Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)
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《美国生理学杂志》
Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, and Renal Section, North Florida/South Georgia Veterans Health System, Gainesville, Florida
Department of Pathology and Laboratory Medicine, University of Pennsylvania, and American Red Cross, Philadelphia, Pennsylvania
ABSTRACT
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 Km of 7.0 ± 1.5 mM and was competitively inhibited by ammonia with a Ki 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
RENAL AMMONIA 1 METABOLISM, which results in new bicarbonate formation, is the primary component of both basal and acidosis-stimulated net acid excretion (13, 23). Ammonia is produced in the proximal tubule as a result of a biosynthetic metabolic pathway that results in equimolar NH4+ and bicarbonate formation. The ammonia is then secreted into the luminal fluid, reabsorbed by the thick ascending limb of the loop of Henle, and, finally, secreted into the luminal fluid by the collecting duct (13, 23). Because ammonia is transported by renal epithelial cells in a number of nephron regions, understanding the mechanisms of renal tubular ammonia transport is an important issue in renal physiology.
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
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 Jtotal is total uptake, Jdiffusive is the diffusive rate coefficient, [MA] is extracellular methylammonia concentration, Jtransporter is the transporter-mediated rate coefficient, and Km is the affinity for transporter-mediated methylammonia transport. Jdiffusive, Jtransporter, and Km were calculated using least-squares minimization (Quattro Pro, version 9; Corel). Because Jtotal 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 Jtotal is total measured uptake, Jdiffusive is diffusive uptake, Jinhibitable is transporter-mediated uptake, [X] is inhibitor concentration, and Ki is the inhibitor concentration that results in 50% inhibition of Jinhibitable. Best estimates for Jdiffusive, Jinhibitable, and Ki 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 (Jdiffusive) as described in Eq. 2. We then calculated the transporter-mediated [14C]MA uptake at each ammonia concentration by subtracting the diffusive component, Jdiffusive, from total uptake. A Dixon plot (reciprocal velocity vs. inhibitor concentration) was used to determine the Ki 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
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 Km of transporter-mediated component was 7.0 ± 1.5 mM (n = 6).
Although mIMCD-3 cells also express a basolateral [14C]MA transport activity (26), the luminal [14C]MA uptake observed is unlikely to be mediated by the basolateral transport activity. The peritubular [14C]MA concentration was quantified in all experiments and never exceeded 2% of the apical concentration. Thus the peritubular [14C]MA concentration is inadequate to enable sufficient basolateral [14C]MA uptake to explain the uptake observed in these studies (26). These results therefore identify that an apical transport activity is present in the mouse collecting duct cell mIMCD-3.
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 Ki 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.
In the studies that follow, we examined the characteristics of the transporter-mediated component of mIMCD-3 apical methylammonia uptake. The inhibitable component of [14C]MA transport is defined as that component inhibited by excess unlabeled methylammonia (20 mM) in parallel studies.
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.
To further confirm the presence of H+ gradient-linked apical [14C]MA transport, we altered intracellular [H+] using a recently described technique that does not involve ammonia exposure (26). Table 1 shows the solutions used and the resultant intracellular pH, and Fig. 4 summarizes the results. Intracellular acidification, decreasing intracellular pH from 7.4 to 7.1, at constant extracellular pH 7.4 increased transport activity significantly, from 0.1 ± 1.4 to 7.9 ± 1.8 pmol [14C]MA·mg protein–1·3 min–1 (P < 0.003, n = 4). Thus increasing intracellular [H+] by two independent methods increases inhibitable apical [14C]MA transport activity.
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An alternative method of increasing the apical membrane H+ gradient is to decrease extracellular [H+] at a constant intracellular [H+]. Increasing extracellular pH from 7.4 to 7.7 while maintaining intracellular pH constant at 7.4 increased mIMCD-3 apical [14C]MA transport activity from 0.1 ± 1.4 to 12.4 ± 0.5 pmol [14C]MA·mg protein–1·3 min–1 (P < 0.03, n = 4). Figure 4 summarizes these results. Thus altering the transmembrane H+ gradient, whether by increasing intracellular [H+] or decreasing extracellular [H+], increases [14C]MA transport activity.
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 Jtransporter, whereas alterations in the affinity for MA+ would alter Km. To differentiate these two possibilities, we determined the effect of acute intracellular acid loading on Jtransporter and Km. 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 Jtransporter 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 Km 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.
