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J. Dairy Sci. 2008. 91:1570-1584. doi:10.3168/jds.2007-0763
© 2008 American Dairy Science Association ®
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Basal Expression of Nucleoside Transporter mRNA Differs Among Small Intestinal Epithelia of Beef Steers and Is Differentially Altered by Ruminal or Abomasal Infusion of Starch Hydrolysate1,2

S. F. Liao, M. J. Alman, E. S. Vanzant, E. D. Miles, D. L. Harmon, K. R. McLeod, J. A. Boling and J. C. Matthews3

Department of Animal and Food Sciences, University of Kentucky, Lexington 40546

3 Corresponding author: jmatthew{at}uky.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
In ruminants, microbial-derived nucleic acids are a major source of N and are absorbed as nucleosides by small intestinal epithelia. Although the biochemical activities of 2 nucleoside transport systems have been described for cattle, little is known regarding the regulation of their gene expression. This study was conducted to test 2 hypotheses: (1) the small intestinal epithelia of beef cattle differentially express mRNA for 3 concentrative (CNT1, 2, 3) and 2 equilibrative (ENT1, 2) nucleoside transporters (NT), and (2) expression of these NT is responsive to small intestine luminal supply of rumen-derived microbes (hence, nucleosides), energy (cornstarch hydrolysate, SH), or both. Eighteen ruminally and abomasally catheterized Angus steers (260 ± 17 kg of BW) were fed an alfalfa cube-based diet at 1.33x NEm requirement. Six steers in each of 3 periods were blocked by BW (heavy vs. light). Within each block, 3 steers were randomly assigned to 3 treatments (n = 6): ruminal and abomasal water infusion (control), ruminal SH infusion/abomasal water infusion, or ruminal water infusion/abomasal SH infusion. The dosage of SH infusion amounted to 20% of ME intake. After a 14-or 16-d infusion period, steers were slaughtered, and duodenal, jejunal, and ileal epithelia were harvested for total RNA extraction and the relative amounts of mRNA expressed were determined using real-time RT-PCR quantification methodologies. All 5 NT mRNA were found expressed by each epithelium, but their abundance differed among epithelia. Specifically, jejunal expression of all 5 NT mRNA was higher than that by the ileum, whereas jejunal expression of CNT1, CNT3, and ENT1 mRNA was higher, or tended to be higher, than duodenal expression. Duodenal expression of CNT2, CNT3, and ENT2 mRNA was higher than ileal expression. With regard to SH infusion treatments, ruminal infusion increased duodenal expression of CNT3 (67%), ENT1 (51%), and ENT2 (39%) mRNA and ileal expression of CNT3 (210%) and ENT2 (65%) mRNA. Abomasal infusion increased (54%) ileal expression of ENT2 mRNA and tended to increase (50%) jejunal ENT2 mRNA expression. This study has uniquely characterized the pattern of NT mRNA expression by growing beef cattle and found that the mRNA abundance for CNT3, ENT1, and ENT2 in small intestinal epithelia can be increased by increasing the luminal supply of nucleotides (CNT3, ENT1, ENT2) or glucose (ENT2).

Key Words: bovine • regulated gene expression • SLC28 • SLC29


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
An adequate cellular supply of nucleosides is critical for many biological processes during animal development and growth, including DNA and RNA syntheses, energy (ATP) production, N and P recycling, cell signaling, and modulation of gene expression (Carver and Walker, 1995; Sánchez-Pozo and Gil, 2002). In the small intestine, which has a large epithelial tissue mass that proliferates very rapidly, the capacity of enterocytes for de novo synthesis of nucleosides is limited (Savaiano and Clifford, 1981; Tsujinaka et al., 1999). Therefore, enterocytes rely on the absorption of nucleosides from the intestinal lumen or the salvage of nucleosides from the extracellular basolateral fluid to meet their metabolic demands (Cosgrove, 1998).

In polarized epithelial cells, the passage of nucleosides across apical and basolateral membranes is mediated by 3 concentrative (CNT) and 4 equilibrative (ENT) nucleoside transporters (NT; Elwi et al., 2006). Although it is believed that NT expression is sensitive to the substrate availability in the small intestinal lumen, data documenting the sensitivity of NT mRNA expression by small intestinal epithelia to increased nucleoside challenge are limited for any species (Casado et al., 2002). Also lacking is experimentation that delineates the effects of increased nucleoside supply from increased nutritional status (increased ruminal energy supply) on epithelial expression of NT mRNA.

In comparison to nonruminants, the small intestinal epithelium of ruminants is exposed to especially large amounts of nucleosides, which result from the digestion of microbial RNA and DNA. Although 2 CNT-like activities have been identified in the brush border membranes of small intestinal epithelia of preruminant and ruminating dairy cattle (Scharrer and Grenacher, 2001; Theisinger et al., 2002), the expression profiles of bovine NT mRNA are unknown. However, the supplementation of high-quality forage diets with energy sources (such as cornstarch) can increase N utilization by rumen microbes (Bach et al., 1999) and, hence, the delivery of microbial nucleosides to the small intestine. Also, the absorption of nucleosides is an energy-demanding event and the expression of NT may be limited by the energy status of cells (Elwi et al., 2006). Therefore, understanding the sensitivity of NT mRNA expression by cattle to the increased nucleoside (NT substrate) load by increasing ruminal starch vs. the increased small intestinal luminal energy supply (without increased substrate supply) is important to optimize N retention in commercially relevant nutritional regimens by matching nutrient load to absorption capacity. Accordingly, the objectives of this study were to (1) determine which NT mRNA are expressed by the small intestinal epithelium of beef cattle and characterize their relative distribution patterns and (2) evaluate the effect of ruminal (increased nucleoside load) vs. abomasal (increased energy status) starch infusion on NT mRNA expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Animal Trial Procedures
Research protocols were approved by the University of Kentucky Institutional Animal Care and Use Committee. Eighteen Angus steers (BW = 260 ± 17 kg) were raised in the University of Kentucky Agricultural Research Center Beef Unit during February to August 2006. Because of facility and technical constraints, the animals were obtained in 3 staggered periods to minimize differences in BW at the initiation of the animal trial. For each of the 3 periods, a randomized complete block experimental design was used. Six steers in each period were blocked by BW (heavy vs. light). Within each of 2 blocks, 3 steers surgically fitted with ruminal and abomasal infusion catheters (Walker and Harmon, 1995) were randomly assigned to 3 infusion treatments: (1) ruminal and abomasal infusion with water (control); (2) ruminal infusion with cornstarch hydrolysate (SH) and abomasal infusion with water; or (3) ruminal infusion with water and abomasal infusion with SH. The dosage of SH infusion amounted to 20% of ME intake (approximately 800 ± g/d). The equalized dosage (20% of ME intake) shared by ruminal and abomasal infusion was based on an assumption that essentially all of the ruminally infused SH would be fermented in the rumen (Walker and Harmon, 1995).

