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Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)

¹Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, and ²Renal Section, North Florida/South Georgia Veterans Health System, Gainesville, Florida; ³Department of Pathology and Laboratory Medicine, University of Pennsylvania, and ⁴American Red Cross, Philadelphia, Pennsylvania

Submitted 8 July 2004 ; accepted in final form 22 March 2005

	ABSTRACT

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The collecting duct is the primary site of urinary ammonia secretion; the current study determines whether apical ammonia transport in the mouse inner medullary collecting duct cell (mIMCD-3) occurs via nonionic diffusion or a transporter-mediated process and, if the latter, presents the characteristics of this apical ammonia transport. We used confluent cells on permeable support membranes and examined apical uptake of the ammonia analog [¹⁴C]methylammonia ([¹⁴C]MA). mIMCD-3 cells exhibited both diffusive and saturable, transporter-mediated, nondiffusive apical [¹⁴C]MA transport. Transporter-mediated [¹⁴C]MA uptake had a K_m of 7.0 ± 1.5 mM and was competitively inhibited by ammonia with a K_i of 4.3 ± 2.0 mM. Transport activity was stimulated by both intracellular acidification and extracellular alkalinization, and it was unaltered by changes in membrane voltage, thereby functionally identifying an apical, electroneutral NH₄⁺/H⁺ exchange activity. Transport was bidirectional, consistent with a role in ammonia secretion. In addition, transport was not altered by Na⁺ or K⁺ removal, not inhibited by luminal K⁺, and not mediated by apical H⁺-K⁺-ATPase, Na⁺-K⁺-ATPase, or Na⁺/H⁺ exchange. Finally, mIMCD-3 cells express the recently identified ammonia transporter family member Rh C glycoprotein (RhCG) at its apical membrane. These studies indicate that the renal collecting duct cell mIMCD-3 has a novel apical, electroneutral Na⁺- and K⁺-independent NH₄⁺/H⁺ exchange activity, possibly mediated by RhCG, that is likely to mediate important components of collecting duct ammonia secretion.

Rh C glycoprotein

To understand renal ammonia transport, one needs to consider that ammonia exists in aqueous solutions in two molecular forms, NH₃ and NH₄⁺. Thus ammonia can be transported as either of the molecular species NH₃ or NH₄⁺. NH₃ is a small, uncharged molecule and is frequently believed to be highly permeable across plasma membranes. However, plasma membrane NH₃ permeability is a significant barrier to ammonia transport in some cells (15, 57). NH₄⁺ is a hydrophilic, cationic molecule that is not diffusible across plasma membranes but can be transported by a variety of proteins.

Renal ammonia metabolism in large part appears to involve NH₄⁺ transport by specific transport proteins. In the proximal tubule, ammonia is secreted in the molecular form, NH₄⁺, by the apical Na⁺/H⁺ exchanger NHE3 (38, 49) and by an apical Ba²⁺-sensitive K⁺ channel (24, 49). In the thick ascending limb of the loop of Henle, luminal ammonia reabsorption involves NH₄⁺ transport by a variety of proteins, including NKCC2 (the apical Na⁺-K⁺-2Cl^– cotransporter), an apical K⁺/NH₄⁺ antiporter, and an amiloride-sensitive NH₄⁺ conductance (2, 5, 28).

However, the mechanism of collecting duct apical ammonia transport is incompletely understood. In vitro microperfused tubule studies have shown that collecting duct ammonia secretion is regulated by both the peritubular-to-luminal ammonia gradient and the luminal-to-peritubular H⁺ gradient (16, 17, 25). These observations have led some to the conclusion that collecting duct ammonia transport occurs primarily, if not completely, through passive, nonionic NH₃ diffusion. However, recent studies have shown that basolateral ammonia transport involves specific transport events, including NH₄⁺ transport via Na⁺-K⁺-ATPase (58, 60) and via a basolateral NH₄⁺/H⁺ exchange activity that may be mediated by the ammonia transporter family member Rh B glycoprotein (RhBG) (26). Thus collecting duct ammonia transport is unlikely to occur solely through passive NH₃ diffusion.

Collecting duct ammonia secretion also involves apical ammonia transport, but the mechanism of collecting duct apical ammonia transport has not been specifically studied. Therefore, the purpose of this study was to determine the mechanism of collecting duct apical ammonia transport. We used [¹⁴C]methylammonia ([¹⁴C]MA) as a radiolabeled ammonia analog and quantified apical ammonia transport using luminal [¹⁴C]MA uptake by mouse inner medullary collecting duct cells (mIMCD-3) grown on permeable support membranes. We first determined whether apical ammonia transport occurs by nonionic NH₃ diffusion or by a saturable and inhibitable transport process. After identifying that a saturable and inhibitable transport activity was present, we determined the functional characteristics of this transport activity. We then determined that this transport activity did not reflect substitution of NH₄⁺ for other cations on either Na⁺ or K⁺ transporters. Finally, we examined whether the recently identified ammonia transporter family member, RhCG, which is expressed in the apical membrane of collecting duct cells in vivo (14, 56, 63), might mediate the apical transport activity.

