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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY
1Faculty of Life Sciences, Stopford Building, The University of Manchester, Manchester; 2School of Cellular and Molecular Biosciences, Division of Physiological Sciences, University Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, United Kingdom; and 3United States Meat Animal Research Center, United States Department of Agriculture/Animal Research Service, Clay Center, Nebraska
Submitted 23 February 2005 ; accepted in final form 12 April 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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facilitative transporter; phloretin; thionicotinamide; transepithelial flux; nitrogen balance
Although initially cloned from kidney (29), facilitative urea transporters have also been identified in several tissues, including brain (2), testis (5), and the gastrointestinal tract (11, 26). Urea transporters expressed in the gastrointestinal tract have been suggested to mediate urea flux into the intestinal lumen as part of the process of urea nitrogen salvaging (UNS) (26). Ruminant animals, such as cattle and sheep, subsist on a diet that is high in cellulose and low in protein nitrogen. As a result, there is a need to recycle nitrogen, and hence UNS is vital for maintaining nitrogen balance in ruminants. During UNS, 40–80% of the urea produced in the liver passes into the digestive tract and is broken down by resident bacteria into ammonia and carbon dioxide (15). The bacteria use ammonia to synthesize amino acids and peptides required for growth, which along with the ammonia can be reabsorbed by the ruminant host, thus completing the "salvaging" of urea nitrogen (7). The molecular basis of ruminal urea entry has to date not been resolved. Because ruminants account for a significant portion of biomass on Earth, understanding how they process nitrogen has widespread implications.
In this study, we determined the structure of the bovine UT-B (bUT-B) gene, identified and characterized bovine UT-B cDNAs, and showed that bUT-B protein is expressed in rumen epithelia. We conclude that UT-B is present in the bovine rumen and may participate in urea transport across rumen epithelia as part of the UNS process.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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12 million reads at the time of screening), was searched via BLASTN using the full insert sequence of bUT-B cDNA (GenBank accession no. AY624602). Trace files whose sequence showed highly significant matches to the cDNA (scores >300), as well as the mate-pair end sequences from the respective clones, were collected in a directory and used to construct initial genomic contigs via phred (3) and phrap (9) algorithims. Contig sequences were masked for repetitive elements using RepeatMasker (21), and they were used to search for overlapping trace files in the archive, which were added to the directory for reconstruction of contigs. The process was repeated until none of the contigs in the phrap output identified trace files not already in the directory. This resulted in construction of three contigs containing portions with exact matches to the exons of the bUT-B1 cDNA, leaving two gaps in the gene sequence. Primers were designed to span the gaps by PCR, and sequence was obtained by amplification of a bovine bacterial artificial chromosome (BAC) (clone 48L6) from the Children’s Hospital Oakland Research Institute (CHORI)-240 library (BACPAC Resources, Oakland, CA) and sequencing of the products with the amplification primers, nested primers, or both. The resulting 27,657-bp contig was edited by manual inspection using the consed viewing program (9), and areas of low sequence quality or areas where read overlap was exclusively from low-complexity sequence were targeted for finishing using additional PCR-based amplification and sequencing. The final genome sequence has been submitted to GenBank with accession number AY838799 and encompasses the entire cDNA sequence with appropriate splice boundaries and poly(A) addition signal. RT-PCR. Poly(A)+ RNA was obtained from bovine rumen (see below) and kidney using an oligo(dT)-cellulose batch method, and 1 µg was used to produce cDNA via RT (Superscript II, Invitrogen). PCR amplification with a Taq polymerase enzyme (Roche) was performed on cDNA from clone AY624602 [GenBank] (positive control), bovine rumen, and bovine kidney using bUT-B-specific primers. The forward primers were 1) primer 1 (5'-TGCCTAACATAACGAGTTC-3') designed against AY624602 [GenBank] , 37–55 bp, and 2) primer 3 (5'-AGGGCTACAACGCTACCCTGGTGG-3') designed against AY624602 [GenBank] , 424–444 bp; the reverse primers were 1) primer 2 (5'-ATAGTACAGTCTTAGTGCCA-3') designed against AY624602 [GenBank] , 1,470–1,489 bp, and 2) primer 4 (5'-GAAGATGCCCCCTGTCCACGG-3') designed against AY624602 [GenBank] , 774–794 bp. Cycling parameters were initial denaturation 94°C for 2 min, followed by 30 cycles at 94°C for 30 sec, 50°C for 30 s, and 72°C for 30 s. The final extension was at 72°C for 8 min. The products obtained from PCR of bovine rumen cDNA were investigated through direct sequencing (Lark Technologies).