The basal diet fed to all steers was "blended" high-quality alfalfa-hay cubes, which contained 17.8% crude protein and 1.31 Mcal of NEM/kg (DM basis) and was provided to steers at 1.33x maintenance energy requirement calculated as 0.077 Mcal of NEM/EBW0.75 (NRC, 1996). The DM content of the alfalfa-hay cubes was 88.1%, and the OM, NDF, ADF, and ADL contents were 90.6, 44.4, 28.1, and 7.8% (DM basis), respectively. A trace mineralized salt (92 to 96% NaCl), which contained (ppm) Zn (5,500), Mn (4,790), Cu (1,835), Fe (9,275), I (115), Co (65), and Se (18), was provided to steers at a dose of 40 g/d. Steers also received 20 g/d of poloxolene (Phibro Animal Health, Ridgefield Park, NJ) to minimize incidence of bloat. Steers were fed daily in 12 equally proportioned meals (once every 2 h) using automatic feeders (Ankom Co., Fairport, NJ), and had ad libitum access to fresh water throughout the trial. Steers were tethered individually in stalls (1.2 x 1.7 m) and housed in an environmentally controlled room (ambient temperature 20°C) with a 24-h light time.

The SH infusate used was a tap water solution of raw cornstarch, partially hydrolyzed by a heat-stable {alpha}-amylase (Bauer et al., 1995). The SH was chosen over raw cornstarch because the digestion characteristics of SH are similar to those of native starch yet is more suspendable in solution and, therefore, facilitates pumping. Stock SH infusate solutions were prepared in 3 to 4 batches for each experimental period and stored at –20°C until use. Before infusion, stock solutions were diluted with tap water to a final weight of 5.5 kg for each animal and infused over 22 h per day. During infusion, the homogeneity of the SH infusate was maintained by rapid continuous mixing of the solution on 6 individual stir-plates. The SH solution or water (5.5 kg) was continuously infused at a rate of 250 mL/h to the animals to help maintain a steady-state SH supply condition for at least 14 d prior to tissue sample collection.

Throughout the course of experimentation, 2 steers were lost from the ruminal SH infusion group. One was due to the factors unrelated to the infusion treatments, and the other one gradually stopped eating during the late phase of the experimental period.

Animal Slaughter and Tissue Collection
After 14- or 16-d SH infusion, steers (3 at a time, by block) were transported to a UDSA-approved slaughter facility per day for slaughter and tissue harvesting. Steers were killed by stunning with a captive-bolt pistol, followed by exsanguination to allow carcass recovery for human consumption. The small intestine was removed, and its total length (from pyloric valve to ileal-cecal junction) was determined by looping the intestine across a wet stationary board that was fitted with metal pegs at 2-m increments. Detailed procedures of animal slaughter and visceral organ collection were previously described (McLeod et al., 2007; Liao et al., 2008).

The sites and protocol for collection of small intestinal epithelial samples for total RNA preparation has been described previously (Howell et al., 2001, 2003). Briefly, 1-m sections of duodenum (0.5 to 1.5 m distal to the pyloric junction), jejunum (middle of the first half non-duodenal small intestine), and ileum (middle of the second half nonduodenal small intestine) were taken after removing the digesta. One-half of each intestinal section was scraped to collect the epithelia (Howell et al., 2001) and the other half snap-frozen in liquid nitrogen for reserve. Each section was cut in half, inverted, and rinsed with ice-cold (4°C) physiological saline (0.9% NaCl), and epithelia scraped off with a glass slide. Approximately 2 g of scraped epithelium were placed into 20 mL of TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA), homogenized immediately, and stored at – 80°C.

RNA Extraction and Reverse Transcription
RNA Extraction and Purification.
The crude RNA was extracted from the frozen epithelial homogenate following the instructions from Invitrogen Corporation for the TRIzol Reagent. After the crude RNA was recovered, a purification procedure was performed using RNeasy Mini Kit (Qiagen, Valencia, CA) to minimize genomic DNA contamination (Applied Biosystems, 2004) and enrich all the mRNA longer than 200 nucleotides in molecular size. Purified RNA was then eluted with 60 µL of RNase-free distilled H2O and stored at –80°C. The integrity of the purified RNA was examined by gel electrophoresis using Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA) at the University of Kentucky Microarray Core Facility. Visualization of the gel images and electropherograms showed that all RNA samples had high quality with RNA integrity number greater than 8.0 and 28S/18S rRNA ratio greater than 1.8. The purity and concentration of the purified RNA samples was analyzed by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), which revealed that all the samples were of high purity with 260/280 absorbance ratios greater than 2.0 and 260/230 absorbance ratios greater than 1.75.

Reverse Transcription (RT).
Approximately 3 µg of crude RNA was first treated with DNase I enzyme (amplification grade) in accordance with the manufacturer’s instructions (Invitrogen). Briefly, one RNA sample was combined with 1 µL of 10x reaction buffer, 1 µL of DNase I (1 U/µL), and DEPC-treated H2O up to 10 µL, incubated at room temperature for 15 min, and then 1 µL of 25 mM EDTA was added to stop the reaction by incubating at 65°C for 10 min. Then the DNase-treated RNA samples were reverse transcribed to cDNA by using SuperScript III First-Strand Synthesis System in accordance with the manufacturer’s instructions (Invitrogen). Briefly, a solution of hexamers (50 ng/µL) and oligo (dT)20 primer (50 µM) mix (1 µL each) was added to one DNase-treated sample (7 µL in volume), incubated at 70°C for 10 min, and then chilled on ice for 1 min. A solution containing 2 µL of RT buffer (10x), 2µL of dithiothreitol (0.1 M), 4 µL of MgCl2 (25 mM), 1 µL of dNTP (10 mM each), and 1 µL of RNAse Out was then added to the reaction. After incubation at 37°C for 2 min, the reaction was incubated with 1 µL reverse transcription at room temperature for 10 min, and then incubated at 50°C for 50 min. To stop the reaction, the reaction mixture was incubated at 70°C for 10 min and then chilled on ice. The resulting reaction products, cDNA, were stored at –20°C until used in real-time PCR.