	METHODS

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mIMCD-3 cells. Confluent, polarized mIMCD-3 cells were used as described recently (26). Briefly, we obtained mIMCD-3 cells from American Type Culture Collection (Manassas, VA) and used them between passages 20 and 40. They were grown to confluence on permeable support membranes (Costar Transwell filters) in 10% FCS-containing DMEM:F-12 medium. FCS was decreased to 0.1% 48 h before study to increase collecting duct-specific transporter expression (9, 51).

Measurement of [¹⁴C]MA transport activity. We measured transporter activity as [¹⁴C]MA uptake (0.275 µCi/ml, 5 µM) from the apical media. Briefly, cells were rinsed with radiotracer-free uptake medium, followed by exposure to uptake medium with [¹⁴C]MA added only to the luminal solution. Cells were rinsed rapidly with ice-cold, radiotracer-free medium to terminate uptake. Because preliminary studies showed that [¹⁴C]MA uptake was linear during the initial 3 min, we terminated uptake at 3 min in all studies. Soluble radioactivity was extracted by precipitating proteins with 10% TCA; cell protein was then solubilized in 0.2% SDS-0.2 N NaOH and quantified using a bicinchoninic acid assay. [¹⁴C]MA uptake is expressed as picomoles of [¹⁴C]MA per milligram of protein per 3 minutes. Appearance of [¹⁴C]MA in the peritubular solution was measured in all experiments and was always <2% of luminal [¹⁴C]MA.

Uptake medium, unless otherwise detailed, contained (in mM) 120 NaCl, 5 KCl, 10 HEPES, 20 choline chloride, 5 glucose, and 1.2 CaCl₂ and was titrated to pH 7.50. Equimolar choline was substituted for Na⁺ or K⁺ when we used Na⁺- or K⁺-free solutions. Methylammonium chloride or ammonium chloride substituted for choline chloride when used.

Methylammonia transport modeling. To determine the relative contributions of diffusive and transporter-mediated transport to total methylammonia uptake, we modeled uptake using the equation (10, 26)

(1)

where J_total is total uptake, J_diffusive is the diffusive rate coefficient, [MA] is extracellular methylammonia concentration, J_transporter is the transporter-mediated rate coefficient, and K_m is the affinity for transporter-mediated methylammonia transport. J_diffusive, J_transporter, and K_m were calculated using least-squares minimization (Quattro Pro, version 9; Corel). Because J_total varied by three orders of magnitude as a function of [MA], we use log-transformed data.

In some studies, we determined the ability of inhibitors to decrease transport activity. To do so, we modeled uptake as the sum of a diffusive and an inhibitable, transporter-mediated component using the formula (10, 26)

(2)

where J_total is total measured uptake, J_diffusive is diffusive uptake, J_inhibitable is transporter-mediated uptake, [X] is inhibitor concentration, and K_i is the inhibitor concentration that results in 50% inhibition of J_inhibitable. Best estimates for J_diffusive, J_inhibitable, and K_i were calculated using least-squares minimization (Quattro Pro, version 9.0).

Competitive inhibition of methylammonia transport. To determine whether ammonia competitively inhibits transporter-mediated methylammonia uptake, we used Dixon plot analysis. Briefly, we measured uptake of either 5 or 10 µM [¹⁴C]MA from the luminal solution in the presence of graded concentrations of extracellular ammonia. Because mIMCD-3 cells have both diffusive and transporter-mediated apical [¹⁴C]MA transport, we estimated the diffusive component of transport (J_diffusive) as described in Eq. 2. We then calculated the transporter-mediated [¹⁴C]MA uptake at each ammonia concentration by subtracting the diffusive component, J_diffusive, from total uptake. A Dixon plot (reciprocal velocity vs. inhibitor concentration) was used to determine the K_i for ammonia to inhibit transporter-mediated methylammonia uptake and to determine whether ammonia was a competitive or noncompetitive inhibitor of transporter-mediated [¹⁴C]MA uptake.

Intracellular acid-loading. We acid-loaded cells using a standard ammonium chloride prepulse technique. Briefly, we incubated cells with ammonium chloride (10 mM, pH 7.50) for 20 min, followed by ammonia washout. Control cells were treated identically, except choline chloride was substituted for ammonium chloride. We substituted choline for sodium in both the incubation and the uptake media to prevent intracellular pH recovery by basolateral Na⁺/H⁺ exchange (50). In preliminary studies we observed that this resulted in intracellular acidification to approximately pH 6.5.

Measurement of [¹⁴C]MA efflux. To examine ammonia, we loaded cells with [¹⁴C]MA and then quantified its secretion into the luminal fluid. We incubated the mIMCD-3 cells for 30 min with 5 µM [¹⁴C]MA; preliminary studies suggested that this results in maximal [¹⁴C]MA loading. They were then rinsed two times in 2.6 ml of room temperature medium and transferred to uptake medium. Parallel filters were treated with unlabeled methylammonia (20 mM) in the luminal and peritubular media during the efflux period. Efflux was terminated by removing the filters from the media and washing the filters with ice-cold uptake medium. [¹⁴C]MA efflux into the luminal solution and remaining cellular [¹⁴C]MA were quantified using standard techniques.