NH4+:H+ coupling ration. If apical MA+ for H+ exchange involves equimolar MA+ for H+ exchange, then varying membrane voltage will not alter transport activity. If the coupling ratio is other than 1:1, then changing the membrane voltage will alter transport activity, and the degree of change in transport activity can be used to predict the transport ratio. Thus, to determine the apparent coupling ratio of NH4+ and H+ exchange, we examined the effect of altering membrane voltage on transport activity. We used the K+ ionophore valinomycin (5 μM) and varied extracellular K+ concentration from 1 to 10 to 100 mM to alter membrane potential over a >100-mV predicted range (26). Extracellular Na+ concentration was maintained constant at 20 mM to avoid possible effects of extracellular Na+ on transport activity. Figure 6 summarizes these results. Varying membrane potential did not alter [14C]MA transport significantly (10.3 ± 0.8, 10.1 ± 0.3, and 11.6 ± 1.2 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). These findings are consistent with mIMCD-3 cells expressing an apical, electroneutral [14C]MA transport activity, most likely 1 MA+ for 1 H+ exchange.
Cation dependence of transport activity. Electroneutral apical ammonia transport activity could involve MA+ exchange with intracellular cations other than H+. Because Na+ and K+ are the cations with the greatest intracellular concentration, we considered the possibility that MA+/Na+ or MA+/K+ exchange contributes to apical [14C]MA uptake. If so, acute luminal Na+ or K+ removal would increase Na+- or K+-coupled MA+ transport. Figure 7 summarizes experiments testing this possibility. Compared with transport in the presence of Na+ and K+, acutely removing either extracellular Na+ or K+ did not alter transport activity significantly (11.5 ± 1.6, 12.4 ± 2.9, and 11.2 ± 0.2 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). Neither Na+ nor K+ appear to be alternative substrates for the apical NH4+/H+ exchange activity. Thus the mouse collecting duct cell mIMCD-3 expresses an apical, electroneutral, Na+- and K+-independent NH4+/H+ exchange activity.
Apical MA+ secretion. Under normal conditions, collecting duct cells secrete ammonia into the luminal fluid. To determine whether mIMCD-3 cell apical MA+/H+ exchange activity might contribute to luminal ammonia secretion, we quantified luminal MA+ secretion. Figure 8 summarizes these results. mIMCD-3 cells rapidly secreted [14C]MA into the luminal fluid. Moreover, extracellular MA+ (20 mM) stimulated [14C]MA efflux. Thus mIMCD-3 cells secrete MA+ across the apical membrane; the observation that extracellular MA+ stimulates apical [14C]MA secretion is consistent with a MA+/[14C]MA exchange mode of the apical transport activity.
Does apical MA+/H+ exchange occur via known apical collecting duct transporters Next, we examined whether apical MA+/H+ exchange activity might be mediated by any of several known collecting duct apical transporters. Because NH4+ and K+ have nearly identical biophysical properties in aqueous solutions, the possibility exists that the transport activity observed might be mediated by substitution of MA+ for K+ on an apical K+ transporter.
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.
Another possibility is that MA+ might substitute for K+ on other apical K+ transporters. If this occurred, then luminal K+ would inhibit transport activity. To test this possibility, we determined the effect of increasing luminal K+ on transport activity. Figure 10 summarizes these results. Increasing luminal K+ concentration from 5 to 60 to 120 mM did not alter transport activity significantly (11.1 ± 2.7, 11.7 ± 1.2, and 11.0 ± 1.9 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 4). mIMCD-3 apical ammonia transport is unlikely to occur through substitution of NH4+ for K+ on a nominal K+ transporter.
