Expression of the ammonia transporter proteins Rh B glycoprotein and Rh C glycoprotein in the intestinal tract
Mary E. Handlogten,2
Seong-Pyo Hong,2
Li Zhang,2
Allen W. Vander,2
Marshall L. Steinbaum,2
Martha Campbell-Thompson,3 and
I. David Weiner1,2
1Medical Service, North Florida/South Georgia Veterans Health System; and 2Departments of Internal Medicine and 3Laboratory and Pathology Medicine, University of Florida College of Medicine, Gainesville, Florida
Submitted 16 September 2004
; accepted in final form 30 November 2004
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ABSTRACT
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Ammonia metabolism is important in multiple aspects of gastrointestinal physiology, but the mechanisms of ammonia transport in the gastrointestinal tract remain incompletely defined. The present study examines expression of the ammonia transporter family members Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) in the mouse gastrointestinal tract. Real-time RT-PCR amplification and immunoblot analysis identified mRNA and protein for both RhBG and RhCG were expressed in stomach, duodenum, jejunum, ileum, and colon. Immunohistochemistry showed organ and cell-specific expression of both RhBG and RhCG. In the stomach, both RhBG and RhCG were expressed in the fundus and forestomach, but not in the antrum. In the forestomach, RhBG was expressed by all nucleated squamous epithelial cells, whereas RhCG was expressed only in the stratum germinativum. In the fundus, RhBG and RhCG immunoreactivity was present in zymogenic cells but not in parietal or mucous cells. Furthermore, zymogenic cell RhBG and RhCG expression was polarized, with apical RhCG and basolateral RhBG immunoreactivity. In the duodenum, jejunum, ileum, and colon, RhBG and RhCG immunoreactivity was present in villous, but not in mucous or crypt cells. Similar to the fundic zymogenic cell, RhBG and RhCG expression in villous epithelial cells was polarized when apical RhCG and basolateral RhBG immunoreactivity was present. Thus the ammonia transporting proteins RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they function cooperatively to mediate gastrointestinal tract ammonia transport.
ammonia; small intestine; large intestine
AMMONIA METABOLISM IS CRITICAL for multiple aspects of normal mammalian physiology. In the liver, ammonia1 uptake and metabolism to either urea or glutamine is essential for prevention of hyperammonemic and hepatic encephalopathy (8, 19, 38). Ammonia metabolism in the central nervous system is essential for normal neuronal function (4, 43) and in the kidney is critical for acid-base homeostasis (10, 17, 54). Finally, the gastrointestinal tract both produces and transports ammonia, which is then absorbed into the portal circulation. Accordingly, the molecular mechanisms underlying ammonia transport are of fundamental importance.
Ammonia exists in aqueous solutions in two molecular forms, NH3 and NH4+, each of which is transported by differing molecular mechanisms. Until recently, ammonia transport was believed to occur predominantly through nonionic NH3 diffusion. However, ammonia may be less lipid soluble than generally believed. In particular, the partition coefficient for ammonia between chloroform and water is
0.04 (1, 37) and between heptane and water is <0.002 (33); a partition coefficient <1.0 indicates relative lipid insolubility. Consistent with these measurements is that the plasma membrane of many cells has a low permeability to NH3 (13, 48). Increasingly, it has been recognized that protein-mediated NH4+ transport may mediate important roles in ammonia metabolism (10, 25, 49, 54, 57). Many proteins, including Na+-K+-ATPase, H+-K+-ATPase, Na+-K+-2Cl cotransporter, K+ channels, KCC proteins, and some Na+/H+ exchange isoforms, have been shown to transport NH4+ (2, 10, 25, 49). For each of these proteins, NH4+ transport is believed to occur through substitution of NH4+ for either K+ or, in the case of Na+/H+ exchangers, H+.
A novel ammonia transporter family, the Rh glycoprotein family, has been identified recently (20, 36, 54, 57). Members of this family include the erythroid Rh-associated glycoprotein (RhAG), and the nonerythroid Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG). RhAG is expressed only in erythrocytes and erythroid precursors (26) where it may function to extrude ammonia (21). RhBG and RhCG are nonerythroid, and are expressed in the kidney, liver, skin, and central nervous system, important sites of ammonia metabolism (27, 28, 41, 47, 57). In the kidney, RhBG and RhCG are found in distal nephron sites that mediate the majority of renal ammonia secretion (11, 41, 47, 54, 57). In the liver, RhBG is expressed by perivenous hepatocytes where it appears to mediate ammonia uptake (56, 57), and RhCG is expressed in bile duct epithelia where it may function to secrete ammonia into biliary fluid (56, 57). Thus specific, novel ammonia transporter family members appear to mediate important roles in mammalian ammonia metabolism in many ammonia transporting organs.
Because the gastrointestinal tract is a major site of ammonia transport, the present studies were designed to determine whether RhBG and RhCG might be expressed in the gastrointestinal tract, and, if so, in which cell populations within specific gastrointestinal segments. We used real-time RT-PCR to examine RhBG and RhCG mRNA expression, immunoblot assay to examine protein expression, and immunohistochemical studies to determine the site(s) of protein expression. Our results identify that RhBG and RhCG exhibit cell-specific, axially heterogeneous, and polarized expression in the intestinal tract suggesting they may function cooperatively to mediate enteric ammonia transport.
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MATERIALS AND METHODS
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Antibodies.
Anti-RhBG and anti-RhCG antibodies were rabbit polyclonal, anti-peptide antibodies generated in our laboratory, which we have characterized previously (47, 56, 57). In some experiments, antibodies were affinity-purified by using the immunizing peptide. Mouse monoclonal antibodies to H+-K+-ATPase (HK 12.18) were graciously provided by Dr. Adam Smolka, (Medical University of South Carolina, Charleston, SC).
RNA isolation.
Female C57/Bl6 mice weighing 1720 g were obtained from Harlan Sprague Dawley (Indianapolis, IN), and maintained on a normal mouse diet and ad libitum water intake until the day of study. At the time of study, mice were anesthetized with pentobarbital sodium (1030 mg/kg ip). Organs were flushed free of erythrocytes with in vivo cardiac perfusion with PBS (pH 7.4). They were then removed, frozen in liquid nitrogen, and stored at 70°C until used. Total RNA was extracted by using RNeasy Midikit (Qiagen, Valencia, CA) and stored at 70°C until used. Endogenous DNA was removed by treatment with RNase-free DNase (Qiagen).