Polymerase Chain Reactions
Real-Time PCR.
Before conducting real-time PCR with ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), primer and probe sets for CNT1, CNT2, CNT3, ENT1, and ENT2 cDNA were designed and manufactured using ABI Assays-by-Design Service (Applied Biosystems). Because no validated bovine NT mRNA were reported in the literature at the initiation of this project, their respective virtual sequences acquired from the Institute for Genomic Research, Rockville, MD (TIGR) and MSU Center for Animal Functional Genomics and National Bovine Functional Genomics Consortium, Michigan State University, East Lansing (NBFGC) public genomic databases were used as templates for primer and probe design (Table 1Go). The bovine 18S rRNA gene sequence was retrieved from GenBank (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD) nucleotide database with an accession number of DQ222453. Each Assays-by-Design primer and probe set consists of 2 unlabeled PCR primers and one TaqMan Minor Groove Binding probe with FAM, a reporter dye labeled at 5' end (Table 1Go). To reduce the noise from genomic DNA contamination, all the primer and probe sets were designed to produce amplicons bridging exon-exon junctions (Cui et al., 2007).


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  		Table 1. Primer and probe sets used for the real-time quantitative PCR analyses of mRNA-derived cDNA for nucleoside transporters and 18S rRNA
 
Components of a 25-µL real-time PCR reaction were an Assays-by-Design Primer and Probe set (1.25 µL), TaqMan Universal PCR Master Mix-No AmpErase UNG (12.5 µL), cDNA template (1.0 to 2.0 µL), and DNase/RNase free H2O (9.25 to 10.25 µL). The PCR conditions used for the amplification and quantification were an initial denaturing stage (95°C for 10 min), followed by 40 cycles of 2 amplification stages for denaturing (95°C for 15 s) and annealing/extension (60°C for 1 min), with a melting curve program (60 to 95°C), a heating rate of 0.15°C/s, and continuous fluorescence measurements.

End-Point PCR.
Because of the inherent limitation of the DNA sequencing-by-synthesis technology, about 15 to 20 bp near the primer site can not be detected after gel electrophoresis (Sambrook et al., 1989). Therefore, a short (<65 bp) real-time PCR product can not be fully sequenced, even with 2 primers from both ends. To generate a longer partial length CNT1 cDNA fragment for validation of the real-time PCR product (59 bp), an end-point PCR procedure was used. Forward and reverse primers were designed based on a virtual bovine CNT1 clone, NBFGC_BE480932, obtained from NBFGC genomic database. The designed forward and reverse primer sequences (not included in Table 1Go) were: 5'-AAGCCCTTCGGAAGTTGG-3' and 5'-GCCAACGAACACGCAGAT-3', respectively. One jejunal and one ileal RT products were used individually as cDNA amplification templates. The PCR reactions were performed with a Hybaid MultiBlock PCR System (Thermo Electron Corporation, Waltham, MA). Each 50-µL reaction mixture contained 4.0 µL of RT product (>200 ng), 0.8 µM each of forward and reverse primers, 1.5 units of Platinum Taq DNA Polymerase, 2.0 mM MgCl2, and 0.2 mM each dNTP (Invitrogen). The thermal cycle program for the PCR consisted of 1 cycle at 94°C (10 min), 35 cycles at [94°C (0.5 min), 54 to 63°C (0.5 min) and 72°C (1.0 min)], and 1 cycle at 72°C (7.5 min).

Development of mRNA Quantification Methodology
Sequence Validation of PCR Products.
To establish mRNA relative quantification methodology, both the real-time PCR and end-point PCR products were validated by DNA sequence verification. To prepare PCR products for sequence verification, a PureLink Quick Gel Extraction Kit (Invitrogen) was used to purify the PCR products from the reaction mixtures, which include unincorporated nucleotides, primers, and DNA polymerase. The real-time PCR mixture also includes FAM dye that was labeled to TaqMan probe by the manufacturer (Applied Biosystems). Approximately 250 µL of pooled PCR reaction mixtures were electrophoresed in a 1.2% agarose slab gel. A single cDNA band at the desired size was identified under a UV light, excised of the gel, placed into a sterile, 1.5-mL polypropylene centrifuge tube, dissolved with the gel solubilization buffer, and then filtered through an extraction column. The column-bound cDNA was cleaned with the washing buffer, eluted with 25 to 40 µL of DNase/RNase free H2O, and concentration was determined using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The cDNA were then sequenced using the flourescent dideoxy-mediated chain termination method and a Perkin Elmer/Applied Bio-systems automated DNA Sequencer by the University of Florida DNA Sequencing Core Laboratory (Gainesville, FL). The resulting sequences were compared with the template sequences retrieved from TIGR, NBFGC, and GenBank genomic/nucleotide databases to confirm their identities.

Relative mRNA Quantification Methods.
For relative quantification of NT mRNA expression levels, real-time quantitative RT-PCR methodology that used 2-step regimen was developed in accordance with ABI guidelines (Applied Biosystems, 2004). In the first step, all the RNA samples were reverse-transcribed to cDNA as described above for RT reaction. In the second, real-time PCR step, 5 relative standard curve methods were established for 5 NT cDNA respectively, and the 18S cDNA (reverse-transcribed from 18S rRNA) was selected as an endogenous control to normalize the variations in sample preparation, mRNA inputs, and RT efficiencies (Liao et al., 2008). Specifically, a cDNA sample was serially diluted 2.5x, 5x, 25x, 125x, 625x, 3,125x, 15,625x, 78,125x, and 390,625x, and the linear range for target mRNA quantification was established to ascertain an appropriate amount of cDNA to be used for a standard curve method. For each cDNA sample the real-time PCR reactions (as described above) were conducted in triplicate to average out the potential pipetting, mixing, or plate setting-up errors. The minimal threshold (CT) values detected using these dilutions were around 35 and 27 for the target and 18S cDNA, respectively. As a result, the optimal detection of NT and 18S cDNA were achieved by using 1:5 and 1:15,625 dilutions of the RT product stocks, respectively.

The potential tissue distribution and SH infusion treatment effects on the expression of 18S rRNA by small intestinal epithelia were evaluated by comparing the CT values obtained from the real-time PCR reactions (Applied Biosystems, 2004). The relative quantities of NT mRNA expression were normalized to the relative 18S quantities by calculating the NT:18S relative quantity ratios, and these 18S-normalized quantity ratios were used for NT tissue distribution pattern analysis (i.e., the expression levels associated with 3 intestinal sections). For SH infusion treatment effect on NT mRNA expression, the 18S-normalized ratio from the control animals (i.e., non-SH infusion) was designated as a calibrator. Then the 18S-normalized ratios for the ruminal, or abomasal infusion animals, as well as the control animals, were divided by the ratio of the calibrator, respectively. The CT values for 18S rRNA quantities, the normalized values for NT tissue distribution pattern analysis, and the calibrated values for SH infusion treatment effect were all subjected to statistical analysis.