Real-time RT-PCR. We performed real-time RT-PCR using standard techniques (26, 62). Briefly, total RNA was extracted using RNeasy MidiKit (Qiagen, Valencia, CA) and stored at –70°C. RNA was reverse-transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and random hexamers. Real-time RT-PCR was performed on an ABI Prism GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA), and results were analyzed using GeneAmp 5700 SDS software (version 1.3; PerkinElmer Applied Biosystems). The forward primer for RhCG mRNA amplification was 5'-GGATACCCCTTCTTGGACTCTTC-3', the reverse primer was 5'-TGCCTTGGAACATGGGAAAT-3', and the probe was 6FAM-AGCCTCCGCCTGCTCCCCAAC-TAMRA.

Antibodies. We used previously characterized polyclonal anti-RhCG and anti-RhBG antibodies (56, 62, 63). For surface biotinylation experiments, antibodies were affinity-purified using the immunizing peptide and a commercially available kit (SulfoLink; Pierce Biotechnology, Rockford, IL).

Immunoblotting. Membrane protein extraction and immunoblotting was performed as described recently (26). Briefly, 20 µg of protein per lane were separated on 10% SDS-PAGE gels, transferred electrophoretically to nitrocellulose membranes, blocked, incubated with primary antibody, washed, and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; Promega, Madison, WI), and sites of antibody-antigen reaction were visualized using enhanced chemiluminescence.

Fluorescent microscopy. Confocal laser scanning microscopy was used to identify RhCG immunoreactivity using techniques described recently (26). Briefly, mIMCD-3 cells were fixed with 4% paraformaldehyde, treated with graded ethanols, rinsed with PBS, blocked with 5% normal goat serum, incubated overnight at 4°C with primary antibody, washed, incubated with FITC-coupled secondary antibody, rinsed with PBS, and coverslipped. We used an Axiovert 100M laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) and LSM 510 software (version 2.8; Carl Zeiss) to image the cells.

Apical plasma membrane biotinylation. mIMCD-3 cells were grown to confluence on permeable support filters as described. Apical plasma membrane proteins were biotinylated and isolated with the use of a commercially available kit (Cell Surface Protein Biotinylation and Purification Kit; Pierce Biotechnology, Rockford, IL) according to the manufacturer's recommended procedure. To identify apical plasma membrane proteins, we added biotin only to the luminal solution. Proteins were then immunoblotted as described. Bands were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology).

Chemicals. All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise detailed. [¹⁴C]methylammonium chloride was obtained from ICN (Irvine, CA).

Statistics. Statistical significance was determined using a paired t-test. In some cases, analysis of variance was used and is specifically noted in the text. In all cases, n refers to the number of separate mIMCD-3 preparations.

	RESULTS

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Apical methylammonia uptake by mIMCD-3 cells. The first set of studies sought to determine whether mIMCD-3 apical ammonia transport occurs via a saturable, transporter-mediated process or exclusively through nonionic NH₃ diffusion. Because direct measures of ammonia movement across a single membrane are not available (26), we quantified apical membrane ammonia transport using the radiolabeled ammonia analog [¹⁴C]MA. To differentiate between diffusive, nonionic uptake and transporter-mediated uptake, we reasoned that unlabeled methylammonia would not alter diffusive, nonionic [¹⁴C]MA uptake, whereas it would inhibit transporter-mediated [¹⁴C]MA uptake (26). As shown in Fig. 1A, unlabeled methylammonia inhibited apical [¹⁴C]MA transport in a concentration-dependent fashion. Another way to view the data is to determine total apical methylammonia uptake as a function of extracellular methylammonia, which is shown in Fig. 1B. As demonstrated, methylammonia uptake is curvilinear with respect to luminal methylammonia concentration. The observation that luminal uptake is not directly proportional to the luminal methylammonia concentration suggests that a saturable methylammonia transport mechanism mediates a significant component of total uptake. Mathematical modeling showed that the K_m of transporter-mediated component was 7.0 ± 1.5 mM (n = 6).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Relationship between methylammonia (MA) uptake and extracellular MA concentration ([MA]). Diffusive and inhibitable, transporter-mediated [14C]MA uptake can be differentiated by the ability of unlabeled MA or ammonia to inhibit uptake. A: MA-inhibitable [14C]MA uptake. MA uptake was determined as a function of extracellular [MA]. To separate the diffusive and the transporter-mediated uptake components, we modeled uptake using the formula Jtotal = Jdiffusive + Jinhibitable x [Ki/([X] + Km)], where Jtotal is the total uptake, Jdiffusive is the diffusive rate coefficient, Jtransporter is the transporter-mediated rate coefficient, and Km is the affinity for transporter-mediated MA transport. The solid line shows total uptake modeled as the sum of diffusive and transporter-mediated components. B: apical MA uptake as a function of luminal [MA]. The relationship between total apical MA uptake and luminal [MA] is shown. The dotted line shows predicted uptake if diffusive MA uptake were the only transport mechanism. The solid line (combined) shows predicted uptake if uptake were modeled as the sum of both diffusive and transporter-mediated uptake mechanisms, using the formula Jtotal = (Jdiffusive x [MA]) + Jtransporter x {[MA]/([MA] + Km)}.