Southern blot analysis. After electrophoresis of PCR products through a 1% agarose gel, the gel was denatured with 0.5 M sodium hydroxide and then neutralized with 1.5 M NaCl plus 0.5 M Tris·HCl. The PCR products were capillary-transferred to Hybond-N filters and then probed with 32P-labeled bovine UT-B1 at high stringency (final wash at 65°C in 0.1x SSC, 0.1% SDS).
Northern blot analysis. To investigate the distribution of bUT-B urea transporter transcripts, poly(A)+ RNA was isolated from bovine kidney, bovine rumen, and mouse kidney. Poly(A)+ RNA was obtained using an oligo(dT)-cellulose batch method (4). Poly(A)+ RNA (3 µg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to Hybond-N filters (Amersham Pharmacia Biotech). Filters were probed with a radioactive 32P-labeled probe consisting of full-length bUT-B1 cDNA (GenBank accession no. AY624602) or mouse UT (mUT)-A1 cDNA (GenBank accession no. AF366052). Hybridization was for 16 h at 42°C (50% formamide) and washing at 65°C (high stringency) in 0.1x SSC, 0.1% SDS. Autoradiographs were produced using Biomax MS Film (Kodak).
Xenopus oocyte expression experiments.
These experiments were performed as previously described (22). A plasmid containing the bovine UT-B1 clone (GenBank accession no. AY624602), obtained from Dr. T. Smith (US Meat Animal Research Center, USDA/ARS, Clay Center, NE), was linearized using NotI. Complementary RNA was prepared using the Sp6 mMessage mMachine RNA kit (Ambion). For bUT-B2, the 1,600-bp ruminal UT-B PCR product was subcloned into the TOPO2.1 vector (Invitrogen), cDNA obtained using the Qiagen Miniprep kit (Qiagen), and then linearized using HindIII. Complementary RNA was prepared using the T7 mMessage mMachine RNA kit (Ambion). Oocytes were injected with water, bUT-B1 cRNA (1 ng per oocyte), bUT-B2 cRNA (1 ng per oocyte), or mouse UT-B1 (mUT-B1) cRNA (1 ng per oocyte), and then they were incubated at 18°C for 3 days. [14C]urea uptake was then measured as previously described (22).
Antisera. To study the distribution of UT-B proteins in bovine rumen, we utilized the characterized polyclonal mouse UT-B antibody MUTB, previously used to detect the mUT-B facilitative urea transporter (26). The antiserum had been affinity purified using Affigel support columns (Bio-Rad) containing immobilized immunizing peptide. Antiserum MUTB was raised in rabbits to amino acids 1–19 (H2N-MEDSPTMVKVDRGENQILS-CONH2) of mouse UT-B (GenBank accession no. AJ420967). Characterized antisera (ML446, MQ2, and ML194), previously used to detect mUT-A facilitative urea transporters (26), were also used.
Immunoblotting.
Rumen samples were obtained from commercial slaughterhouses and were excised from the fore stomachs of cattle within 15–40 min of slaughter. Approximately 100 cm2 of rumen from the ventral sac, in the region 10 cm from the left longitudinal groove, were immediately washed and then transported in ice-cold organ transplantation preservation solution (140 mM sucrose, 42.3 mM Na2HPO4, 26.7 mM NaH2PO4; pH 7.4). The epithelial mucosae were then stripped from the muscle layers using arterial forceps scissors and scalpel, snap frozen in liquid nitrogen, and stored at –80°C. Male adult NMR1 mice were killed by cervical dislocation, and kidneys were removed immediately. Rumen and kidney samples were homogenized in ice-cold buffer with a handheld dounce homogenizer. The homogenization buffer (pH 7.6) contained 12 mM HEPES, 300 mM mannitol, and several peptidase inhibitors added immediately before use [1 µg/ml pepstatin, 2 µg/ml leupeptin, and 1 µg/ml phenylmethylsulfonyl fluoride (Sigma)]. Homogenates were initially centrifuged at 2,500 g for 15 min at 4°C. The resulting supernatant was centrifuged at 200,000 g for another 30 min at 4°C. These plasma membrane enriched pellets were retained and resuspended in homogenization buffer. Total protein concentrations were determined using a Bio-Rad Protein Assay Reagent Kit (Bio-Rad). 5x reducing Laemmli sample buffer (5% SDS, 25% glycerol, 0.32 M Tris, pH 6.8, bromophenol blue, 5% 
-mercaptoethanol) was added to protein samples in a ratio of 1:4, which were then heated at 60°C for 15 min. SDS-PAGE was performed on minigels of 10% polyacrylamide by loading 20 µg/lane of protein. Proteins were then transferred electrophoretically to nitrocellulose membranes (Gelman Sciences). After blocking with 5% nonfat dry milk in washing buffer (15 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.01% Tween 20) for 1 h, the membranes were probed with affinity-purified MUTB antiserum for 16 h at 4°C. Membranes were rinsed in washing buffer for 3 x 10 min., and then they were probed with goat anti-rabbit horseradish peroxidase-linked secondary antiserum (Dako) at 1:5,000 dilution in 5% nonfat milk in washing buffer for 1 h. After another 3 x 10-min rinse in washing buffer, detection of protein was performed using the enhanced chemiluminescence (ECL) Western Blotting Detection Reagents and ECL film (Amersham Pharmacia).