Statistical Analysis
The effects of 3 small intestinal sections (duodenum, jejunum, and ileum), and the effects of SH infusion (control, ruminal, or abomasal) treatment, on the expression of 18S rRNA and NT mRNA were analyzed by a split-plot design ANOVA for a randomized complete block design using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). The SH infusion treatment effects were tested in the main plot with individual steers as experimental units, and the tissue distribution effects were tested in the subplot with individual intestinal sections as experimental units. Sums of squares were partitioned to period, block nested in period, treatment, intestinal section, treatment x section interaction, and the residual error. Random effects were specified for 2 model terms, period, and block nested in period. The treatment x intestinal section interaction was used as the error term for the main plot, and the residual error was used as the error term for the subplot. When the interaction of treatment x section was not significant (P > 0.10), the least square means associated with infusion treatment were separated by Fisher’s protected LSD (P ≤ 0.10), and the least square means associated with intestinal section were similarly separated within each infusion treatment. When the treatment x section interaction was significant (P ≤ 0.10), the least square means associated with infusion treatment were separated by Fisher’s protected LSD (P ≤ 0.10) within each intestinal section. The probability levels of P ≤ 0.10 and 0.10 < P ≤ 0.20 were defined as significant differences and tendencies toward differences, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Development of mRNA Quantification Methodology
Generation of a Partial-Length CNT1 cDNA.
Because the CNT1 real-time PCR product was too short (56 bp) to be fully sequenced using the fluorescent dideoxy-mediated chain termination method, a longer cDNA fragment (448 bp) for CNT1 was generated from one jejunal and one ileal cDNA samples of the control steers using end-point PCR method. The resulting sequence was designated as UK-bCNT1, and the alignment of UK-bCNT1 with a reported virtual sequence for CNT1 cDNA revealed that UK-bCNT1 shares 99.6% (446/448) nucleotide identity with the corresponding region of NBFGC_BE480932 (Figure 1Go). The sequence of UK-bCNT1 now resides in GenBank (accession number EF446671).


Figure 1
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  		Figure 1. Alignment of UK-bCNT1 sequence (UKseq; GenBank accession number EF446671) with NBFGC_BE480932 sequence (NBFGC). The alignment was performed by using BLASTN computer program (version 2.2.12, at http://www.ncbi.nlm.nih.gov/BLAST). The : symbol indicates identical base pairs between the 2 sequences, whereas spaces indicate heterogeneity of gene expression. The percentage of nucleotide identities between these 2 sequences is at least 99.6% (446/448). The positions of forward (bp 1 to 18) and reverse (bp 431 to 448) primers are underlined, and the position of the real-time PCR product (bp 39 to 94) is highlighted.

 
Validation of Real-Time RT-PCR Products.
Sequence analyses validated all the real-time RT-PCR products generated in this study. Specifically, the sequences of the CNT1, CNT2, CNT3, ENT1, and ENT2 products have 98.2% (55/56), 100% (92/92), 100% (83/ 83), 97.3% (72/74), and 100% (73/73) identities, respectively, with their corresponding expected sequences that were acquired from their virtual template sequences reported in the genomic databases (i.e., NBFGC and TIGR; Table 1Go and Figure 2Go). The sequences of these real-time PCR products have resided in GenBank with accession numbers of EF446671, EF469827, EF469828, EF469829, and EF469830, respectively. The sequence of 18S real-time PCR product was validated previously in this laboratory and resides in GenBank with an accession number of EF469831 (Liao et al., 2008).


Figure 2
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  		Figure 2. Comparison of the real-time RT-PCR products (Product) sequences to their respective template sequences retrieved from NBFGC, TIGR, and GenBank databases. The : symbol indicates identical base pairs between the corresponding sequences, whereas spaces indicate heterogeneity of gene expression. The underlines indicate the positions of forward and reverse primers, and the highlights mark the probe positions. These product sequences now reside in GenBank with accession numbers EF446671 for CNT1, EF469827 for CNT2, EF469828 for CNT3, EF469829 for ENT1, EF469830 for ENT2, and EF469831 for 18S.

 
Validation of 18S as an Endogenous Expression Control Gene.
Using the relative mRNA quantification methodology to compare the treatment effects on the steady-state expression (mRNA abundance) of a given gene depends on the identification of an appropriate quantity control gene, which can be used to normalize the relative quantity of target gene expression. Numerous studies have revealed that expression of 18S rRNA is very stable and that the content of 18S rRNA can be used as an endogenous control to normalize the expression of other genes in response to various stimuli (Bustin et al., 2005). Accordingly, we chose to normalize the relative expression of CNT1, CNT2, CNT3, ENT1, and ENT2 to 18S expression. Before normalization, however, the use of 18S was validated by evaluating the potential treatment effects on the expression levels of 18S rRNA among small intestinal epithelia. No intestinal section distribution effect (0.25 < P < 0.64) nor SH infusion treatment effect (0.52 < P < 0.71) on 18S rRNA expression was observed (data not shown). Accordingly, the 18S rRNA expression levels were used in this study to normalize the relative quantities of the target NT mRNA expression by small intestinal epithelia.

Basal Expression of Nucleoside Transporter mRNA Differed Among Small Intestinal Epithelia
To establish the normal tissue distribution pattern of CNT1, CNT2, CNT3, ENT1, and ENT2 mRNA expressed by the small intestine of normal beef steers, the relative mRNA abundance levels obtained from 6 control animals (non-SH infusion) were compared among duodenal, jejunal, and ileal epithelia (Figure 3Go). The mRNA for these 5 NT were all expressed by the epithelia from all 3 intestinal sections. However, the abundance levels of each NT mRNA typically differed among the 3 sections. Specifically, for the concentrative nucleoside transporters, the jejunal expression of CNT1 mRNA was approximately 1.2- or 2.5-fold higher (P = 0.007) than the duodenal or ileal expression, respectively, whereas no difference (P = 0.35) between the duodenal and the ileal expression of CNT1 mRNA was found. For CNT2 mRNA, the duodenal and the jejunal expression were approximately 10.0- and 11.7-fold higher (P < 0.001) than the ileal expression, respectively, whereas no difference (P = 0.46) between the duodenal and the jejunal expression of CNT2 mRNA was found. For CNT3 mRNA, the duodenal and the jejunal expression were approximately 1.9- and 3.1-fold higher (P < 0.02) than the ileal expression, respectively, whereas the jejunal expression tended to be higher (approximately 0.4-fold, P = 0.11) than the duodenal expression.