To examine this apical transport activity further, we examined whether ammonia inhibited apical [¹⁴C]MA uptake. Figure 2A shows the results of a representative experiment. Ammonia inhibited apical [¹⁴C]MA transport in a concentration-dependent fashion. To determine whether this inhibition was mediated through changes in intracellular pH, we clamped intracellular pH at 7.1 with extracellular pH 7.4, using techniques previously described (26). Under these conditions, ammonia (10–20 mM) inhibited [¹⁴C]MA transport significantly ( = 6.6 ± 0.9 pmol [¹⁴C]MA·mg protein^–1·3 min^–1; P < 0.0005, n = 4). Thus ammonia's inhibition of mIMCD-3 apical [¹⁴C]MA occurs through mechanisms independent of changes in intracellular pH. Finally, Dixon plot analysis of ammonia's inhibition of 5 and 10 µM [¹⁴C]MA uptake showed that ammonia was a competitive inhibitor of [¹⁴C]MA uptake, with a K_i of 4.3 ± 2.0 mM (n = 4). Figure 2B shows a representative Dixon plot. Thus these results are the first to identify that mIMCD-3 cells have an apical ammonia-sensitive methylammonia transport activity.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Inhibition of [14C]MA transport activity by ammonia. A: the ability of luminal ammonia to inhibit [14C]MA uptake is shown. As in Fig. 1A, uptake was modeled as a combination of diffusive and inhibitable, transporter-mediated uptake, and the sum is plotted as the solid line. B: Dixon plot of inhibitable transport of either 5 µM () or 10 µM () luminal [14C]MA. Lines were calculated using least-squares linear regression. Lines intersect at –Ki.

Effect of intracellular and extracellular H⁺ on transport activity. Transepithelial H⁺ gradients regulate collecting duct ammonia secretion (16, 25, 53), which has led to the conclusion that transport involves nonionic NH₃ diffusion. However, the observation that a saturable, transporter-mediated component of ammonia transport occurs suggests other interpretations. In particular, these previous studies also are consistent with the exchange of NH₄⁺ for H⁺. To test this possibility, we examined whether changing the apical membrane H⁺ gradient would alter inhibitable transport activity.

First, we acutely acidified cells using a standard ammonia prepulse technique. Figure 3 summarizes these results. Intracellular acidification increased transport activity significantly, from 15.5 ± 1.0 to 47.5 ± 2.6 pmol [¹⁴C]MA·mg protein^–1·3 min^–1 (P < 0.003, n = 3). Thus increasing intracellular [H⁺] stimulates inhibitable [¹⁴C]MA transport.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effect of intracellular acidification on MA transport activity. Cells were acid-loaded by an NH4Cl prepulse, followed by a change to a Na+-free solution. Control cells were treated identically, except that NH4Cl-free solutions were used during the acid-loading period. Intracellular acid loading increased inhibitable transport activity significantly (P < 0.003, n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effect of separately altering intracellular (pHi) and extracellular pH (pHe) on transport activity. pHi and pHe were separately altered using a modification of the nigericin-K+ technique (26). Increasing the intracellular to extracellular H+ gradient, whether through intracellular acidification (pH 7.1) or extracellular alkalinization (pH 7.7), increased [14C]MA transport activity compared to cells with intracellular and extracellular pH 7.4.

Changes in apical transport activity as a result of increasing the transmembrane H⁺ gradient could occur either because H⁺ functions as a countertransported ion, e.g., MA⁺/H⁺ exchange, or because H⁺ alters the transporter's MA⁺ affinity. If [¹⁴C]MA uptake occurs via MA⁺/H⁺ exchange, then acute intracellular acidification will increase the transporter-mediated rate coefficient J_transporter, whereas alterations in the affinity for MA⁺ would alter K_m. To differentiate these two possibilities, we determined the effect of acute intracellular acid loading on J_transporter and K_m. Figure 5A shows results of a representative experiment, and Fig. 5, B and C, summarizes these results. Acute intracellular acidification increased the transporter rate coefficient J_transporter significantly, from 4,960 ± 1,490 to 29,740 ± 840 pmol MA⁺·mg protein^–1·3 min^–1 (P < 0.001 by paired t-test, n = 4). There was a tendency for K_m to increase, indicating a decreased MA⁺ affinity, but this did not reach statistical significance [P = not significant (NS) by paired t-test, n = 4]. However, decreases in MA⁺ affinity cannot account for the increased transport observed. These results confirm that mIMCD-3 cells express an apical, H⁺ gradient-driven MA⁺ transport activity.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of acute intracellular acidification on apical [14C]MA uptake kinetics. Mouse inner medullary collecting duct cells (mIMCD-3) were acid-loaded with an ammonia prepulse technique and compared with controls cells treated in parallel. The transporter rate coefficient (Jtransporter) and MA affinity (Km) were determined as described in Fig. 1B. Lines connect paired measurements made in cells treated in parallel. A: apical MA uptake as a function of luminal [MA]. Solid curves represent predicted uptake after total uptake was modeled as described in Fig. 1B. B: effect of acid loading on Jtransporter. C: effect of acid loading on Km.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effect of membrane potential on MA transport activity. Cells were incubated with the K+ ionophore valinomycin (5 µM) plus varying concentrations of extracellular K+ to clamp membrane potential over a wide range of membrane potentials. Altering membrane potential did not significantly alter transport activity (P = NS by ANOVA, n = 3).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of extracellular Na+ or K+ removal on MA transport activity. [14C]MA uptake was measured after an acute change to either control (normal) or to Na+-free (zero sodium) or K+-free (zero potassium) solutions. Neither Na+ nor K+ removal altered inhibitable MA transport activity significantly (P = NS by paired t-test and by ANOVA, n = 3).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Apical MA efflux. Cells were incubated with 5 µM [14C]MA for 30 min for loading. They were then rapidly washed, and [14C]MA secretion into the luminal fluid was quantified. Apical secretion was measured in the absence () and presence () of extracellular MA (20 mM) in parallel experiments. A: cellular [14C]MA rapidly decreased, indicating [14C]MA secretion. Extracellular MA stimulated [14C]MA efflux, suggestive of an MA/MA+ exchange mode. B: luminal [14C]MA secretion. mIMCD-3 cells rapidly secreted [14C]MA into the luminal fluid. Extracellular MA stimulated [14C]MA secretion, consistent with an MA/MA+ mode of transport. Results are shown as means ± SE of 3 separate experiments; when not visible, SE bars are within the size of the data symbol. *P < 0.05 by paired t-test.