Another family of transporter proteins that can transport ammonia is the Na+/H+ exchanger family. In particular, the proximal tubule apical Na+/H+ exchanger NHE3 secretes ammonia due to substitution of NH4+ for H+ at the cytoplasmic binding site, resulting in apical Na+/NH4+ exchange (29, 37). To determine whether similar mechanisms are involved in mIMCD-3 apical ammonia transport, we examined whether Na+/H+ exchange inhibitors would alter transport activity. Figure 11 summarizes these results. Compared with transport in the absence of inhibitors, neither of the Na+/H+ exchange inhibitors, amiloride (1 mM) or ethylisopropyl amiloride (EIPA; 1 μM), altered transport activity significantly (12.1 ± 1.8, 13.4 ± 0.2, and 13.1 ± 2.1 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). mIMCD-3 apical ammonia transport appears to be mediated by an amiloride- and EIPA-insensitive transporter, most likely not Na+/H+ exchange. These results, combined with the Na+ independence of transport activity (Fig. 7), indicate that apical NH4+/H+ exchange is not mediated by an apical Na+/H+ exchanger. Thus mIMCD-3 apical NH4+/H+ exchange activity appears to be mediated by a novel, Na+- and K+-independent transporter unrelated to H+-K+-ATPase, Na+-K+-ATPase, other K+ transporters, or Na+/H+ exchange.
Expression of ammonia transporter family member RhCG. A novel ammonia transporter family that includes the Rh glycoproteins RhAG, RhBG, and RhCG was recently identified (39, 63). RhAG is an erythroid-specific protein that mediates Na+ - and K+-independent NH4+/H+ exchange (64, 65). RhBG and RhCG are expressed in the kidney (14, 44, 56, 63), and collecting duct cells express apical RhCG immunoreactivity (44, 56, 63). This suggests that RhCG is a candidate to mediate the apical NH4+/H+ exchange activity observed in mIMCD-3 cells.
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.
DISCUSSION
The majority of urinary ammonia excretion involves collecting duct ammonia secretion (23, 47), making understanding the mechanisms of ammonia transport across the apical and basolateral membranes of collecting duct cells important. The current study, with the use of [14C]MA as a radiolabeled ammonia analog, shows for the first time that collecting duct mIMCD-3 cells expresses an apical, Na+- and K+-independent, electroneutral NH4+/H+ exchange activity. This transport activity is not mediated by the apical transporters H+-K+-ATPase, Na+-K+-ATPase, or Na+/H+ exchange, is not mediated by substitution of NH4+ for K+ on nominal K+ transporters, and thus involves transport by a novel apical ammonia transporter. Finally, mIMCD-3 cells express apical RhCG, which may mediate the apical NH4+/H+ exchange activity observed.
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
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
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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Department of Pathology and Laboratory Medicine, University of Pennsylvania, and American Red Cross, Philadelphia, Pennsylvania
ABSTRACT
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 Km of 7.0 ± 1.5 mM and was competitively inhibited by ammonia with a Ki 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
RENAL AMMONIA 1 METABOLISM, which results in new bicarbonate formation, is the primary component of both basal and acidosis-stimulated net acid excretion (13, 23). Ammonia is produced in the proximal tubule as a result of a biosynthetic metabolic pathway that results in equimolar NH4+ and bicarbonate formation. The ammonia is then secreted into the luminal fluid, reabsorbed by the thick ascending limb of the loop of Henle, and, finally, secreted into the luminal fluid by the collecting duct (13, 23). Because ammonia is transported by renal epithelial cells in a number of nephron regions, understanding the mechanisms of renal tubular ammonia transport is an important issue in renal physiology.
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
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 Jtotal is total uptake, Jdiffusive is the diffusive rate coefficient, [MA] is extracellular methylammonia concentration, Jtransporter is the transporter-mediated rate coefficient, and Km is the affinity for transporter-mediated methylammonia transport. Jdiffusive, Jtransporter, and Km were calculated using least-squares minimization (Quattro Pro, version 9; Corel). Because Jtotal 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 Jtotal is total measured uptake, Jdiffusive is diffusive uptake, Jinhibitable is transporter-mediated uptake, [X] is inhibitor concentration, and Ki is the inhibitor concentration that results in 50% inhibition of Jinhibitable. Best estimates for Jdiffusive, Jinhibitable, and Ki 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 (Jdiffusive) as described in Eq. 2. We then calculated the transporter-mediated [14C]MA uptake at each ammonia concentration by subtracting the diffusive component, Jdiffusive, from total uptake. A Dixon plot (reciprocal velocity vs. inhibitor concentration) was used to determine the Ki 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
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 Km of transporter-mediated component was 7.0 ± 1.5 mM (n = 6).