Immunocytochemistry. Rumen papillae were isolated directly from stripped rumen epithelial mucosae using scissors. Papillae were immersion fixed in –20°C methanol, acetone or ice-cold 3% paraformaldehyde in PBS for a minimum of 4 h or overnight. Papillae were then incubated for 24 h in 30% sucrose in PBS at 4°C overnight before embedding in OCT (Tissue-Tek, Miles Laboratories, Naperville, IL) and cryosectioning. Then, 5-µm sections were washed three times with PBS, permeabilized with 0.1% saponin in PBS for 20 min, and then blocked with 10% serum (species dependent on host animal used for secondary antibody production) in PBS. Sections were then incubated overnight at 4°C with primary antibody (typically diluted 1:50 in 10% serum-PBS), washed three times, and then incubated for 1 h at room temperature with an appropriate Alexa Fluor-conjugated secondary antibody (Molecular Probes). Typically, sections were counterstained with ethidium homodimer-1 (Molecular Probes) to highlight the nuclei. Immunofluorescence signals were detected using confocal laser scanning microscopy, and all immunocytochemistry was repeated for material obtained from at least three animals.
Ussing chamber experiments. Isolated bovine ruminal sheets were mounted in thermostated (37°C) Ussing chambers (1.76-cm2 exposed area) for measurement of transepithelial [14C]urea and [3H]mannitol fluxes in the absorptive (Jlumen to blood) and secretory (Jblood to lumen) directions. A modified Ringer solution was used (all mM): 80 NaCl, 25 NaHCO3, 40 Na acetate, 2.5 CaCl2, 3 MgSO4, 2.8 KH2PO4-K2HPO4 and glucose (10), with gassing by 95% O2-5% CO2 (pH 7.4, 37°C). Na acetate is present to partially mimic rumen anion composition and provide an alternative energy source (8). Mannitol and urea were added to give a total unlabeled concentration of 1 mM. The composition of the Ringer in the apical (lumen) and basal (blood) chambers (7-cm3 total volume) was identical. [14C]urea (0.1 µCi/ml) and [3H]mannitol (0.2 µCi/ml) were added to either the apical or basal chamber, and in each case an equivalent concentration of unlabeled substrate was present in the contralateral chamber. The tissues were equilibrated for 10 min, when 0.2-cm3 samples from both apical and basal chambers were taken. Two flux periods of 30 min with further sampling were then undertaken. Phloretin (0.1 M in DMSO) was added at 40 min to either apical or basal bathing solutions to give a final concentration of 1 mM. Fluxes were expressed as nanomoles per squared centimeter per hour.
Statistics.
For statistical analysis of oocyte expression experiments, one-way ANOVA was used. If the ANOVA indicated a difference, treatment comparison between groups with the Student-Newman-Keuls post hoc test was performed. Groups were deemed statistically significant if P 
0.05. Linear regression was performed by the method of least squares (Sigmaplot, SPSS).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1,600- and 900-bp ruminal products, generated by primer sets 1 and 2 and primer sets 1 and 4, respectively, showed that they corresponded to bUT-B2. In addition, a second, weaker signal 150 bp smaller was present with primer sets 1 and 4 (Fig. 3D) and corresponds to bUTB1. It therefore appears that although the predominant isoform in the rumen is bUT-B2, bUT-B1 is also present.
Northern blot analysis.