mIMCD-3 cells express both the gastric and the colonic isoforms of H⁺-K⁺-ATPase (42). If MA⁺ substituted for K⁺ on either of these H⁺-K⁺-ATPases, this could result in ATPase-mediated MA⁺/H⁺ exchange. To test this possibility, we examined the effect of H⁺-K⁺-ATPase inhibitors on transport activity. Figure 9 summarizes these results. Neither SCH-28080 (10 µM), an inhibitor of "gastric-type" H⁺-K⁺-ATPases, nor ouabain (1 mM), an inhibitor of "colonic-type" H⁺-K⁺-ATPases, altered transport activity significantly (8.5 ± 1.5 and 14.8 ± 3.1, respectively, vs. 10.2 ± 1.6 pmol [¹⁴C]MA·mg protein^–1·3 min^–1 in the absence of inhibitors, P = NS by ANOVA, n = 3). Thus apical H⁺-K⁺-ATPases are unlikely to mediate the apical NH₄⁺/H⁺ exchange activity observed. Because this ouabain concentration also inhibits Na⁺-K⁺-ATPase, these findings also demonstrate that an apical Na⁺-K⁺-ATPase does not mediate mIMCD-3 apical ammonia transport.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Effect of H+-K+-ATPase inhibitors on [14C]MA transport activity. These studies examined whether apical H+-K+-ATPases might mediate luminal [14C]MA uptake. Inhibiting H+-K+-ATPase with luminal SCH-28080 (10 µM) or ouabain (1 mM) did not significantly alter inhibitable [14C]MA uptake (P = NS by paired t-test and by ANOVA, n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 10. Effect of luminal K+ on [14C]MA transport activity. These studies examined whether luminal K+ might inhibit luminal [14C]MA uptake. Luminal Na+ was maintained constant at 12.5 mM, and changes in luminal KCl were balanced by equimolar choline chloride changes. Luminal K+ did not alter inhibitable MA transport activity significantly (P = NS by ANOVA and by paired t-test, n = 4), indicating that K+ is not a competitive inhibitor of MA transport activity.

View larger version (23K): [in this window] [in a new window]   Fig. 11. Effect of Na+/H+ exchange inhibitors on [14C]MA transport activity. In some cells, an apical Na+/H+ exchange activity mediates ammonia transport. Therefore, the effect of Na+/H+ exchange inhibitors on mIMCD-3 MA transport was determined. Amiloride (1 mM) and ethylisopropyl amiloride (EIPA; 1 µM) were added to the luminal solution, and apical [14C]MA transport activity was determined. Neither amiloride nor EIPA altered [14C]MA transport activity significantly (P = NS by ANOVA, n = 3). mIMCD-3 apical MA transport does not appear to occur via apical Na+/H+ exchange activity.

To test this possibility, we examined mIMCD-3 RhCG expression. mRNA amplification with the use of real-time RT-PCR and RhCG-specific primers and fluorescent probes demonstrated that mIMCD-3 cells express RhCG mRNA (data not shown). Amplification was not observed when reverse transcription was not performed or when sample RNA was not added, confirming the specificity of RhCG mRNA amplification. Immunoblot analysis confirmed RhCG protein expression with an apparent molecular weight similar to that observed in the mouse kidney in vivo (56). Confocal laser scanning fluorescent microscopy identified that mIMCD-3 cells express apical RhCG immunoreactivity. Figure 12 shows representative results. This pattern of RhCG immunoreactivity clearly differs from that of the related protein RhBG, which, in studies performed simultaneously with these immunofluorescence microscopy studies and previously reported (26), was shown to exhibit basolateral immunoreactivity. Finally, we isolated apical plasma membranes using biotinylation of mIMCD-3 cells grown on permeable support membranes. Immunoblot analysis demonstrated RhCG expression when luminal biotin was used but not when luminal biotin was omitted (Fig. 12), thereby confirming apical plasma membrane RhCG protein expression. Thus mIMCD-3 cells express apical RhCG, consistent with RhCG mediating the apical NH₄⁺/H⁺ exchange activity observed.

