and Good (6) have observed a decrease in the interstitial NH3 concentration and therefore a reduction in the NH3 gradient from the interstitium to the collecting duct lumen. However, in rats which received a high-K+ diet, no net transfer of ammonium from the interstitium to the lumen was observed, despite the presence of an NH3 gradient that should favor NH3 diffusion into the collecting duct lumen. This observation suggested a defect in NH+4 transfer from the interstitium and the collecting duct lumen. The present study shows that this transfer defect can be explained, at least in part, by competition between NH+4 and K+ for the extracellular binding site on the Na+ pump. Increasing extracellular K+ reduces NH+4 uptake by the tIMCD cell and hence reduces acid secretion.

In conclusion, NH+4 uptake across the basolateral membrane of the tIMCD is mediated by the Na+ pump. NH+4 uptake provides a source of H+ for apical H+ secretion and the titration of luminal buffers. The reduction in acid secretion in the rat tIMCD observed following the administration of amiloride or upon dietary K+ loading can be explained, at least in part, by reduced NH+4 uptake by the Na+-K+-ATPase.

    ACKNOWLEDGEMENTS

I thank Drs. Steve Sansom, Andrew Kahn, and Roger O'Neil for helpful suggestions. I am again grateful to Dr. Thomas D. DuBose, Jr., for suggestions and continued support.

    FOOTNOTES

This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46493.

1 Our laboratory has demonstrated that removal of extracellular Na+ results in a prompt reduction in pHi and Na+i, mediated by Na+/H+ exchange (35).

2 In experiments that explored the effect of Ba2+ on pHi, SO<SUP>2−</SUP><SUB>4</SUB> and PO<SUP>2−</SUP><SUB>4</SUB> were removed from the perfusate and bath. These anions were removed, since both BaSO4 and BaPO4 are poorly soluble in aqueous solution.

3 Changes in pHi are dependent on cellular buffering capacity. An increase in buffering capacity should attenuate pHi changes measured following changes in net proton transport. However, buffering capacity is greater in the presence of NH+4 than in its absence (22). Thus the NH+4-dependent, ouabain-induced increase in pHi observed in the present study differs directionally from expected pHi changes that represent an NH+4-induced change in buffering capacity. The observation that ouabain-induced alkalinization occurs only in the presence of NH4Cl therefore cannot be explained by differences in cellular H+ buffering capacities in cells exposed to NH4Cl.

4 In the presence of NH4Cl, luminal amiloride reduces JtCO2 and increases pHi through inhibition of Na+ pump-mediated NH+4 uptake. However, we cannot exclude an additional mechanism responsible for the amiloride-induced reduction in JtCO2.

Address for reprint requests: S. M. Wall, Division of Renal Diseases and Hypertension, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030.

Received 3 January 1997; accepted in final form 24 July 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Renal Physiol 273(6):F857-F868
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