Identification of two cell culture models to study bovine CAT1 activity and expression.

Although L-Lysine (Lys) is a dietarily-essential amino acid (AA) for cattle fed a high corn diet, proteins responsible for absorption of Lys by cattle have not been described. CAT1 is a major intestinal transporter of cationic AA. CAT1 demonstrates a high-affinity (μM) System y⁺ activity that differs from b^0,+, y⁺L, and B^0,+ cationic AA transporter systems due to its independence of Na⁺ (-Na⁺) and/or insensitivity to neutral AA. This project was conducted to determine if Madin-Darby Bovine Kidney (MDBK) cells and steer hepatocytes express CAT1 activity and/or mRNA. Putative System y⁺ activity was assessed in wells (n = 8-12) of 2-d cultured MDBK cells (250,000/2 cm² well) by characterizing the -Na⁺ uptake (pmol/mg protein) of Lys (10 μM; [H³]Lys, radiotracer) in the presence and absence of 2 mM L-Arg or L-Leu. System y⁺ Lys uptake accounted (P < 0.001) for 50% of total -Na⁺ Lys uptake, as did Systems B^0,+ and/or y⁺L. K_m determination for -Na⁺ Lys in the presence of 5 mM L-Leu was 250 μM, consistent with CAT1 activity. RT-PCR of total RNA extracted from MDBK cells (and bovine kidney, and ileal epithelium), using the full-length pig CAT1 as a template, produced a single cDNA product of about 700 bp. Sequencing of the MDBK and kidney products revealed a 695-bp cDNA that possessed 89, 87, 99% homology with corresponding regions of the pig, human, and predicted bovine CAT1 mRNA, respectively. The expression of System y⁺ activity and CAT1 mRNA next was evaluated in wells (4-6) of 2-d cultured hepatocytes (200,000/2 cm² well) isolated (collagenase perfusion) from the caudal lobe of 30-d old Angus steer livers (n = 4), using transport and RT-PCR parameters identical to those used for MDBK cells. Systems y⁺, b^0,+, and y⁺L accounted (P < 0.034) for 19, 34, and 47% of -Na⁺ Lys uptake, respectively, and RT-PCR yielded a product of about 700 bp. These results demonstrate that MDBK cells and steer hepatocytes express CAT1 activity and mRNA and indicate their usefulness to study bovine CAT1 function and expression. [ABSTRACT FROM AUTHOR]