View larger version (26K): [in this window] [in a new window]   Fig. 12. mIMCD-3 Rh C glycoprotein (RhCG) protein expression. A: immunoblot of mIMCD-3 proteins using anti-peptide and anti-RhCG antibodies previously characterized (56, 62). An 52-kDa protein was present in mIMCD-3 cells, and immunoreactivity was blocked by preincubating the antibody with the immunizing peptide. B: confocal laser scanning fluorescent micrograph of mIMCD-3 cells stained for RhCG. Apical immunoreactivity is present and is confirmed by XZ reconstruction (top). The blue line at top shows the level of the section shown at bottom. C: confocal laser scanning fluorescent micrograph of mIMCD-3 cells treated in parallel with cells in B but labeled for RhBG. RhBG expression differs from RhCG expression and is predominantly basolateral, as shown by XZ reconstruction (top). The blue line at top shows the level of the section shown at bottom. D: apical plasma membrane mIMCD-3 cell proteins were isolated using luminal biotinylation. Immunoblot analysis identified RhCG expression when apical biotinylation was performed; when biotin was not added to the luminal fluid, RhCG protein was not isolated. Affinity-purified antibodies were used in this protocol, eliminating the nonspecific band present in A.

	DISCUSSION

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The current study adds important new information to prior evidence that collecting duct ammonia transport is mediated, at least in part, by specific ammonia transport mechanisms and not solely by nonionic NH₃ diffusion. For example, the toad bladder, an amphibian analog of the mammalian collecting duct, secretes ammonia against an NH₃ gradient, thereby excluding nonionic NH₃ diffusion as the primary ammonia transport mechanism and implicating the need for specific ammonia transporters (36). Turtle urinary bladder transepithelial ammonia secretion is inhibited by methylammonia, and transepithelial methylammonia secretion is inhibited by ammonia (4, 55). These observations implicate the need for one or more ammonia-methylammonia transporters to mediate ammonia secretion and, again, exclude nonionic NH₃ diffusion as the primary ammonia transport mechanism. Studies using isolated perfused rat inner medullary collecting duct suggest that peritubular ammonia uptake by a basolateral Na⁺-K⁺-ATPase contributes to transepithelial ammonia secretion (58, 60), and studies using the mIMCD-3 cell identify a basolateral NH₄⁺/H⁺ exchange activity (26). The current study adds to these previous studies by demonstrating, for the first time, that a Na⁺- and K⁺-independent NH₄⁺/H⁺ exchange activity is present in the apical membrane of the mIMCD-3 cell. Thus collecting duct transepithelial ammonia secretion appears to involve specific transport processes at both the apical and basolateral membranes.

The identification that mIMCD-3 cells express an apical NH₄⁺/H⁺ exchange activity is both consistent with and provides a new interpretation of previous studies that have examined the mechanism of mammalian collecting duct ammonia secretion. In vitro microperfused tubule studies demonstrate that collecting duct transepithelial ammonia secretion is determined by both the ammonia concentration and the pH gradient (16, 17, 25). In these studies, increasing luminal H⁺ concentration stimulates transepithelial ammonia secretion and counterbalances transepithelial ammonia gradients. Because changes in luminal and/or peritubular pH alter NH₃ concentration much more substantially than they alter NH₄⁺ concentration, one interpretation of these results has been that transepithelial ammonia secretion occurs through nonionic NH₃ diffusion. However, the current study, by identifying that apical membrane ammonia transport occurs, at least in part, through a saturable and inhibitable mechanism, excludes nonionic NH₃ diffusion as the sole apical transport mechanism and, instead, suggests that a specific ammonia transporter contributes to apical ammonia transport. At physiological pH, >98% of total ammonia is present as NH₄⁺. Thus the identification that this apical ammonia transport activity can be functionally identified as an NH₄⁺/H⁺ exchange is fully consistent with the observation that transepithelial ammonia secretion is determined by the total ammonia concentration gradient and is counterbalanced by the transepithelial H⁺ gradient.

The current study provides two independent lines of evidence that the apical transport activity observed is likely to contribute to collecting duct ammonia secretion. First, direct measurements demonstrate apical [¹⁴C]MA secretion. Second, the observation that apical MA⁺:H⁺ coupling ratio is 1:1, and thus electroneutral, suggests that this transport activity contributes to collecting duct ammonia secretion. Collecting duct luminal NH₄⁺ concentrations can exceed 200 mM. Electroneutral NH₄⁺/H⁺ exchange enables NH₄⁺ secretion against this NH₄⁺ gradient by coupling NH₄⁺ secretion to H⁺ movement from the highly acidic luminal fluid to the relatively alkaline collecting duct cytoplasm. NH₄⁺ transport, if not coupled to exchange for H⁺, in contrast, would likely result in NH₄⁺ reabsorption. The cytoplasm of collecting duct cells is negatively charged relative to the luminal fluid, yielding an electrical gradient for NH₄⁺ reabsorption. This, in combination with high luminal NH₄⁺ concentrations, would result in an electrochemical gradient favoring NH₄⁺ reabsorption. Instead, the identification of MA⁺ for H⁺ exchange enables MA⁺ and, by extension, NH₄⁺ secretion by collecting duct cells and supports the conclusion that this transport activity mediates an important role in transepithelial ammonia secretion.