Real-time RT-PCR.
Real-time RT-PCR amplification of RhBG and RhCG was performed as described in detail previously (56). Briefly, total RNA was reverse-transcribed by using SuperScript First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) and either random hexamer or oligo-dT primers. For real-time PCR amplification of RhBG, we used forward primer, 5'-GCCTGCAGAGTGTGTTTCCA-3', reverse primer, 5'-GAGCTGATACACGGCCTGAGA-3', and fluorescent probe, 6Fam-TGGCACTCCGCTGACCCTTGG-Tamra. For RhCG, the forward primer was 5'-GGATACCCCTTCTTGGACTCTTC-3', the reverse primer was 5'-TGCCTTGGAACATGGGAAAT-3', and the fluorescent probe was 6Fam-AGCCTCCGCCTGCTCCCCAAC-Tamra. Real-time RT-PCR was performed on an ABI Prism GeneAmp 5700 Sequence Detection System. We used a two-step cycle protocol including an initial 95°C denaturation step for 15 s and then 60°C for 1 min, and then 4050 cycles of alternating temperatures. 18S RNA was amplified in all experiments as an internal control using commercially available primers and probes (Applied Biosystems). All experiments included samples not treated with reverse transcription and samples to which RNA was not added as internal controls. All results were confirmed by repeating on specimens from at least three separate preparations.
Membrane protein preparation.
Female C57/Bl6 mice weighing 1720 g were obtained from Harlan Sprague Dawley and maintained on a normal mouse diet and ad libitum water intake until the day of study. On the day of study, mice were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Gastrointestinal tract tissues were rinsed by in vivo cardiac perfusion with PBS (pH 7.4); rapidly removed; divided into stomach, duodenum, jejunum, ileum, and colon; and stored frozen at 70°C until used. Tissues were homogenized in buffer A (in mM: 50 sucrose, 10 TRIS buffer, 1 EDTA, pH7.4) with a glass dounce and then diluted in buffer B (in mM: 250 sucrose, 10 Tris buffer, and 1 EDTA, pH 7.4) containing PMSF, leupeptin, aprotinin, and pepstatin. The sample was then centrifuged at 1,000 g for 5 min at 4°C. The pellet was resuspended in buffer B and again centrifuged at 1,000 g. The combined supernatants were then centrifuged at 21,000 g for 30 min at 4°C. The 21,000 g pellet was then resuspended in buffer B. An aliquot was obtained for protein determination using a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL), and the remainder was stored at 70°C until used.
Immunoblotting procedure.
Immunoblotting was performed as described in detail previously (47, 56). Briefly, 20 µg of membrane proteins were electrophoresed on 10% PAGE ReadyGel (Bio-Rad, Hercules, CA), transferred electrophoretically to nitrocellulose membranes, blocked with 5 g/dl nonfat dry milk, and incubated for 2 h with primary antibody diluted in Blotto buffer (in mM: 50 Tris, 150 NaCl, 5 Na2EDTA, and 0.05% Tween 20, pH 7.6) with 5 g/dl nonfat dry milk. After washing, membranes were exposed to secondary antibody (goat anti-rabbit IgG; Promega, Madison, WI) at a dilution of 1:5,000. Sites of antibody-antigen reaction were visualized with enhanced chemiluminescence (SuperSignal West Pico Substrate; Pierce, Rockford, IL) and a Kodak Image Station 440CF digital imaging system. All results were confirmed by repeating on specimens from at least three separate preparations.
Immunohistochemistry.
Female C57/Bl6 mice weighing 1720 g (n = 6) were anesthetized with pentobarbital sodium (1030 mg/kg ip). Tissues were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate-lysine-2% paraformaldehyde and then cut transversely into 2- to 4-mm thick slices and immersed overnight at 4°C in the same fixative. Samples of stomach, duodenum, jejunum, ileum, and colon were embedded in polyester wax (polyethylene glycol 400 distearate; Polysciences, Warrington, PA), and 5-µM thick sections were cut and mounted on gelatin-coated glass slides.
Immunohistochemical detection of RhBG and RhCG was performed by using standard immunoperoxidase procedures (Vectastain Elite kit; Vector Laboratories, Burlington, CA) as previously described (47, 57). Briefly, sections were dewaxed, rehydrated, and incubated with 0.6% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity. Antigen retrieval was performed by using Trilogy (Cell Marque; Hot Springs, Arkansas) according to the manufacturer's instructions for examination of the duodenum, jejunum, ileum, and colon. After blocking with 5% normal goat serum, sections were incubated with affinity-purified RhBG and RhCG antibodies overnight at 4°C. Sections incubated without primary antibody served as negative control. The sections were rinsed in PBS, incubated with the biotinylated secondary antibody against rabbit IgG for 30 min, and subsequently rinsed with Vector avidin-biotin complex reagent for 30 min. After being rinsed with PBS, the sections were incubated with the peroxidase substrate solution diaminobenzidine for 4 min. In some cases, tissues were counterstained with hematoxylin. Finally, sections were dehydrated, mounted with Permount (Fisher Scientific, Fairlawn, NJ) and examined on a Nikon E600 microscope equipped with differential interference contrast optics and photographed by using a DXM1200F digital camera and ACT-1 software (Nikon). All results were confirmed by repeating on specimens from at least three separate preparations.
Colocalization of RhBG and RhCG with H+-K+-ATPase.