Using a full-length bUT-B1 cDNA probe (AY624602
[GenBank]
), high stringency northern analysis revealed a 3.5-kb mRNA signal in bovine kidney and a slightly larger 3.7-kb signal in bovine rumen (see Fig. 3E). These results indicate that bUT-B is expressed in both kidney and rumen, although the predominant ruminal transcript appears to be 0.2 kb longer, corresponding to the difference between bUT-B1 and bUT-B2. These results also suggest that the cDNA AY624602
[GenBank]
is lacking up to 0.5 kb of untranslated sequence. In contrast to these results, no signals were detected when the mouse UT-A probe AF366052
[GenBank]
was used (data not shown).
Xenopus oocyte expression. To test that bUT-B proteins were functional transporters, we expressed bUT-B1 and bUT-B2 cRNA in Xenopus oocytes. Expression of bUT-B1 cRNA induced a fourfold increase in urea transport compared with water-injected controls (P < 0.001, ANOVA, see Fig. 4A). This increase in urea transport was significantly inhibited by 500 µM phloretin (P < 0.01, ANOVA, see Fig. 4A). Expression of bUT-B2 cRNA induced a twofold increase in urea transport compared with water-injected controls (P < 0.01, ANOVA; see Fig. 4A), which was again significantly inhibited by 500 µM phloretin (P < 0.05, ANOVA). Thionicotinamide (2 mM), another known inhibitor of facilitative urea transporters (13), also inhibited the urea transport induced by bUT-B1 (P < 0.01, ANOVA, see Fig. 4B) to a similar degree as phloretin. Indeed, the results obtained for bUT-B1 were very similar to those obtained with mUT-B1 (see Fig. 4B). Therefore, both bUT-B1 and bUT-B2 cDNAs encode functional, phloretin-inhibitable urea transporters.
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0.05 mg/ml of the initial immunizing peptide abolished the 43-to 54-kDa signal in mouse kidney and markedly reduced the ruminal signal (see Fig. 5). The residual signal indicated that, whereas the majority of the 43- to 54-kDa UT-B smear corresponded to bUT-B, a minor component was due to a nonspecific, non-peptide-blockable signal. Differential centrifugation was used to separate plasma membranes and intracellular vesicles from cytosolic proteins. Using semiquantitative immuoblotting, the ruminal UT-B signal was found to be stronger in the 200,000 g pellet fraction than in the 200,000-g supernatant fraction, indicating that the UT-B protein was predominantly located in the plasma membranes and intracellular vesicles rather than the cytoplasm. Indeed, the majority of the supernatant signal was not associated with UT-B. Finally, in contrast to MUTB, the use of a battery of UT-A targeted antibodies (ML446, MQ2, ML194) detected no signals (data not shown).
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DISCUSSION |
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We determined the structure of the UT-B gene and discovered that it was very similar to the human UT-B gene. Comparative genomic analysis indicated that exon 2 had not been described previously and that it was present in the human UT-B gene. This raised the possibility that UT-B may exist as two isoforms in humans. Clearly, this needs further investigation, possibly making use of primer sets homologous to those that we have developed in the present study.
By performing BLASTn database searches we identified a 5' EST with homology to human UT-B. Direct sequencing of this cDNA and functional analysis using Xenopus oocytes confirmed that, characteristic of facilitative urea transporters, the encoded protein transported urea and was phloretin-inhibitable. Although a previous study by Ritzhaupt et al. (18) reported that a cDNA fragment amplified from sheep rumen cDNA was homologous to rat kidney UT-B, they did not report isolation of a full-length clone. Therefore, our study is the first to characterize a functional urea transporter from a ruminant animal. In the present study, we also discovered a splice variant of bUT-B, namely bUT-B2. bUT-B2 was predicted to be identical to bUT-B1, but due to the splicing-in of an in-frame ATG has an additional 55 NH2-terminal amino acids. This novel 55-amino acid sequence does not appear to contain any additional phosphorylation sites or any targeting motifs, so its precise function is as yet unclear. Importantly, however, expression of bUT-B2 cDNA in Xenopus oocytes confirmed that this novel isoform encoded a functional, phloretin-sensitive transporter. Previously, the human UT-B gene has been suggested to encode a 4.4- and a 2.0-kb UT-B transcripts that differ in length due to the use of alternative polyadenylation but that encode the same protein (16). Using Northern blot analysis, we showed the presence of a 3.7 kb mRNA UT-B transcript in the bovine rumen and a 3.5-kb UT-B transcript in the bovine kidney. The presence of additional sequence in the ruminal transcript compared with bUT-B cDNA was also confirmed by RT-PCR. We suggest that bUT-B1 is likely to be encoded by the 3.5-kb transcript and that it is the predominant renal UT-B isoform. Furthermore, the predominant ruminal UT-B transcript is 3.7 kb and is likely to encode UT-B2.