Figure 3
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  		Figure 3. The mRNA tissue distribution patterns of 5 nucleoside transporters (NT), 3 concentrative NT (CNT1, CNT2, and CNT3) and 2 equilibrative NT (ENT1 and ENT2), expressed by duodenal (Duo), jejunal (Jej), and ileal (Ile) epithelia of Angus steers without starch hydrolysate infusion (control steers). Bars represent the least square means ± SEM (n = 6) of the mRNA relative quantities (normalized to 18S relative quantities) for each individual small intestinal section. The SEM represents the pooled standard error of the means. Means within a NT that have different letters differ (a–cP ≤ 0.10) or tend to differ (x,yP < 0.20).

 
For the equilibrative nucleoside transporters, the jejunal expression of ENT1 mRNA was approximately 0.9-fold higher (P = 0.06) than the duodenal expression and tended to be higher (approximately 0.5-fold, P = 0.15) than the ileal expression (Figure 3Go). No difference (P = 0.63) between the duodenal and the ileal expression of ENT1 mRNA was found. For ENT2 mRNA, the duodenal and the jejunal expression were approximately 2.5- and 2.7-fold higher (P < 0.001) than the ileal expression, respectively, whereas no difference (P = 0.69) was found between the duodenal and the jejunal expression of ENT2 mRNA.

In general, jejunal epithelium expression of all 5 NT mRNA was higher than ileal epithelium. Jejunal expression of CNT1, CNT3, and ENT1 mRNA was also higher or tended to be higher than duodenal expression. In contrast, no difference was found between jejunal and duodenal expression of CNT2 and ENT2 mRNA. Duodenal expression of CNT2, CNT3, and ENT2 mRNA was higher than the ileal expression, whereas no difference was found between the duodenal and the ileal expression of CNT1 and ENT1 mRNA.

Starch Hydrolysate Infusion Altered CNT3, ENT1, and ENT2, But Not CNT1 and CNT2, mRNA Expression by Small Intestinal Epithelia
Neither ruminal nor abomasal SH infusion altered the CNT1 mRNA expression by duodenal (P = 0.28), jejunal (P = 0.48), or ileal (P = 0.31) epithelium of small intestine (Figure 4Go), and there was no SH infusion treatment by intestinal section interaction (P = 0.37). Similar to CNT1, ruminal or abomasal SH infusion did not alter the CNT2 mRNA expression by duodenal (P = 0.29), jejunal (P = 0.77), or ileal (P = 0.48) epithelium of the small intestine (Figure 4Go), and there was no SH infusion treatment x intestinal section interaction (P = 0.47). In contrast to CNT1 and CNT2, there was a SH infusion treatment x intestinal section interaction (P = 0.06) for CNT3. Ruminal infusion of SH increased the duodenal CNT3 mRNA expression by 67% (P = 0.04) and ileal expression by 210% (P = 0.02), but not the jejunal (P = 0.31) expression (Figure 4Go). However, abomasal SH infusion did not alter the CNT3 mRNA expression by duodenal (P = 0.92), jejunal (P = 0.35), or ileal (P = 0.41) epithelium of small intestine (Figure 4Go).


Figure 4
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  		Figure 4. Comparison of concentrative nucleoside transporter (CNT) mRNA expression (CNT1, CNT2, and CNT3) by duodenal (Duo), jejunal (Jej), or ileal (Ile) epithelium among 3 infusion treatment groups: ruminal and abomasal infusion with water (Ctrl); ruminal infusion with cornstarch hydrolysate and abomasal infusion with water (Rum); ruminal infusion with water and abomasal infusion with cornstarch hydrolysate (Abom). Bars represent the least square means ± SEM (n = 4 or 6) of the relative quantities (the normalized quantities of the infusion groups calibrated to the control group). The SEM represents the pooled standard error of the means. Means within a small intestinal section that have different letters differ (a–cP ≤ 0.10).

 
Similar to CNT3, ruminal infusion of SH increased (P = 0.05) duodenal ENT1 mRNA expression by 51%, whereas the abomasal infusion of SH did not (P = 0.95; Figure 5Go). Neither ruminal nor abomasal SH infusion altered jejunal (P = 0.58) or ileal (P = 0.46) expression of ENT1 mRNA. However, the apparent SH infusion treatment x intestinal section interaction was not significant (P = 0.41).


Figure 5
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  		Figure 5. Comparison of equilibrative nucleoside transporter (ENT: ENT1 and ENT2) mRNA expression by duodenal (Duo), jejunal (Jej), or ileal (Ile) epithelium among 3 infusion treatment groups: ruminal and abomasal infusion with water (Ctrl); ruminal infusion with cornstarch hydrolysate and abomasal infusion with water (Rum); ruminal infusion with water and abomasal infusion with cornstarch hydrolysate (Abom). Bars represent the least square means ± SEM (n = 4 or 6) of the relative quantities (the normalized quantities of the infusion groups calibrated to the control group). The SEM represents the pooled standard error of the means. Means within a small intestinal section that have different letters differ (a–cP ≤ 0.10) or tend to differ (x,yP < 0.20).

 
Similar to CNT3 and ENT1, ruminal infusion of SH increased the duodenal expression of ENT2 mRNA by 39% (P = 0.09), whereas abomasal infusion of SH did not increase the ENT2 mRNA expression by duodenal epithelium (P = 0.38; Figure 5Go). Similar to CNT3, ruminal infusion of SH also increased (P = 0.05) ileal ENT2 mRNA expression by 65%. Different from CNT3, however, abomasal SH infusion increased (P = 0.07) ileal expression of ENT2 mRNA by 54%. Ruminal SH infusion did not alter (P = 0.53) the expression of ENT2 mRNA by the jejunal epithelium, although abomasal SH infusion tended to increase (P = 0.19) the jejunal expression of ENT2 mRNA by 50%. The apparent SH infusion treatment x intestinal section interaction was not significant (P = 0.22).

Overall, neither ruminal nor abomasal SH infusion altered CNT1 and CNT2 mRNA expression by small intestinal epithelia (P > 0.28). Ruminal, but not abomasal, infusion of SH increased CNT3, ENT1, and ENT2 mRNA expression by duodenal epithelium of the small intestine (P < 0.09; Table 2Go). The ruminal infusion of SH also increased CNT3 and ENT2 mRNA expression by ileal epithelium (P < 0.05), but not jejunal epithelium (P = 0.53). Abomasal infusion of SH increased (P = 0.07) ENT2 mRNA expression by ileal epithelium and had a tendency (P = 0.19) to increase ENT2 mRNA expression by jejunal epithelium. However, abomasal infusion of SH did not alter any NT mRNA expression by duodenal epithelium (P > 0.38).


