The Mechanism Of Small Peptide Absorption In Bovine Omasal Epithelial Cells And Charactrization Of Bovine Peptide Transporter 1

Small peptide is an important source of nitrogen of dairy cows and one of important precursors of milk protein. Small peptides can be absorbed largely firom gastrointestine of dairy cows. In this study, the omasal epithelial cells (OEC) of dairy cow were used to study the mechanisum of small peptide absorption in omasum, and the role and functional expression of peptide transporter 1 (PepTl) in small peptide absorption in OEC. There are three parts of this study. Firstly, to establish and characterize an omasal epithelial cell model derived from dairy calves for the study of small peptide absorption. Secondly, to study the role of PepTl in the absorption of dipeptides in bovine OEC. Thirdly, to study the functional expression of the bovine PepTl in Chinese hamster ovary (CHO) cells. The main results are shown as below:1. Establishment and characterization of an omasal epithelial cell model derived from dairy calves for the study of small peptide absorption. The objective of this study was to establish a primary culture of OEC derived from 4 dairy calves and to characterize its function in small peptide absorption. Bovine omasal tissues were obtained from newborn Chinese Holstein calves and digested with a 2.5% trypsin solution to obtain OEC. The isolated cells were later cultured in DMEM containing 10% fetal bovine serum,10 ng/mL epidermal growth factor,5μg/mL insulin,100 U/mL penicillin,100 μg/mL streptomycin,50 μg/mL gentamycin and 2.5 μg/mL amphotericin B. Hematoxylin and eosin staining of omasal tissue after digestion indicated that the cultured cells originated from the epithelial strata. Pure epithelial cells displayed an epithelial cell-like morphology, similar to cobblestone, with few visible fibroblasts and were cytokeratin 18-positive and PepTl-positive according to immunocytochemical analyses. The OEC were morphologically characterized with desmosomes, tight junctions and microvilli. These cells exhibited normal growth properties, as assessed using a cell growth curve, and were stably cultured for 10 passages. The OEC expressed the PepTl mRNA and absorbed intact glycylsarcosine (Gly-Sar). The uptake of Gly-Sar by OEC was pH-dependent with an optimal pH of 5.5-6.5. Furthermore, the uptake of Gly-Sar was also time-dependent, concentration-dependent and temperature-dependent. Moreover, PepTl was saturated with Gly-Sar at a concentration of 2.5 mM. The uptake via PepTl was higher compared with that via passive route at low substrate concentrations (< 1.5 mM). This result suggested that PepT1 contributed more to total small peptide absorption at low concentrations. In addition, this uptake could be competitively inhibited by methionine-glycine. Taken together, these data suggested that PepTl contributes to small peptide absorption in OEC. Thus, OEC may serve as a useful culture model for the study of the absorption of small peptides in bovine omasum.2. Role of peptide transporter 1 in the absorption of dipeptides in bovine omasal epithelial cells,In this study, the role of proton-coupled PepTl in the absorption of dipeptides by bovine OEC was investigated. Primary OEC derived from newborn Chinese Holstein male calves were strongly immunopositive for anti-junction protein (zonula occludens 1 and occludin) antibodies. In the transwells of primary OEC culture, the transepithelial electrical resistance reached a plateau after one week, at which the permeability of OEC layers was 0.63% as measured by transmittance of fluorescein sodium. Next, the OEC layers were incubated with the non-metabolized dipeptide glycylsarcosine (Gly-Sar) under various conditions. The results showed that the accumulation of Gly-Sar on the basolateral side (pH 7.4) was significantly higher at 37℃ than at 4℃, with an optimal pH of 6.0-6.5 in the apical medium, and was inhibited significantly by diethylpyrocarbonate, an inhibitor of PepTl, so was the uptake of Gly-Sar into OEC. Moreover, the absorption of Gly-Sar was inhibited by the competitive dipeptide Met-Gly but not by the free