Immunofluorescent localization and confocal laser scanning microscopy were used in some studies to colocalize RhBG and RhCG with H+-K+-ATPase using techniques previously described in detail (47, 56). Briefly, sections were dewaxed with graded ethanols, rehydrated, rinsed in PBS, and then treated for 30 min with 50 mM NH4Cl. They were then rinsed, blocked with 5% normal goat serum (Vector Laboratories) in PBS, and incubated at 4°C overnight with primary antibodies diluted with PBS. The sections were rinsed and incubated for 30 min with a fluorescently tagged species-specific secondary antibodies [FITC anti-mouse IgG, 1:50 dilution (Sigma), and Alexa Fluor 546 goat anti-rabbit IgG, 1:100 dilution]. Sections were then rinsed and mounted by using Fluoromount (Southern Biotechnology Associates, Birmingham, AL). We then visualized the tissue by using an Axiovert 100 M laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) with LSM 510 Software, version 2.8 (Carl Zeiss). All results were confirmed by repeating procedures on specimens from at least three separate preparations.
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RESULTS
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RhBG and RhCG mRNA expression.
The first set of studies examine whether the gastrointestinal tract expresses RhBG mRNA using real-time RT-PCR. Figure 1A shows representative results. RhBG mRNA expression was detected in the stomach, duodenum, jejunum, ileum, and colon. The slopes of amplification were similar in all tissues. No significant amplification was observed in samples that did not undergo reverse transcription or when sample RNA was omitted (data not shown).

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Fig. 1. Real-time RT-PCR amplification of Rh B glycoprotein (RhBG) and Rh C glycoprotein (RhCG) mRNA in intestinal tissues. A: RhBG RNA from gastric, duodenum, jejunum, ileum, and colon was reverse transcribed and amplified by using real-time RT-PCR with RhBG-specific primers and TaqMan probe. No amplification was seen in samples not treated with reverse transcription or to which sample RNA was not added (not shown). B: RhCG mRNA from gastric, duodenum, jejunum, ileum, and colon was amplified using RhCG-specific primers and TaqMan probe as described in MATERIALS AND METHODS (Real-time RT-PCR). No amplification was seen in samples not treated with reverse transcription or to which sample RNA was not added (not shown).
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Similar techniques were used to examine gastrointestinal tract RhCG mRNA expression. RhCG mRNA was amplified from the stomach, duodenum, jejunum, ileum, and colon. Figure 1B shows representative results. Again, the slopes of amplification were similar in all tissues. Samples not treated with reverse transcription or in which sample RNA was not added did not develop significant amplification. These studies therefore identify that mRNA for the Rh glycoprotein ammonia transporter family members RhBG and RhCG are present in the gastrointestinal tract.
RhBG protein expression.
We then examined the distribution of RhBG protein using RhBG-specific antibodies previously characterized (47, 56). Figure 2A demonstrates representative results in the stomach, and Fig. 2B shows representative results from duodenum, jejunum, ileum, and colon. An
50 kDa protein was present in each of the gastrointestinal tract tissues examined. The apparent molecular weight differed slightly in different organs, consistent with organ-specific variations reported previously (41, 56). Thus RhBG protein expression parallels RhBG mRNA expression in the gastrointestinal tract. RhBG protein was also detectable in whole cell lysates but at lower relative abundance (data not shown) consistent with RhBG being an integral membrane protein.

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Fig. 2. RhBG and RhCG protein expression in the gastrointestinal tract. A: RhBG protein expression in the stomach. The stomach and the kidney used as a "positive control" both express RhBG protein by immunoblot analysis. Immunoreactivity was blocked by preincubation of the antibody with the immunizing peptide ("+ Peptide"). B: RhBG protein expression in duodenum, jejunum, ileum, and colon. Membrane proteins from duodenum, jejunum, ileum, and colon were analyzed for RhBG protein expression using immunoblot analysis. Liver was used as a positive control. C: RhCG protein expression in the stomach. Both the stomach and the kidney express RhCG protein. Immunoreactivity was blocked by preincubation of the antibody with the immunizing peptide ("+ Peptide"). D: RhCG protein expression in the duodenum, jejunum, ileum, and colon. Immunoblot analysis of proteins from the duodenum, jejunum, ileum, and colon. Liver and kidney are used as positive controls.
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RhCG protein expression.
Similar to RhBG, RhCG protein expression paralleled RhCG mRNA expression. Immunoblot analysis of stomach, jejunum, ileum, and colon proteins using previously characterized anti-RhCG antibodies (47, 56) demonstrated expression of a
54 kDa protein in each of these organs. Figure 2C shows a representative immunoblot of gastric proteins and Fig. 2D shows representative results from the duodenum, jejunum, ileum, and colon. In some experiments, faint immunoreactivity at
64 kDa, was variably present, consistent with variations in glycosylation of the glycosylated RhCG in different tissues. Immunoreactivity was prevented when the antibody was preincubated with the immunizing peptide. RhCG protein was also detectable in whole cell lysates, but at lower relative abundance (data not shown), consistent with RhCG being an integral membrane protein. These results indicate that both mRNA and protein for RhBG and RhCG are widely expressed in the gastrointestinal tract.
Gastric RhBG immunolocalization.
We then examined RhBG cellular expression in the stomach. The pattern of RhBG immunoreactivity differed in the fundus, forestomach, and antrum. In the fundus, basolateral RhBG immunoreactivity was present in a subpopulation of cells in the deep portion of gastric glands. Cells with RhBG immunoreactivity appeared to have a cuboidal appearance, suggestive of zymogenic cell morphology. Cells lacking RhBG immunoreactivity were larger and more oval in shape suggesting they were parietal cells. Figure 3, A and B show representative micrographs. Immunoreactivity was blocked by preincubating the antibody with the immunizing peptide, and no immunoreactivity was observed when the primary antibody was omitted (Figure 4A).

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Fig. 3. RhBG immunolocalization in mouse stomach. Fixed sections were processed for immunohistochemistry using diaminobenzidine (brown) as described. A: cross-section of gastric fundus area showing positivity in deep-gland regions. B: higher power image of gastric fundus deep glands showing predominant basolateral expression with lighter, diffuse cytoplasmic staining. Large, pale cells are parietal cells. C and D: squamous mucosa was positive for RhBG throughout nucleated layers of the gastric forestomach. The section was counterstained with hematoxylin (blue). E: antral glands of the gastric antrum did not express identifiable RhBG immunoreactivity.
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Fig. 4. Immunoreactivity in the absence of primary antibody. In the absence of primary antibody, no significant immunoreactivity was observed in stomach (A), jejunum (B), or colon (C).