Although mIMCD-3 cells also express a basolateral [14C]MA transport activity (26), the luminal [14C]MA uptake observed is unlikely to be mediated by the basolateral transport activity. The peritubular [14C]MA concentration was quantified in all experiments and never exceeded 2% of the apical concentration. Thus the peritubular [14C]MA concentration is inadequate to enable sufficient basolateral [14C]MA uptake to explain the uptake observed in these studies (26). These results therefore identify that an apical transport activity is present in the mouse collecting duct cell mIMCD-3.
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 Ki 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.
In the studies that follow, we examined the characteristics of the transporter-mediated component of mIMCD-3 apical methylammonia uptake. The inhibitable component of [14C]MA transport is defined as that component inhibited by excess unlabeled methylammonia (20 mM) in parallel studies.
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.
To further confirm the presence of H+ gradient-linked apical [14C]MA transport, we altered intracellular [H+] using a recently described technique that does not involve ammonia exposure (26). Table 1 shows the solutions used and the resultant intracellular pH, and Fig. 4 summarizes the results. Intracellular acidification, decreasing intracellular pH from 7.4 to 7.1, at constant extracellular pH 7.4 increased transport activity significantly, from 0.1 ± 1.4 to 7.9 ± 1.8 pmol [14C]MA·mg protein–1·3 min–1 (P < 0.003, n = 4). Thus increasing intracellular [H+] by two independent methods increases inhibitable apical [14C]MA transport activity.
View this table:
An alternative method of increasing the apical membrane H+ gradient is to decrease extracellular [H+] at a constant intracellular [H+]. Increasing extracellular pH from 7.4 to 7.7 while maintaining intracellular pH constant at 7.4 increased mIMCD-3 apical [14C]MA transport activity from 0.1 ± 1.4 to 12.4 ± 0.5 pmol [14C]MA·mg protein–1·3 min–1 (P < 0.03, n = 4). Figure 4 summarizes these results. Thus altering the transmembrane H+ gradient, whether by increasing intracellular [H+] or decreasing extracellular [H+], increases [14C]MA transport activity.
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 Jtransporter, whereas alterations in the affinity for MA+ would alter Km. To differentiate these two possibilities, we determined the effect of acute intracellular acid loading on Jtransporter and Km. 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 Jtransporter 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 Km 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.
NH4+:H+ coupling ration. If apical MA+ for H+ exchange involves equimolar MA+ for H+ exchange, then varying membrane voltage will not alter transport activity. If the coupling ratio is other than 1:1, then changing the membrane voltage will alter transport activity, and the degree of change in transport activity can be used to predict the transport ratio. Thus, to determine the apparent coupling ratio of NH4+ and H+ exchange, we examined the effect of altering membrane voltage on transport activity. We used the K+ ionophore valinomycin (5 μM) and varied extracellular K+ concentration from 1 to 10 to 100 mM to alter membrane potential over a >100-mV predicted range (26). Extracellular Na+ concentration was maintained constant at 20 mM to avoid possible effects of extracellular Na+ on transport activity. Figure 6 summarizes these results. Varying membrane potential did not alter [14C]MA transport significantly (10.3 ± 0.8, 10.1 ± 0.3, and 11.6 ± 1.2 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). These findings are consistent with mIMCD-3 cells expressing an apical, electroneutral [14C]MA transport activity, most likely 1 MA+ for 1 H+ exchange.
Cation dependence of transport activity. Electroneutral apical ammonia transport activity could involve MA+ exchange with intracellular cations other than H+. Because Na+ and K+ are the cations with the greatest intracellular concentration, we considered the possibility that MA+/Na+ or MA+/K+ exchange contributes to apical [14C]MA uptake. If so, acute luminal Na+ or K+ removal would increase Na+- or K+-coupled MA+ transport. Figure 7 summarizes experiments testing this possibility. Compared with transport in the presence of Na+ and K+, acutely removing either extracellular Na+ or K+ did not alter transport activity significantly (11.5 ± 1.6, 12.4 ± 2.9, and 11.2 ± 0.2 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). Neither Na+ nor K+ appear to be alternative substrates for the apical NH4+/H+ exchange activity. Thus the mouse collecting duct cell mIMCD-3 expresses an apical, electroneutral, Na+- and K+-independent NH4+/H+ exchange activity.