amino acid, Gly. The inhibitor profiles and pH dependence of the transport process were consistent with the properties of PepT1. Gly-Sar accumulation of apical-to-basolateral transport across OEC layers was significantly higher than that of basolateral-to-apical transport (3-fold difference). Furthermore, the knockdown of the PepTl gene by small interfering siRN A resulted in less uptake of Gly-Sar by OEC. Overexpression of the PepTl gene by transfection with a PepTl expression vector pcDNA3.1/PepTl resulted in higher uptake of Gly-Sar by OEC. In addition, the expression of PepTl protein in OEC was upregulated significantly by treatment with various dipeptides (Gly-Sar, Lys-Lys and Met-Lys) at different concentrations (0.25,2.5 and 10 mM). In addition, the result also showed that LPS can decrease the transepithelial electrical resistance (TEER) of OEC layer, increase the expression of PepT1 mRNA and the transport of Gly-Sar of OEC layer. We also cultured the rumen epithelial cells (REC) in vitro successfully, and found that the TEER of REC layer was higher than TEER of OEC layer. Moreover, the results also showed that OEC had stronger ability to transport Gly-Sar than REC. In summary, these results demonstrate that PepT1 may play an important role in dipeptide absorption in bovine OEC, and the uptake and transport of Gly-Sar can be affected by many factors.3. Functional expression of the bovine gastrointestinal peptide transporter (bPepT1) in Chinese hamster ovary cells. We cloned and determined the primary structure, tissue distribution and in vitro functional characterization of the bPepT1. The cDNA ofbPepT1 is 2742 bp long, encoding a protein of 707 amino acid residues with an isoelectric point of 7.86 and an estimated molecular size of 78.4 kDa. bPepT1 AA sequence is high identical to PepT1 from goat (96.2%), sheep (95.2%), horse (85.9%), pig (85.6%), dog (83.4%), human (83.0%), mouse (82.7%), rat (81.6%), monkey (81-83%), rabbit (78.6%), gray short-tailed opossum (74.8%), platypus (71.9%), poultry (64-65%), fish (56-61%). Western blot analysis of body tissue distribution showed the highest levels of bPepT1 protein in jejunum and ileum in the gastrointestinal tract of dairy cows (P< 0.05):Jejunum> Ileum> Duodenum> Omasum> Rumen, suggesting intestine has the stronger ability to absorb small peptides than forestomach and jejunum is the major part to absorb small peptides of dairy cow. To investigate the kinetics of peptide transport by bPepT1, Chinese hamster ovary cells were transfected with an expression vector containing cloned bPepT1 cDNA. Transport was assessed by uptake studies using the radiolabeled d:peptide, [3H]-Gly-Sar. Uptake of [3H]-Gly-Sar by bPepT1 was pH-dependent with an optimal pH of 6.5-7.0, concentration-dependent and saturable with an apparent Km value of 0.94±0.06 mM and a maximum velocity of 20.80±1.74 nmol/(mg protein± 5 min). Competition studies with nonradiolabeled peptides that contained the essential amino acids Met, Lys and (or) Trp were used for functional analysis of bPepT1 showed that all di-, tri- and tetrapeptides inhibited uptake of [3H]-Gly-Sar. Ten dipeptides and one tripeptide had IC50 values (the concentration of unlabeled peptide that inhibited 50% of [3H]-Gly-Sar uptake) that ranged from 0.014 to 0.096 mM. Four peptides, Lys-Lys, Leu-Gly-Gly, Lys-Trp-Lys and Met-Gly-Met-Met, had IC50 values greater than 2.069 mM and seemed to be poor substrates for bPepT1. Among them, Lys-Lys is the poorest substrates for bPepT1.In addition, there was no inhibition of [3H]-Gly-Sar uptake detected in the presence of nonradiolabeled free Gly. These results demonstrate for the first time the functional characteristics of bPepT1, which can transport a variety of small peptides, including di- and tripeptides, and has species specificity.In summary, we have established and characterized an OEC model successfully, using which finding out that the important role of PepT1 in small peptide absorption. Finally, we demonstrated the functional characteristics of bPepT1, which can transport a variety of small peptides, including di- and tripeptides, and has species specificity.