Extracellular ammonia stimulates collecting duct H⁺ secretion (19, 20, 31, 58), and this involves, at least in the cortical collecting duct, stimulation of apical H⁺-K⁺-ATPase (20). Moreover, this occurs through effects of ammonia that are independent from ammonia's effects as a transported molecule (18–20). The identification that collecting duct cells also express an apical NH₄⁺/H⁺ exchange activity suggests that ammonia may stimulate its transepithelial secretion by stimulating H⁺-K⁺-ATPase, that the secreted H⁺, at least in part, recycles through apical NH₄⁺/H⁺ exchange activity, and that the net effect is ATPase-dependent NH₄⁺ for K⁺ exchange. This can then mediate ammonia's effect to decrease cortical collecting duct net K⁺ secretion, at least in part, by stimulating active K⁺ reabsorption (20, 22).

The current study in conjunction with a recent report (26) shows that mIMCD-3 cells have both apical and basolateral NH₄⁺/H⁺ exchange activity. The presence of both apical and basolateral NH₄⁺/H⁺ exchange activities are well positioned to mediate coordinated mechanism of H⁺ gradient-regulated transepithelial ammonia secretion. For example, luminal acidification stimulates collecting duct ammonia secretion (30, 31, 52, 53). The current studies suggest that luminal acidification stimulates apical NH₄⁺/H⁺ exchange activity by increasing the gradient for apical H⁺ entry, thereby increasing apical NH₄⁺ secretion. This decreases intracellular NH₄⁺ concentration, which would increase basolateral NH₄⁺ uptake via the basolateral NH₄⁺/H⁺ transporter. Other studies have shown that transepithelial total ammonia gradients stimulate ammonia secretion. Because the majority of total ammonia is present as NH₄⁺, it is likely that peritubular NH₄⁺ uptake via basolateral NH₄⁺/H⁺ exchange coupled with apical NH₄⁺ secretion via apical NH₄⁺/H⁺ exchange mediates an important role in transepithelial ammonia secretion under these conditions. In addition, a basolateral Na⁺-K⁺-ATPase also may contribute to peritubular NH₄⁺ uptake (59, 61).

Because the mIMCD-3 has both apical and basolateral NH₄⁺/H⁺ exchange activities, it is important to compare and contrast these activities. Both are Na⁺ and K⁺ independent, electroneutral, and are not mediated by previously identified apical and basolateral transporters (26). The major differences reside in their basal activity, in their sensitivity to extracellular and intracellular pH, and in their affinities for ammonia and methylammonia. First, mIMCD-3 basolateral ammonia transport exhibits greater activity under basal conditions than the apical transport activity. Whether this is due to intrinsic differences in transport efficiency, differences in the level of expression of these transporters, or differences in the cellular regulation of apical and basolateral transport cannot be determined at present. Second, the apical transport activity is more sensitive to changes in intracellular and extracellular pH than the basolateral transport activity. Third, the basolateral transport activity has a greater affinity for both ammonia and methylammonia (26) than does the apical transport activity. A preliminary report has identified similar differences in the ammonia affinity of RhBG and RhCG, the proteins possibly responsible for mIMCD-3 cell basolateral and apical NH₄⁺/H⁺ exchange activity, respectively (66). The similar overall transport characteristics, in combination with the differences noted, are consistent with the apical and basolateral transport activities being mediated by separate members of the same protein family.

The molecular mechanism of the apical NH₄⁺/H⁺ exchange activity may be mediated by the ammonia transporter family member RhCG. Apical RhCG immunoreactivity is present in the renal connecting segment and collecting duct (44, 56, 63) and in the mouse collecting duct mIMCD-3 cell line. Complete confirmation that RhCG mediates this apical transport activity would require the availability of specific inhibitors, which are not available at present, the availability of a RhCG-gene knockout animal model, which is not available at present, or the ability to "knock down" RhCG mRNA expression in vitro, perhaps with the small interfering RNA technique (11). Unfortunately, attempts to knock down RhCG mRNA expression using constructs designed according to "Tuschl" criteria have not succeeded in decreasing mRNA expression reliably (Handlogten ME and Weiner ID, unpublished observations). Another approach is to express RhCG in heterologous expression systems. This approach has resulted in inconsistent data in the literature. One report suggests that RhCG, when expressed in the Xenopus oocyte, mediates both electroneutral and electrogenic ammonia transport (7). Two preliminary reports have reached different, and opposing, conclusions, with one suggesting that RhCG mediates only electroneutral NH₄⁺/H⁺ exchange (66) and the other suggesting that RhCG mediates only electrogenic NH₄⁺ transport (40). Thus all three reports confirm that RhCG can transport ammonia but differ in the extent to which transport is electrogenic NH₄⁺ transport vs. electroneutral NH₄⁺/H⁺ exchange. Whether these differences reflect differences in the technical procedures used in these different reports, variations in protein-protein interactions between RhCG and endogenous Xenopus oocyte proteins, or other etiologies is unclear at present. Determining the specific transport characteristics of RhCG and reconciling these various observations are important areas for future study.