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Four main cell populations (zymogenic, parietal, mucous, and neuroendocrine cells) are present in gastric glands. RhBG immunoreactivity was not present in mucous cells, and the distribution of RhBG immunoreactivity was not consistent with expression in either progenitor cells, which are present only in the midregion of gastric glands or in neuroendocrine cells. The morphological appearance of RhBG positive cells is similar to that of the zymogenic cell, and, moreover, the presence of RhBG immunoreactivity only in deep gastric glands and not in midregions differs from the distribution of the parietal cell. To confirm this morphological appearance, we performed colocalization of RhBG with H+-K+-ATPase. Figures 5, AC shows representative results. Cells expressing RhBG immunoreactivity did not express H+-K+-ATPase immunoreactivity, and cells expressing H+-K+-ATPase immunoreactivity did not express RhBG immunoreactivity. Thus RhBG and H+-K+-ATPase are expressed in different cell populations, indicating that the gastric gland cells that express RhBG immunoreactivity are zymogenic cells.

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Fig. 5. Colocalization of RhBG and RhCG with H+-K+-ATPase using confocal laser scanning fluorescent microscopy. A: a subset of gastric fundic cells express basolateral RhBG immunoreactivity. B: same section labeled for H+-K+-ATPase immunoreactivity to identify parietal cells. C: merged image of A and B showing gastric fundic cells that express RhBG immunoreactivity (red) do not express H+-K+-ATPase immunoreactivity (green) and vice versa. D: a subset of gastric fundic cells express apical RhCG immunoreactivity. E: same section labeled for H+-K+-ATPase to identify parietal cells. F: merged image of D and E showing that gastric fundic cells that express RhCG immunoreactivity (red) do not express H+-K+-ATPase immunoreactivity (green) and vice versa.
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In the forestomach, RhBG immunoreactivity was observed in all three nucleated layers, the stratum germinativum, stratum spinosum, and stratum granulosum. Figure 3, C and D shows representative results. In the antrum, RhBG immunoreactivity was not observed. Figure 3E shows a representative micrograph.
Gastric RhCG immunoreactivity.
We then examined RhCG cellular expression in the stomach. In the fundus, RhCG immunoreactivity was present in a subset of cells in the deep portions of gastric glands. Similar to RhBG, RhCG-expressing cells had a cuboidal appearance, suggestive of zymogenic cell morphology. However, in contrast to the basolateral RhBG immunoreactivity, RhCG immunoreactivity was apical. In addition, surface epithelial cells expressed RhCG immunoreactivity. Figure 6, A and B shows representative immunohistochemical findings.

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Fig. 6. RhCG immunoreactivity in the mouse stomach. A: low-power cross section of the gastric fundus showing apical RhCG immunoreactivity in a subset of cells in deep-gland regions. B: apical RhCG immunoreactivity in a subset of cells of gastric fundus. Cells with apical immunoreactivity (arrows) had cuboidal appearance suggestive of zymogenic cell morphology. Cells lacking RhCG immunoreactivity (arrowhead) were larger and more oval or polygonal in shape suggesting that they were parietal cells. C and D: squamous mucosa region (gastric forestomach) has RhCG immunoreactivity only in the stratum germinativum. These sections are serial sections of the sections used in Fig. 3, C and D: section was counterstained with hematoxylin (blue). E: the gastric antrum of the gastric forestomach does not have identifiable RhCG immunoreactivity.
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As with RhBG, the localization of RhCG in deep gastric glands is consistent with zymogenic cell expression. To exclude expression in a subset of parietal cells, we colocalized RhCG with H+-K+-ATPase. Figure 5, DF shows representative results. RhCG-positive cells did not express detectable H+-K+-ATPase immunoreactivity, and H+-K+-ATPase-positive cells did not express detectable RhCG immunoreactivity. Thus parietal cells do not express RhCG, thereby identifying that fundic zymogenic cells express RhCG.
RhCG immunoreactivity was present in the forestomach, but its cellular distribution differed from that of RhBG. Stratum germinativum cells expressed RhCG immunoreactivity, with no detectable immunoreactivity in stratum spinosum, granulosum, or corneum. The difference of RhBG and RhCG immunoreactivity was confirmed in analysis of serial sections. Figure 6, C and D, which are serial sections of the micrographs shown in Fig. 3, C and D, demonstrates representative findings. Thus both RhBG and RhCG are expressed in squamous epithelia of the mouse forestomach but are expressed in different cell populations. Detectable RhCG immunoreactivity was not observed in the gastric antrum. Figure 6E shows representative results.
RhBG immunoreactivity in the duodenum, jejunum, ileum, and colon.
We then examined the expression of RhBG in the duodenum, jejunum, ileum, and colon using immunohistochemistry. Figures 7, A and B; 8, A and B; 9, A and B; and 10, A and B demonstrate representative findings in the duodenum, jejunum, ileum, and colon, respectively. In each of these intestinal regions, RhBG immunoreactivity was observed in villous cells but not in mucous cells or in crypt cells. Colonic RhBG immunoreactivity was less intense, and the basolateral pattern of immunoreactivity was less distinct than in other intestinal regions. RhBG immunoreactivity in the distal colon was slightly less intense than in the proximal colon. Faint immunoreactivity was variably observed in the lamina muscularis externa. No immunoreactivity was observed when the primary antibody was omitted or when the primary antibody was preincubated with the immunizing peptide (Fig. 4).

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Fig. 7. RhBG and RhCG immunoreactivity in the duodenum. A: low-power magnification of duodenum showing basolateral RhBG immunoreactivity in villous epithelial cells. B: high-power magnification of duodenum demonstrating basolateral RhBG immunoreactivity in villous epithelial cells but not in crypt cells or in mucous cells. C: low-power magnification of RhCG immunoreactivity in duodenum demonstrating predominantly apical immunoreactivity in villous epithelial cells. D: high-power magnification photomicrograph of RhCG immunoreactivity in duodenal villous epithelial cells demonstrating apical immunoreactivity.