Immunolocalization studies provided an interesting insight into the possible role of UT-B in bovine rumen. bUT-B was present in the plasma membranes of cells in the stratum basale, spinosum, and granulosum, but it was not found in the outermost cell layer, the stratum corneum. Recently, a functional model of the rumen epithelium has been suggested on the basis of the expression of junctional markers, gap junctions and the Na+-K+ ATPase; it is likely that the epithelial permeability barrier (zonae occludentes) occurs at the level of the stratum granulosum, that the cells of the stratum granulosum, spinosum and basale form a functional syncitium interconnected by gap junctions, and that the Na+-K+ ATPase is concentrated in the stratum basale (10). The overall distribution of bUT-B in the plasma membrane of all three cell layers suggests that it will mediate flux of urea across the functional thickness of the rumen epithelium (stratum basale, spinosum, and granulosum); lack of expression of bUT-B in the stratum corneum correlates with an absence of occluding junctions in this cell layer, indicating that the stratum corneum may not restrict trans-ruminal urea flux. The stratum corneum is subject to mechanical abrasion by rumen contents, so that its primary role is that of mechanical protection.
Using the Ussing chamber method, functional analysis showed that transepithelial urea flux was bidirectional, as would be expected if transcellular urea transport were via facilitated diffusion through UT-B proteins. Importantly, the flux of urea was reduced by phloretin, implicating a UT-A or UT-B transporter in the movement of urea. Indeed, our findings agree with a previous report on sheep rumen epithelia (17), in which urea transport was reduced by phloretin and by thiourea, another known inhibitor of facilitative urea transporters (28). Our data suggest that ruminal urea transport occurs via UT-B proteins, because bovine UT-A proteins could not be detected by either Northern or Western blot analysis.
Metabolism of urea by resident bacteria plays an important role in maintaining nitrogen balance in ruminants (15). The rumen is a specialized organ anterior to the small intestine. The rumen is home to a plethora of commensal bacteria that facilitate digestion of a diet that is high in fiber. In addition, these bacteria metabolize urea, and in so doing they make urea nitrogen available for absorption by the host. In ruminants, 40–80% of the urea produced in the liver passes into the digestive tract and is broken down by resident bacterial into ammonia and carbon dioxide (15). Our results suggest that UT-B could play a role in this entry of urea into the ruminant digestive tract.
The action of bacterial urease in the rumen renders ruminal urea to low levels and, combined with the maintained high levels of urea in the blood, implies that the net urea concentration gradient would be expected to be from blood to rumen. Interestingly, because facilitative transporters only mediate net flux down a concentration gradient, the normal physiological situation would promote urea movement from blood into the rumen. In Ussing chambers in which isolated tissue is mounted, leak pathways are inevitably introduced most notably from edge damage. It is likely that in vivo, the magnitude of the diffusive mediated cellular pathway will exceed that of the paracellular pathway. Further work is required to investigate the nature of additional, phloretin-insensitive urea pathway(s) in bovine rumen and to define the direction of urea transport in vivo.
It should be noted that because there is continual loss of rumen contents to the omasum and abomasum, regeneration of rumen microflora is from a loosely adherent flora on rumen papillae. Thus delivery of urea across the rumen epithelium may play an essential role in maintaining rumen function. Potentially, UT-B is an important regulator of ruminal bacterial growth. It may provide a means for the host to regulate the flux of urea into the rumen and hence govern bacterial metabolism. Finally, the fact that both UT-A6 (24) and UT-B (11) have also now been detected in the human colon highlights the possible relevance of urea transporters to gastrointestinal function and the host-commensal microflora interaction in monogastric species.
In conclusion, we have determined the structure of the bUT-B gene, identified two bovine cDNAs encoding UT-B orthologs, and showed that they are expressed in bovine rumen. bUT-B is functionally similar to previously characterized UT-B transporters, and it is located on plasma membranes of cells throughout the rumen. As such, it is likely to be responsible for the phloretin-sensitive urea trans-epithelial flux found across bovine rumen tissue. bUT-B may play a role in mediating urea flux into the rumen as part of the UNS process.
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ACKNOWLEDGMENTS |
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Part of this work was presented in abstract form at the Focused Meeting of The Physiological Society at The University of Newcastle, Newcastle-upon-Tyne, United Kingdom, July 22–23, 2004. 559P.
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FOOTNOTES |
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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.
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