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  		Table 2. Summary of the effects of ruminal and abomasal starch hydrolysate infusion on nucleoside transporter mRNA expression by small intestinal epithelia
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Expression of Nucleoside Transporter mRNA by Small Intestinal Epithelia
Nucleotides are thought to be conditionally essential or semiessential nutrients, especially for the development of gut and immune system function (Sánchez-Pozo and Gil, 2002). Although the nutritional significance of nucleotides for domestic animal species is poorly elaborated (Mateo and Stein, 2004), an adequate cellular supply of nucleosides is critical for many biological processes during animal development and growth, including DNA and RNA syntheses, energy (ATP) production, N and P recycling, cell signaling, and modulation of gene expression (Carver and Walker, 1995; Sánchez-Pozo and Gil, 2002).

In mammals, diet- and endogenous-derived nucleic acids are degraded into nucleotides by pancreatic nucleases and intestinal phosphodiesterases. These nucleotides then are hydrolyzed into nucleosides by purine/pyrimidine nucleotidases and nonspecific alkaline phosphatases. It has long been recognized that nucleic acids constitute a major part (5.2 to 9.5%) of the total N in grasses and hay forages fed to ruminants and that nearly all these dietary nucleic acids are totally degraded in the rumen (Huntington, 1986; Hobson and Stewart, 1997). Moreover, ruminal bacterial cell mass contains approximately 16% RNA and 3.2% DNA on DM basis (Russell, 2002) and the amount of rumen microbe-derived nucleic acids entering the duodenum via the abomasum is estimated to be about 15 to 35 g/ kg (of DM) for beef cattle, of which RNA constitutes about 60 to 70% of total nucleic acids (McAllan, 1980). For dairy cattle, amounts ranging from 100 g/d (Scharrer and Grenacher, 2001) to about 250 g/d (Clark et al., 1992) have been reported. In growing lambs about 10% of total rumen microbial N is in the form of RNA-N, and 4.5% in the form of DNA-N (Storm and Ørskov, 1983). Under typical ruminant dietary regimens, nucleic acids appear to be very efficiently digested and absorbed in the upper small intestinal tract of ruminants, as nucleosides appear in the duodenal digesta in appreciable amounts, rapidly disappear from digesta between the duodenum and ileum, and are only nominally detectable at the terminal ileum (McAllan, 1980, 1982).

In the small intestine, which has a large epithelial tissue mass that proliferates very rapidly, the capacity of enterocytes to generate nucleosides by de novo synthesis of nucleosides is limited (Savaiano and Clifford, 1981; Tsujinaka et al., 1999). Therefore, enterocytes rely on their capacity to absorb nucleosides from the intestinal lumen, or the salvage of nucleosides from the extracellular basolateral fluid, to meet their great metabolic demands (Cosgrove, 1998). Because nucleosides are hydrophilic, mediated transport is required to achieve vectorial movement across plasma membranes. The absorption of nucleosides is performed by 2 families of transporters, CNT (SLC28 family; substrates can be absorbed against their concentration gradient, Na+-dependent) and ENT (SLC29 family; transmembrane substrate concentrations dictate bidirectionality of transport, ion-independent). Different family members possess different recognition patterns and affinities for nucleosides (Griffith and Jarvis, 1996; Van Aubel et al., 2000; Gray et al., 2004; Elwi et al., 2006). CNT1 (SLC28A1/system N2 or cit activity) prefers pyrimidine nucleosides (cytidine, uridine, and thymidine) and adenosine, whereas CNT2 (SLC28A2/system SPNT, N1 or cif activity) prefers purine nucleosides (guanosine, adenosine) and uridine. CNT3 (SLC28A3/system N3 or cib activity) is broadly selective, thus transporting both purine and pyrimidine nucleosides. The CNT1 and CNT3 activities have been identified in apical membranes of polarized epithelia, whereas CNT2 appears localized in both apical and basolateral membranes (Mangravite et al., 2001).

To date, 4 members of the ENT mammalian family have been characterized: ENT1 (SLC29A1, system es activity), ENT2 (SLC29A2 system ei activity), ENT3 (SLC29A3), and ENT4 (SLC29A4; Griffith and Jarvis, 1996; Baldwin et al., 2004; Elwi et al., 2006). The ENT1, ENT2, and ENT3 are broadly selective, transporting both pyrimidine and purine nucleosides with a low affinity for substrates, whereas ENT4 is thought to function primarily as a monoamine transporter, recognizing adenosine as its only nucleoside substrate. Two primary differences between ENT1 and ENT2 are that ENT1 recognizes nucleosides with a greater affinity than does ENT2 and that ENT2 transports purine and pyrimidine nucleobases, whereas ENT1 does not. Although incompletely characterized for enterocytes, ENT1 and ENT2 are thought to be predominantly located in the basolateral membrane of polarized epithelial cells (Pastor-Anglada et al., 2001). However, the localization of ENT1 in the apical membrane has been documented in renal epithelial cells (Baldwin et al., 2004). In contrast to ENT1 and ENT2, ENT3 is localized to intracellular membranes/organelles (e.g., lysosome), whereas the membrane localization of ENT4 in epithelial cells is poorly defined (Elwi et al., 2006).

The results from our study show for the first time that the bovine small intestine epithelium expresses mRNA for 3 CNT and 2 ENT (Figures 3Go, 4Go). The overall pattern of expression for CNT and ENT mRNA by duodenal, jejunal, and ileal small intestinal epithelia is consistent with that expressed by rat and mice (Lu et al., 2004). However, differences appear to exist between the relative abundance of NT mRNA species in rodents and cattle. Specifically, in terms of concentrative nucleoside transport capacity, the expression of CNT1 mRNA by male and female mice and rats is greater by duodenal or jejunal than by ileal epithelium, whereas we found that jejunal expression was greatest among the 3 epithelia in steers. For CNT2, whereas expression among the 3 small intestinal epithelia does not differ for mice and rats, the ileal expression of CNT2 mRNA was lowest in steers. For CNT3, however, the pattern of expression among these 3 species essentially does not differ. In terms of interspecies patterns of ENT mRNA expression, differences also appear to exist with steers displaying a greater jejunal content of ENT1 and a greater content of ENT2 in duodenal and jejunal epithelia, whereas no differences in ENT mRNA content was found among small intestinal epithelia of mice and rats (Lu et al., 2004).