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Fig. 8. RhBG and RhCG immunoreactivity in the jejunum. A: low-power magnification of jejunum showing basolateral RhBG immunoreactivity in villous epithelial cells. B: high-power magnification of jejunum demonstrating basolateral RhBG immunoreactivity in villous epithelial cells but not in crypt cells or in mucous cells. C: low-power magnification of RhCG immunoreactivity in jejunum demonstrating predominantly apical immunoreactivity in villous epithelial cells and in lamina muscularis externae (arrowhead). D: high-power magnification photomicrograph of RhCG immunoreactivity in villous epithelial cells demonstrating apical immunoreactivity (arrows) and immunoreactivity in lamina muscularis mucosae (arrowheads).
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Fig. 9. RhBG and RhCG immunoreactivity in the ileum. A: low-power magnification of ileum showing basolateral RhBG immunoreactivity in villous epithelial cells. B: high-power magnification of ileum demonstrating basolateral RhBG immunoreactivity in villous epithelial cells but not in crypt cells or in mucous cells. C: low-power magnification of RhCG immunoreactivity in ileum demonstrating predominantly apical immunoreactivity in villous epithelial cells and in lamina muscularis externae (arrowhead). D: high-power magnification photomicrograph of RhCG immunoreactivity in villous epithelial cells demonstrating apical immunoreactivity (arrow) and immunoreactivity in lamina muscularis mucosae (arrowhead).
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Fig. 10. RhBG and RhCG immunoreactivity in the colon. A: low-power magnification of proximal colon showing basolateral RhBG immunoreactivity in villous epithelial cells. B: high-power magnification of proximal colon demonstrating basolateral RhBG immunoreactivity in villous epithelial cells but not in crypt cells or in mucous cells. C: low-power magnification of RhCG immunoreactivity in proximal colon demonstrating predominantly apical immunoreactivity in villous epithelial cells and in lamina muscularis externae. D: high-power magnification photomicrograph of RhCG immunoreactivity in proximal colon villous epithelial cells demonstrating apical immunoreactivity and immunoreactivity in lamina muscularis mucosae.
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RhCG immunoreactivity in the gastrointestinal tract.
Similar studies were performed to examine RhCG immunoreactivity in the duodenum, jejunum, ileum, and colon. Figures 7, C and D; 8, C and D; 9, C and D; and 10, C and D demonstrate representative findings in the duodenum, jejunum, ileum, and colon, respectively. Similar to RhBG, RhCG immunoreactivity was present in villous cells, but not in mucous or crypt cells. In contrast to the basolateral pattern of RhBG immunoreactivity, RhCG immunoreactivity was apical. RhCG apical immunoreactivity appeared less intense in the colon than in the duodenum, jejunum, or ileum, consistent with the decreased expression in the colon by immunoblot. RhCG immunoreactivity in the distal colon was slightly less intense than in the proximal colon. We also observed RhCG immunoreactivity in the lamina muscularis mucosa and in the muscularis externae in the jejunum and ileum, and in skeletal muscle from the lateral gastrocnemius (data not shown). No immunoreactivity was observed when the primary antibody was omitted or when the primary antibody was preincubated with the immunizing peptide (Fig. 4).
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DISCUSSION
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These studies are the first to examine expression of the Rh glycoprotein mammalian ammonia transporter family members RhBG and RhCG in the gastrointestinal tract. Both RhBG and RhCG mRNA and protein are expressed in the stomach, duodenum, jejunum, ileum, and colon. In the stomach, RhBG and RhCG are expressed in zymogenic cells, whereas in the duodenum, jejunum, ileum, and colon, RhBG and RhCG are expressed in villous epithelial cells. Throughout the intestinal tract, RhBG and RhCG exhibit polarized expression, with basolateral RhBG and apical RhCG immunoreactivity.
Although not widely recognized, the stomach both secretes and absorbs ammonia (14, 44). Several characteristics suggest that specific proteins and not passive NH3 diffusion mediate gastric ammonia transport. First, direct measurements of gastric apical membranes show that diffusive NH3 permeability is low (48). Second, if diffusive NH3 transport were the only mechanism of gastric ammonia transport, then changes in transepithelial ammonia secretion should parallel changes in the NH3 gradient, which parallels the pH gradient. However, ammonia secretion parallels neither gastric pH or gastric H+ secretion. For example, individuals with pernicious anemia, who cannot acidify their gastric contents, have gastric fluid ammonia concentrations similar to normal individuals (12), and H2 receptor activation stimulates gastric ammonia secretion with a time course different from that observed for changes in luminal H+ concentration (12). The differing time course of H2-receptor stimulated ammonia and H+ secretion is consistent with the observation that the ammonia transporter family members RhBG and RhCG are expressed by the fundic zymogenic cell and not by the parietal cell. Finally, H2 receptor activation stimulates gastric ammonia secretion even in individuals with pernicious anemia who are unable to secrete H+ (12). Thus identification of the ammonia transporters RhBG and RhCG in the mouse stomach is both consistent with previous studies and provides a potential biological mechanism for gastric ammonia transport.
A second major finding in this study is that there are high levels of RhBG and RhCG expression in the small intestine. Ammonia metabolism in the small intestine is an important biological process. Quantitatively, 5070% of total intestinal ammonia absorption occurs in the small intestine (52, 53). The small intestine uses glutamine and other amino acids through the glutaminase pathway as the primary respiratory fuel, producing ammonia as a result of this metabolic pathway (31, 5153). Consistent with the high rate of small intestinal ammonia production is that glutaminase, the rate-limiting enzyme for small intestinal ammonia production, is expressed at higher levels in the small intestine than in other regions of the intestinal tract (22, 23). Finally, ammonia produced by small intestinal epithelial cells can be either secreted into the luminal fluid or absorbed into the portal circulation (39).