RhAG and RhBG are unlikely to mediate the apical NH₄⁺/H⁺ exchange activity observed in the current study. RhAG is a component of the erythrocyte Rh complex (6), and seminal studies show that it mediates NH₄⁺/H⁺ exchange (64, 65). However, RhAG appears to be expressed only by erythrocytes and erythroid progenitor cells (33). RhBG is unlikely to mediate collecting duct apical ammonia transport activity because RhBG exhibits basolateral immunoreactivity both in vivo in the collecting duct (44, 56, 63) and in vitro in the mIMCD-3 cell (26).

The current study also identifies that mIMCD-3 apical plasma membrane methylammonia diffusional permeability is limited. Ammonia and methylammonia have the unusual properties of existing in equilibrium between the neutral, lipophilic molecular species NH₃ and CH₃NH₂, respectively, and the lipophobic molecular species NH₄⁺ and CH₃NH₃⁺, respectively. Small, neutral molecules, such as NH₃ and CH₃NH₂, are generally believed to diffuse rapidly across lipid membranes. However, both the current study and several previous studies (16, 25, 26, 53, 67) show that collecting duct cell NH₃ and CH₃NH₂ permeability is finite and limiting to total transport. Furthermore, direct measurements show that ammonia may be less lipid soluble than generally believed. In particular, the partition coefficient for ammonia between chloroform and water is 0.04 (8, 41), and that between heptane and water is <0.002 (35); a coefficient >1.0 is generally necessary to indicate significant lipid solubility.

The current study used the mIMCD-3 cell as a cultured collecting duct cell model system. The mIMCD-3 cell was derived from the collecting duct of mice transgenic for the early region of simian virus SV40 (Large T antigen) (45). mIMCD-3 cells possess multiple characteristics of collecting duct cells, including high transepithelial resistance (45), amiloride-inhibitable Na⁺ transport (45), and expression of the epithelial sodium channel (45), NHE-1 and NHE-2 (50), ATP-sensitive K⁺ channels (48), Na⁺-K⁺-ATPase (61), the basolateral Na⁺-K⁺-2Cl^– cotransporter NKCC1 (12, 27), both gastric and colonic H⁺-K⁺-ATPases (42), and H⁺-ATPase (1, 3). Finally, the mIMCD-3 cell expresses both apical RhCG and basolateral RhBG, characteristics found in vivo in the collecting duct (44, 56). Thus this cell line is well suited to serve as a model system in which to examine collecting duct ammonia transport.

One possible limitation of this study is that transport was measured using methylammonia, not ammonia. Direct measurements of transmembrane ammonia transport are difficult. Radiolabeled ammonium (¹⁵NH₄⁺) is not commercially available and, even if it were, can be quantified only by mass spectrometry. Intracellular voltage and pH measurements can indirectly assess electrogenic NH₄⁺ transport but might not detect electroneutral transport. Intracellular NH₃- or NH₄⁺-sensitive electrodes and fluorescent NH₃- or NH₄⁺-sensitive dyes are not available. As a result, direct NH₄⁺ transport measurement is difficult and generally not practical. Instead, methylammonia is widely used as an ammonia surrogate because it appears to be transported through the same pathways as ammonia (21, 32, 34, 43, 46, 54).

In summary, these studies are the first to identify that an apical ammonia transport activity is present in collecting duct cells. This transport activity is identified functionally as an electroneutral, Na⁺- and K⁺-independent NH₄⁺/H⁺ exchange activity and may be mediated by the ammonia transporter family member RhCG. As such, it has electrochemical characteristics suggesting that it plays a central role in collecting duct ammonia transport. Moreover, the identification that the collecting duct cell mIMCD-3 expresses both apical and basolateral NH₄⁺/H⁺ exchange activities (current study and Ref. 26) suggests that transepithelial ammonia secretion involves sequential basolateral and apical carrier-mediated ammonia transport. Identifying the factors that regulate apical and basolateral plasma membrane ammonia transport are important areas for future study.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
These studies were supported by National Institutes of Health Grants DK-45788, DK-02751 and NS-47624, the Department of Veterans Affairs Merit Review Program, and the Florida Affiliate of the American Heart Association.

	ACKNOWLEDGMENTS

 
We thank Gina Cowsert for secretarial assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: I. D. Weiner, Univ. of Florida College of Medicine, PO Box 100224, Gainesville, FL 32610-0224 (e-mail: weineid{at}ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Ammonia and methylammonia exist in aqueous solutions in two molecular forms. Ammonia exists in an equilibrium between NH₃ and NH₄⁺ and methylammonia in an equilibrium between CH₃NH₂ and CH₃NH₃⁺. In this report, the terms "ammonia" and "methylammonia" refer to the combination of their two molecular forms. The term "ammonium" specifically refers to the molecular species NH₄⁺, and the term "methylammonium" (MA⁺) specifically refers to the molecular species CH₃NH₃⁺. When referring to either NH₃ or CH₃NH₂, we specifically state "NH₃" or "CH₃NH₂."

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