Capacity for Small Intestinal Nucleoside Absorption in Cattle
In nonruminants, as noted above, CNT1 and CNT3 activities have been identified in apical membranes, ENT1 and ENT2 in basolateral membranes, and ENT1 and CNT2 in both. This apparent subcellular distribution pattern of nucleoside transporters in polarized epithelial cells allows for both concentrative and equilibrative nucleoside substrate uptake across apical and basolateral membranes. Accordingly, this differential pattern of transporter localization would accommodate a transepithelial flux of nucleosides to occur from apical to basolateral membranes. However, it is thought that the intracellular metabolism of nucleosides is too fast to allow for a transcellular flow of nucleosides (Barcells et al., 1992; Casado et al., 2002; Aymerich, et al., 2004). Therefore, this arrangement of transporters most likely ensures a capacity to absorb nucleosides from either luminal or plasma sources. Our finding that the mRNA for 5 nuceloside transporters are expressed in each of the 3 small intestinal epithelia of steers indicates that ruminants may also possess these nucleoside uptake capacities, assuming that the identified mRNA are translated and the subsequent transporter proteins are localized and active in the same membranes as seen for nonruminants.

In terms of cattle-specific nucleoside uptake activities/proteins, the seminal work of Scharrer and colleagues with jejunal brush-border membrane vesicles from dairy cows (Scharrer and Grenacher, 2001, 2002) and veal calves (Theisinger et al., 2002) revealed that nucleosides are transported across the small intestine epithelial brush-border membrane by at least 2 distinct active Na+-dependent nucleoside transport systems: system N1, which transported purines (including guanosine and inosine), and system N2, which transported pyrimidines (including thymidine and cytidine). The recent expression cloning of nonruminant CNT orthologs has revealed that system N1 and N2 activities are achieved by the function of CNT2 and CNT1, respectively (Van Aubel et al., 2000). Importantly, the Km values for CNT2/system N2 and CNT2/system N1 activities are consistent with those derived from bovine brush-border vesicles (Scharrer and Grenacher, 2001, 2002; Theisinger et al., 2002). A third CNT protein has been cloned (CNT3), which possesses system N3 activity and recognition of a broad range substrates, including both purines and pyrimidines. Like CNT1 and CNT2, CNT3 shows a high affinity (Km in 15 to 53 µM range) for its natural substrates (Casado et al., 2002; Kong et al., 2004), but unlike CNT1 and CNT2, evidence for CNT3 activity in bovine apical membranes was not found in the above bovine studies.

Our finding that both CNT1 and CNT2 mRNA are expressed by beef steers is consistent with the CNT1/ N2 and CNT2/N1 activities measured by Scharrer and coworkers for ruminating and nonruminating dairy cattle (Scharrer and Grenacher, 2001, 2002, 2005; Theisinger et al., 2002). However, our finding that CNT3 mRNA is expressed by all 3 small intestinal epithelia is not consistent with that of Scharrer and coworkers (Scharrer and Grenacher, 2001; Theisinger et al., 2002), who failed to find evidence for a distinct CNT3 (N3)-like activity in jejunal-derived apical membrane vesicles. This apparent discrepancy between measured CNT3 mRNA and lack of functional activity awaits clarification.

Two additional understandings drawn from the seminal studies of Scharrer and colleagues (Scharrer and Grenacher, 2001, 2002, 2005; Theisinger et al., 2002) were that (1) the small intestine epithelial activities of N1 and N2 for both mature cows and veal calves were about 10 times that measured for nonruminants, such as rats and mice, and (2) the relative pattern of nucleoside uptake activity decreased from proximal-to-distal jejunal epithelia. Our finding that both CNT1 and CNT2 mRNA abundance was lesser in the ileal epithelium than the duodenal and jejunal epithelia of beef steers (Figure 3Go) is generally consistent with the proximal-to-distal jejunal epithelium gradient of CNT2/N1 and CNT2/N2 uptake activity measured in the small intestine of dairy cows and veal calves. The relative pattern of bovine CNT mRNA expression among small intestinal epithelia found in the current study also is generally consistent with the greater proximal than distal CNT uptake activities measured in nonruminants, especially for CNT1, which shows the greatest functional activity in the jejunum of nonruminants (Ngo et al., 2001; Casado et al., 2002).

In contrast to CNT family members, ENT family members transport nucleosides across plasma membrane bidirectionally, do not cotransport Na+, display a lower affinity for substrates (Km in 0.04 to 5.61 mM range), are more permissive in terms of substrate recognition (Casado et al., 2002; Kong et al., 2004), and are predominantly located in the basolateral membrane of epithelial cells (Pastor-Anglada et al., 2001). Within the ENT family, ENT1 possesses system "es" activity, whereas ENT2 possesses system "ei" activity. Both es and ei systems transport both pyrimidine and purine nucleosides (Casado et al., 2002; Lu et al., 2004). To our knowledge, the expression of ENT activities by bovine small intestinal epithelia has not been evaluated or reported.

Regulated Expression of Nucleoside Transporter mRNA in Small Intestinal Epithelia of Cattle
Thorough evaluations on the substrate regulation of NT expression by the small intestinal epithelium have not been conducted. However, from nonruminant studies, it is generally thought that NT expression and activities in polarized epithelia are sensitive to luminal substrate availability (Casado et al., 2002), are cell-type specific (Cabrita et al., 2002; Kong et al., 2004), and occur by various regulatory pathways (Pastor-Anglada et al., 2001; Aymerich et al., 2004). Given these understandings, and the special importance of nucleoside transport capacity to support the relatively high rate of nucleotide synthesis in the rapidly dividing intestinal epithelium by supplying the nucleotide salvage pathway with substrate (Casado et al., 2002; Gray et al., 2004; Baldwin et al., 2004), the lack of research in this area is notable.

This deficit of knowledge seems particularly relevant to ruminants, which are challenged with high levels of microbe-derived nucleic acids arriving in the small intestine, and for which relatively high capacity for nucleoside uptake (as compared with nonruminants) has been measured in brush border membranes isolated from cows and veal calves (Theisinger et al., 2002; Scharrer and Grenacher, 2005). Because the nucleoside challenge to the small intestine of veal calves is low compared with mature (ruminating) cows, and because NT uptake capacity was as least as great in veal as for mature cows, Scharrer and colleagues concluded that CNT2/N1 and CNT1/N2 activity was not likely to be regulated by substrate (nucleosides). However, this conclusion considers only developmental regulation and does not consider the additive possibility that "basal" NT transporter expression and activity can be acutely regulated by substrate supply.