Small intestinal ammonia transport appears to involve specific transport processes. Specifically, small intestinal ammonia transport is energy-dependent, saturable, and exhibits Michaelis-Menton constant kinetics (34, 35), and recalculation of published data suggest that the Km for luminal ammonia absorption is
7 mM (35). Small intestinal ammonia transport may be mediated, at least in part, by the NH4+/H+ exchange activity present in small intestinal brush-border vesicles that has a Km for ammonia of
1 mM (32). This apical NH4+/H+ exchange activity is consistent with the observation that increasing luminal H+ concentration increases small intestinal ammonia secretion (34, 35, 45, 46). RhCG is a candidate protein to mediate this apical NH4+/H+ exchange activity. RhCG when expressed in the Xenopus oocyte mediates NH4+/H+ exchange activity, and has a similar Km (58); and renal epithelial cells that express apical RhCG exhibit an apical NH4+/H+ exchange activity with a Km for ammonia of
4 mM (55). Thus luminal acidification appears to stimulate apical ammonia secretion, at least in part, by stimulating luminal H+ for cytosolic NH4+ exchange that may be mediated by apical RhCG.
Although small intestinal basolateral ammonia transport mechanisms have not been functionally characterized, the observation that small intestine ammonia transport parallels transepithelial H+ gradients is consistent with the presence of both apical and basolateral NH4+/H+ exchangers. Moreover, RhBG mediates NH4+/H+ exchange activity when expressed in the Xenopus oocyte (29), and renal epithelial cells that express basolateral RhBG express a basolateral NH4+/H+ exchange activity (18). Thus basolateral RhBG is likely to contribute to ammonia transport into the intestinal villi, resulting in net absorption.
Colonic ammonia metabolism has both similarities to and differences from small intestinal ammonia metabolism. In the colon, luminal ammonia is produced predominantly from urea metabolism by enteric bacteria (50, 59, 60), with a minor contribution by enterocyte amino acid metabolism (53). Functional studies show that the colon absorbs luminal ammonia, and that the rate and direction of transport are related to the luminal pH (3, 7, 9, 15, 40). Although some studies (5, 9, 46) have suggested that colonic ammonia transport occurs through nonionic NH3 diffusion, changes in luminal pH, which exponentially alters luminal NH3 concentration (10pH), do not proportionally change the rate of ammonia transport. These observations are thus inconsistent with nonionic NH3 diffusion as the primary mechanism of colonic ammonia metabolism. Instead, they suggest that specific, saturable mechanisms mediate significant components of colonic ammonia transport. The present studies, by identifying the presence of apical RhCG and basolateral RhBG immunoreactivity in colonic enterocytes provides a mechanistic explanation for saturable, H+-gradient-stimulated colonic ammonia transport.
Although the present studies suggest that RhBG and RhCG function cooperatively to mediate intestinal ammonia absorption, these proteins may not be the only mechanisms of intestinal ammonia absorption. In hydrated solutions, NH4+ and K+ have nearly identical biophysical properties. Many proteins, including Na+/H+ exchanger, Na+-K+-ATPase, K+ channels, and Na+-K+-2Cl cotransporters can transport NH4+. Consistent with this mechanism is the observation that both furosemide and Ba+2 inhibit small intestinal ammonia absorption (16). Other studies (6) suggest that the colonic H+-K+-ATPase might contribute to colonic NH4+ absorption. Defining the specific roles of RhBG and RhCG compared with that of other transporters in intestinal ammonia transport will be an important area for future studies.
Both RhBG and RhCG are glycosylated proteins, presumably related to an N-linked glycosylation site on the first extracellular loop (11, 27, 28, 41). The apparent molecular weight of RhBG varies slightly in the kidney and liver (47, 56) and, as shown in the current manuscript, in the stomach, duodenum, jejunum, ileum, and colon. Moreover, the apparent molecular weight of RhBG can even vary in different regions of the same organ, as previously shown in the rat kidney (41). These differences may reflect differences in the glycosylation patterns of RhBG, which may reflect subtle differences in ion transport characteristics. In particular, alterations in glycosylation alters the ion transport characteristics of the yeast ammonia transporter family member Mep2 (30).
The polarized expression of RhBG and RhCG in gastrointestinal tract epithelial cells provides new insights into possible mechanisms of vectorial ammonia transport. For example, luminal acidification in the colon inhibits ammonia absorption (3, 15, 40) and is used clinically to induce net ammonia secretion (15, 42). The increased luminal proton concentration in this condition would tend to inhibit ammonia absorption via apical NH4+/H+ exchange, and could result in cytoplasm-to-lumen ammonia secretion. The resulting decrease in intracellular NH4+ concentration might then accelerate basolateral NH4+ uptake via basolateral NH4+/H+ exchange.
Previous studies (27, 28) using Northern blot analysis did not identify either RhBG or RhCG mRNA expression in either the small intestine or the colon. The explanation for the differing results in the present study is not clear, but may reflect the differing sensitivity of real-time RT-PCR and Northern blot analysis for detecting mRNA expression.
Recent studies have identified important new insights regarding the bacterial ammonia transporter family member, AmtB. Khademi et al, (24) using X-ray crystallographic analysis identified that AmtB exists as a trimer, with 11 membrane-spanning
-helixes that form a right-handed helical bundle around each channel. AmtB has structurally similar carboxy- and amino-terminal halves with opposite polarity that span the plasma membrane, consistent with an ability to mediate bidirectional transport. A critical finding, based on both crystallographic analysis and reconstitution studies, was that AmtB transports NH3, not NH4+. This appears to occur because the bacterial ammonia transporter family member AmtB has a narrow central hydrophobic pore element
20 µm long, which allows passage of NH3 but not of NH4+. An important avenue for future studies will be to determine both the tertiary structure of mammalian ammonia transporter family members and to determine whether they function as NH3 channels, NH4+/H+ exchangers or have other functional characteristics. It is important to recognize, however, that an NH3 channel and an NH4+/H+ exchanger mediate the same net transport.
In summary, the present study identifies that the ammonia transporter proteins RhBG and RhCG are expressed in the stomach, jejunum, ileum, and colon. Their localization and cellular expression suggests that they mediate important roles in intestinal ammonia transport and metabolism. Understanding the molecular mechanisms of intestinal ammonia transport by RhBG and RhCG in the intestine may lead in the future to novel therapies for conditions, such as hepatic and hyperammonemic encephalopathy, which result when intestinal ammonia absorption exceeds hepatic ammonia metabolism.
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GRANTS
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These studies were supported by funds from the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45788, National Institute of Neurological Disorders and Stroke Grant NS-47624, Department of Veterans Affairs Merit Review Program, and the Florida Affiliate of the American Heart Association.