Apical MA+ secretion. Under normal conditions, collecting duct cells secrete ammonia into the luminal fluid. To determine whether mIMCD-3 cell apical MA+/H+ exchange activity might contribute to luminal ammonia secretion, we quantified luminal MA+ secretion. Figure 8 summarizes these results. mIMCD-3 cells rapidly secreted [14C]MA into the luminal fluid. Moreover, extracellular MA+ (20 mM) stimulated [14C]MA efflux. Thus mIMCD-3 cells secrete MA+ across the apical membrane; the observation that extracellular MA+ stimulates apical [14C]MA secretion is consistent with a MA+/[14C]MA exchange mode of the apical transport activity.
Does apical MA+/H+ exchange occur via known apical collecting duct transporters Next, we examined whether apical MA+/H+ exchange activity might be mediated by any of several known collecting duct apical transporters. Because NH4+ and K+ have nearly identical biophysical properties in aqueous solutions, the possibility exists that the transport activity observed might be mediated by substitution of MA+ for K+ on an apical K+ transporter.
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.
Another possibility is that MA+ might substitute for K+ on other apical K+ transporters. If this occurred, then luminal K+ would inhibit transport activity. To test this possibility, we determined the effect of increasing luminal K+ on transport activity. Figure 10 summarizes these results. Increasing luminal K+ concentration from 5 to 60 to 120 mM did not alter transport activity significantly (11.1 ± 2.7, 11.7 ± 1.2, and 11.0 ± 1.9 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 4). mIMCD-3 apical ammonia transport is unlikely to occur through substitution of NH4+ for K+ on a nominal K+ transporter.
Another family of transporter proteins that can transport ammonia is the Na+/H+ exchanger family. In particular, the proximal tubule apical Na+/H+ exchanger NHE3 secretes ammonia due to substitution of NH4+ for H+ at the cytoplasmic binding site, resulting in apical Na+/NH4+ exchange (29, 37). To determine whether similar mechanisms are involved in mIMCD-3 apical ammonia transport, we examined whether Na+/H+ exchange inhibitors would alter transport activity. Figure 11 summarizes these results. Compared with transport in the absence of inhibitors, neither of the Na+/H+ exchange inhibitors, amiloride (1 mM) or ethylisopropyl amiloride (EIPA; 1 μM), altered transport activity significantly (12.1 ± 1.8, 13.4 ± 0.2, and 13.1 ± 2.1 pmol [14C]MA·mg protein–1·3 min–1, respectively, P = NS by ANOVA, n = 3). mIMCD-3 apical ammonia transport appears to be mediated by an amiloride- and EIPA-insensitive transporter, most likely not Na+/H+ exchange. These results, combined with the Na+ independence of transport activity (Fig. 7), indicate that apical NH4+/H+ exchange is not mediated by an apical Na+/H+ exchanger. Thus mIMCD-3 apical NH4+/H+ exchange activity appears to be mediated by a novel, Na+- and K+-independent transporter unrelated to H+-K+-ATPase, Na+-K+-ATPase, other K+ transporters, or Na+/H+ exchange.
Expression of ammonia transporter family member RhCG. A novel ammonia transporter family that includes the Rh glycoproteins RhAG, RhBG, and RhCG was recently identified (39, 63). RhAG is an erythroid-specific protein that mediates Na+ - and K+-independent NH4+/H+ exchange (64, 65). RhBG and RhCG are expressed in the kidney (14, 44, 56, 63), and collecting duct cells express apical RhCG immunoreactivity (44, 56, 63). This suggests that RhCG is a candidate to mediate the apical NH4+/H+ exchange activity observed in mIMCD-3 cells.
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.
DISCUSSION
The majority of urinary ammonia excretion involves collecting duct ammonia secretion (23, 47), making understanding the mechanisms of ammonia transport across the apical and basolateral membranes of collecting duct cells important. The current study, with the use of [14C]MA as a radiolabeled ammonia analog, shows for the first time that collecting duct mIMCD-3 cells expresses an apical, Na+- and K+-independent, electroneutral NH4+/H+ exchange activity. This transport activity is not mediated by the apical transporters H+-K+-ATPase, Na+-K+-ATPase, or Na+/H+ exchange, is not mediated by substitution of NH4+ for K+ on nominal K+ transporters, and thus involves transport by a novel apical ammonia transporter. Finally, mIMCD-3 cells express apical RhCG, which may mediate the apical NH4+/H+ exchange activity observed.
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
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
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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