To evaluate this possibility under physiologically and commercially relevant conditions, we used the well-proven ruminal SH infusion experimental model to challenge the small intestinal epithelium with an increased supply of nucleotides. The supplementation of high quality forage diets with SH (such as used in our experiment) is known to increase the production of rumen microbes and, hence, the load of microbial nucleic acids (RNA and DNA) to the small intestine (Bach et al., 1999; Elizalde et al., 1999). The breakdown of RNA and DNA in the small intestine of cattle produces pyrimidine and purine nucleosides, including uridine, thymidine, cytidine, adenosine, and guanosine (McAllan, 1980). Because the absorption of nucleosides is an energy-demanding event, and because the expression of NT may be limited by the energy status of cells (Elwi et al., 2006), we included the infusion of SH into the abomasum to provide an increased energy supply to the small intestine. Abomasal SH infusion is known to increase in (1) carbohydrate supply to the small intestinal lumen, (2) glucose appearance in the blood, and (3) small intestinal epithelial mass (Weser et al., 1985; Walker and Harmon, 1995; McLeod et al., 2007). In this state, the proliferating epithelial cells likely require more nucleosides for DNA and RNA syntheses (Aymerich et al., 2004), and the absorption of nucleosides from the basolateral extracellular fluid or blood likely is the major pathway for the entry of purines and pyrimidines into epithelial cells (Sanderson and He, 1994; Cosgrove, 1998).

In the present study, we found that the presence of an increased NT substrate load (ruminal SH infusion treatment) increased the potential to transport nucleosides (increased content of NT mRNA) across both apical and basolateral membrane (ENT1, ENT2) in the duodenum (CNT3, ENT1, ENT2) and ileum (CNT3, ENT2). Conversely, increasing the supply of carbohydrate-derived energy (glucose) to the small intestinal lumen (abomasal SH infusion treatment) increased the potential for increased flux of nucleosides across the basolateral (ENT2) membrane. Why CNT3 mRNA expression, but not CNT1 or CNT2, was sensitive to the luminal increase of nucleotides is not obvious, especially because CNT1 and CNT2 expression is known to be sensitive to substrate regulation. That is, the expression of CNT1 and CNT2 proteins and activities are upregulated in the rat small intestinal epithelia when nucleosides are reduced by starvation or feeding of nucleoside-depleted diets (Valdés et al., 2000).

However, differences in substrate affinity and transport capacity between CNT members may provide another explanation. That is, given the broader substrate specificities of CNT3 (both pyrimidine and purine nucleosides, Gray et al., 2004) and its apparently much greater capacity to concentrate substrate against a concentration gradient (Elwi et al., 2006), it is convenient to speculate that an increased expression of CNT3 represents a more "efficient" upregulation of transporter capacity. Our data suggest that CNT3 expression is sensitive to substrate supply and insensitive to epithelial energy status. Reports to support or refute claims about nutrient regulation of CNT3 expression are unknown to us.

Compared with the CNT family, even less is known about the substrate or energy status regulation of ENT mRNA and protein expression and their transport activities in animal small intestinal epithelium, except for limited pharmacological studies with several in vitro cell lines derived from humans and laboratory animals (Mun et al., 1998; Podgorska et al., 2005). Our in vivo study with beef steers, however, demonstrated that ruminal infusion of SH (thus an increased supply of nucleic acids to the lumen of the small intestine) increased steady-state ENT1 mRNA expression by duodenal epithelium, and ENT2 mRNA expression by duodenal and ileal epithelia. Thus, despite the understanding that intracellular metabolism of absorbed luminal nucleosides might negate the need for an increased intracellular-to-blood basolateral capacity (Barcells et al., 1992; Casado et al., 2002; Aymerich, et al., 2004), this finding indicates that the increased expression of ENT family members may be a cellular response to the increased cytosolic concentration of nucleosides absorbed across the apical membrane, and that this response may be a necessity for the intestinal epithelium to supply the surplus nucleosides, nucleobases, or both to the basolateral extracellular fluid and then blood. Alternatively or simultaneously, differences in substrate specificity between ENT1 and ENT2 may provide a different explanation. That is, if ENT1 can be localized on the apical membrane of enterocytes, as is reported for cultured renal epithelia (Baldwin et al., 2004), then an increase in ENT1 mRNA content may represent a response to increased luminal substrate. In contrast, the increase in ENT2 mRNA may represent a response to increased intracellular nucleobases that result from an increased absorption of luminal nucelosides because ENT2 has a unique capacity to transport nucleobases (such as hypoxanthine and adenine), in contrast to CNT1, 2, 3 or ENT1 (Baldwin et al., 1999; Lu et al., 2004).

In the present study we found that the abomasal infusion of SH increased ENT2 mRNA expression by the ileal epithelium and tended to increase jejunal epithelial expression of ENT2 mRNA, respectively. Why only ENT2, but not ENT1 or CNT3, mRNA expression is responsive to the luminal increase of glucose supply is not obvious. The differential regulation of NT expression and functional activity in response to endocrine and intracellular signaling pathways has been well documented, at least for cells from blood (Griffith and Jarvis, 1996), bone (Stolk et al., 2005), and liver (Fernández-Veledo et al., 2007). However, investigation of substrate regulation of NT expression or activity by intestinal epithelia appears limited to a single report on the effect of starvation and nucleotide-deficient models on CNT1 and CNT2 expression and activity (Valdés et al., 2000). Accordingly, our findings that the expression of CNT3, ENT1, and ENT2 mRNA by duodenal and ileal epithelia was upregulated by an increased luminal supply of nucleosides (substrate), whereas an increased small intestine luminal supply of starch (energy) only upregulates ENT2 mRNA expression by small intestinal epithelia, are unique and enable future research to further elaborate the relationship between nutrient supply and NT uptake capacity in commercially relevant cattle models.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study found that the mRNA for 5 mammalian NT (3 CNT and 2 ENT) are expressed throughout the small intestinal epithelia of growing beef steers, in a pattern of mRNA abundance that is consistent with and extends previously reported pattern of NT functional activities in veal calves (nonruminating) and mature dairy cows. Furthermore, this study uniquely demonstrated that the small intestinal epithelial expression of CNT3, ENT1, and ENT2 mRNA is upregulated in response to increased substrate supply, whereas only ENT2 mRNA was upregulated in response to increased supply of energy to the small intestinal epithelium.


   FOOTNOTES
 
1 This research was supported by a USDA-ARS Special Cooperative Agreement Grant, the University of Kentucky, and Kentucky Agricultural Experiment Station (publication no. 07-07-120). Back

2 Six partial-length cDNA sequences associated with this publication were deposited in GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html), and their accession numbers are specified in the manuscript. Back

Received for publication October 9, 2007. Accepted for publication December 14, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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S. F. Liao, E. S. Vanzant, D. L. Harmon, K. R. McLeod, J. A. Boling, and J. C. Matthews
Ruminal and abomasal starch hydrolysate infusions selectively decrease the expression of cationic amino acid transporter mRNA by small intestinal epithelia of forage-fed beef steers
J Dairy Sci, March 1, 2009; 92(3): 1124 - 1135.
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