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ACKNOWLEDGMENTS
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The authors thank Gina Cowsert for secretarial assistance.
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FOOTNOTES
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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 exists in aqueous solutions in an equilibrium between NH3 and NH4+. In this report, the term "ammonia" refers to the combination of these two molecular forms. The term "ammonium" specifically refers to the molecular species NH4+. When referring specifically to NH3, we specifically state "NH3." 
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REFERENCES
|
---|
- Bell JM and Feild AL. The distribution of ammonia between water and chloroform. J Am Chem Soc 33: 940943, 1911.[CrossRef]
- Bergeron MJ, Gagnon E, Wallendorff B, Lapointe JY, and Isenring P. Ammonium transport and pH regulation by K+-Cl cotransporters. Am J Physiol Renal Physiol 285: F68F78, 2003.[Abstract/Free Full Text]
- Bown RL, Sladen GE, Clark ML, and Dawson AM. The production and transport of ammonia in the human colon. Gut 12: 863, 1971.[ISI][Medline]
- Butterworth RF. Pathophysiology of hepatic encephalopathy: a new look at ammonia. Metab Brain Dis 17: 221227, 2002.[CrossRef][ISI][Medline]
- Castell DO and Moore EW. Ammonia absorption from the human colon. The role of nonionic diffusion. Gastroenterology 60: 3342, 1971.[ISI][Medline]
- Codina J, Pressley TA, and DuBose TD Jr. The colonic H+-K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon. J Biol Chem 274: 1969319698, 1999.[Abstract/Free Full Text]
- Cohen RM, Stephenson RL, and Feldman GM. Bicarbonate secretion modulates ammonium absorption in rat distal colon in vivo. Am J Physiol Renal Fluid Electrolyte Physiol 254: F657F667, 1988.[Abstract/Free Full Text]
- Dasarathy S and Mullen KD. Hepatic encephalopathy. Curr Treat Options Gastroenterol 4: 517526, 2001.[Medline]
- Down PF, Agostini L, Murison J, and Wrong OM. The interrelations of faecal ammonia, pH and bicarbonate: evidence of colonic absorption of ammonia by non-ionic diffusion. Clin Sci 43: 101114, 1972.[ISI][Medline]
- DuBose TD Jr, Good DW, Hamm LL, and Wall SM. Ammonium transport in the kidney: new physiological concepts and their clinical implications. J Am Soc Nephrol 1: 11931203, 1991.[Abstract]
- Eladari D, Cheval L, Quentin F, Bertrand O, Mouro I, Cherif-Zahar B, Cartron JP, Paillard M, Doucet A, and Chambrey R. Expression of RhCG, a new putative NH3/NH4+ transporter, along the rat nephron. J Am Soc Nephrol 13: 19992008, 2002.[Abstract/Free Full Text]
- Fleshler B and Gabuzda GJ. Effect of ammonium chloride and urea infusions on ammonium levels and acidity of gastric juice. Gut 6: 349356, 1965.[ISI][Medline]
- Flessner MF and Knepper MA. Ammonium and bicarbonate transport in isolated perfused rodent ascending limbs of the loop of Henle. Am J Physiol Renal Fluid Electrolyte Physiol 264: F837F844, 1993.[Abstract/Free Full Text]
- Gips CH, Que GS, and Wibbens-Alberts M. The arterial ammonia curve after oral and intraduodenal loading with ammonium acetate. Absorption in the stomach. Neth J Med 16: 1417, 1973.[Medline]
- Haemmerli UP and Bircher J. Wrong idea, good results (the lactulose story). N Engl J Med 281: 441442, 1969.[Medline]
- Hall MC, Koch MO, and McDougal WS. Mechanism of ammonium transport by intestinal segments following urinary diversion: evidence for ionized NH4+ transport via K+-pathways. J Urol 148: 453457, 1992.[ISI][Medline]
- Hamm LL and Simon EE. Roles and mechanisms of urinary buffer excretion. Am J Physiol Renal Fluid Electrolyte Physiol 253: F595F605, 1987.[Abstract/Free Full Text]
- Handlogten ME, Hong SP, Westhoff CM, and Weiner ID. Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol 287: F628F638, 2004.[Abstract/Free Full Text]
- Haussinger D, Lamers WH, and Moorman AF. Hepatocyte heterogeneity in the metabolism of amino acids and ammonia. Enzyme 46: 7293, 1992.[ISI][Medline]
- Heitman J and Agre P. A new face of the Rhesus antigen. Nat Genet 26: 258259, 2000.[CrossRef][ISI][Medline]
- Hemker MB, Cheroutre G, van Zwieten R, Maaskant-van Wijk PA, Roos D, Loos JA, van der Schoot CE, and dem Borne AE. The Rh complex exports ammonium from human red blood cells. Br J Haematol 122: 333340, 2003.[CrossRef][ISI][Medline]
- James LA, Lunn PG, and Elia M. Glutamine metabolism in the gastrointestinal tract of the rat assess by the relative activities of glutaminase (EC 3.512) and glutamine synthetase (EC 6312). Br J Nutr 79: 365372, 1998.[ISI][Medline]
- James LA, Lunn PG, Middleton S, and Elia M. Distribution of glutaminase and glutamine synthetase activities in the human gastrointestinal tract. Clin Sci (Lond) 94: 313319, 1998.[ISI][Medline]
- Khademi S, O'Connell J III, Remis J, Robles-Colmenares Y, Miercke LJ, and Stroud RM. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305: 15871594, 2004.[Abstract/Free Full Text]
- Knepper MA. NH4+ transport in the kidney. Kidney Int 40: S95S102, 1991.[ISI]
- Liu Z and Huang CH. The mouse Rhl1 and Rhag genes: sequence, organization, expression, and chromosomal mapping. Biochem Genet 37: 119138, 1999.[CrossRef][ISI][Medline]
- Liu Z, Chen Y, Mo R, Cc Hui Cheng JF, Mohandas N, and Huang CH. Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis. J Biol Chem 275: 2564125651, 2000.[Abstract/Free Full Text]
- Liu Z, Peng J, Mo R, Cc Hui, and Huang CH. Rh type B glycoprotein is a new member of the Rh superfamily and a putative ammonia transporter in mammals. J Biol Chem 276: 14241433, 2001.[Abstract/Free Full Text]
- Ludewig U. Electroneutral ammonium transport by basolateral Rhesus B glycoprotein. J Physiol 559: 751759, 2004.[Abstract/Free Full Text]
- Marini AM and Andre B. In vivo N-glycosylation of the mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus. Mol Microbiol 38: 552564, 2000.[CrossRef][ISI][Medline]
- McCauley R, Kong SE, Heel K, and Hall JC. The role of glutaminase in the small intestine. Int J Biochem Cell Biol 31: 405413, 1999.[CrossRef][ISI][Medline]
- McDougal WS, Stampfer DS, Kirley S, Bennett PM, and Lin CW. Intestinal ammonium transport by ammonium and hydrogen exchange. J Am Coll Surg 181: 241248, 1995.[Medline]
- Melton LB. Control and mechanism of transport of ammonia by the urinary bladder of the toad, Bufo marinus. (PhD thesis). Dallas, TX: University of Texas Graduate School of Biomedical Sciences, 1978.
- Mossberg SM. Ammonia absorption in hamster ileum: effect of pH and total CO2 on transport in everted sacs. Am J Physiol 213: 13271330, 1967.[Free Full Text]
- Mossberg SM and Ross G. Ammonia movement in the small intestine: preferential transport by the ileum. J Clin Invest 46: 490498, 1967.[ISI][Medline]
- Nakhoul NL and Hamm LL. Non-erythroid Rh glycoproteins: a putative new family of mammalian ammonium transporters. Pflügers Arch 447: 807812, 2004.[CrossRef][ISI][Medline]
- Occleshaw VJ. The distribution of ammonia between chloroform and water at 25°C. J Chem Soc 14361437, 1931.
- Olde Damink SW, Deutz NE, Dejong CH, Soeters PB, and Jalan R. Interorgan ammonia metabolism in liver failure. Neurochem Int 41: 177188, 2002.[CrossRef][ISI][Medline]
- Plauth M, Raible A, Vieillard-Baron D, Bauder-Gross D, and Hartmann F. Is glutamine essential for the maintenance of intestinal function? A study in the isolated perfused rat small intestine. Int J Colorectal Dis 14: 8694, 1999.[CrossRef][ISI][Medline]
- Price JB Jr, Sawada M, and Voorhees AB Jr. Clinical significance of intraluminal pH in intestinal ammonia transport. Am J Surg 119: 595598, 1970.[CrossRef][ISI][Medline]
- Quentin F, Eladari D, Cheval L, Lopez C, Goossens D, Colin Y, Cartron JP, Paillard M, and Chambrey R. RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron. J Am Soc Nephrol 14: 545554, 2003.[Abstract/Free Full Text]
- Riordan SM and Williams R. Treatment of hepatic encephalopathy. N Engl J Med 337: 473479, 1997.[Free Full Text]
- Suarez I, Bodega G, and Fernandez B. Glutamine synthetase in brain: effect of ammonia. Neurochem Int 41: 123142, 2002.[CrossRef][ISI][Medline]
- Summerskill WH, Aoyagi T, and Evans WB. Ammonia in the upper gastrointestinal tract of man: quantitations and relationships. Gut 7: 497501, 1966.[ISI][Medline]
- Swales JD, Papadimitriou M, and Wrong OM. Observations upon ammonia absorption from the human ileum. Gut 14: 697700, 1973.[ISI][Medline]
- Swales JD, Tange JD, and Wrong OM. The influence of pH, bicarbonate and hypertonicity on the absorption of ammonia from the rat intestine. Clin Sci 39: 769779, 1970.[Medline]
- Verlander JW, Miller RT, Frank AE, Royaux IE, Kim YH, and Weiner ID. Localization of the ammonium transporter proteins RhBG and RhCG in the mouse kidney. Am J Physiol Renal Physiol 284: F323F337, 2003.[Abstract/Free Full Text]
- Waisbren SJ, Geibel JP, Modlin IM, and Boron WF. Unusual permeability properties of gastric gland cells. Nature 368: 332335, 1994.[CrossRef][ISI][Medline]
- Wall SM. Ammonium transport and the role of the Na,K-ATPase. Miner Electrolyte Metab 22: 311317, 1996.[ISI][Medline]
- Walser M and Bodenlas L. Urea metabolism in man. J Clin Invest 53: 16171626, 1959.
- Weber FL Jr, Friedman DW, and Fresard KM. Ammonia production from intraluminal amino acids in canine jejunum. Am J Physiol Gastrointest Liver Physiol 254: G264G268, 1988.[Abstract/Free Full Text]
- Weber FL Jr and Veach GL. GI production of ammonia. Gastroenterology 77: 1166, 1979.[ISI][Medline]
- Weber FL Jr and Veach GL. The importance of the small intestine in gut ammonium production in the fasting dog. Gastroenterology 77: 235240, 1979.[ISI][Medline]
- Weiner ID. The Rh gene family and renal ammonium transport. Curr Opin Nephrol Hypertens 13: 533540, 2004.[ISI][Medline]
- Weiner ID and Handlogten ME. Apical ammonia transport by the mouse collecting duct, mIMCD-3, cell (Abstract). FASEB J 18: A1017, 2004.
- Weiner ID, Miller RT, and Verlander JW. Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein in the mouse liver. Gastroenterology 124: 14321440, 2003.[CrossRef][ISI][Medline]
- Weiner ID and Verlander JW. Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein. Acta Physiol Scand 179: 331338, 2003.[CrossRef][ISI][Medline]
- Westhoff CM, Weiner ID, and Foskett JK. Transport characteristics of the putative ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, when expressed in the Xenopus oocyte (Abstract). J Am Soc Nephrol 14: 304A, 2003.
- Wrong OM. Intestinal handling of urea and ammonia. Proc R Soc Med 64: 10251026, 1971.[ISI][Medline]
- Wrong OM and Vince A. Urea and ammonia metabolism in the human large intestine. Proc Nutr Soc 43: 7786, 1984.[ISI][Medline]