The TR-iBRB2 cells were cultured, and the [
3H]L-ornithine uptake was investigated as described previously.
28,29,35,36 The cells on BioCoat Collagen I Cellware 24-well dishes (BD Bioscience, Franklin Lakes, NJ, USA) were washed with extracellular fluid (ECF)-buffer three times, and then incubated in 200 μL ECF-buffer containing [
3H]L-ornithine (0.1 μCi, 8.33 nM) at 37°C. The RPE-J cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (Nissui Pharmaceuticals, Tokyo, Japan) with 4% fetal bovine serum, 20 mM NaHCO
3, 25 mM D-glucose, 0.1 mM nonessential amino acids, and antibiotics at 33°C under an atmosphere of 5% CO
2 in air.
30 The RPE-J cells were cultured on Not TC–treated culture dishes (Corning, Corning, NY, USA). Before the transport study, RPE-J cells were cultured on Transwell polyester membrane inserts (surface area 0.33 cm
2 and pore size 0.4 μm; Corning) for 6 days at 33°C, and maintained in culture medium containing 10 nM all-trans retinoic acid for another 2 days at 40°C. The integrity of the cell monolayer was evaluated by the transepithelial electrical resistance (TEER) using a voltohmmeter (Millicell ERS-2; Merck Millipore, Billerica, MA, USA). The directional transport in RPE-J cells was studied at 37°C and pH 7.4. After washing the cells with ECF buffer three times, the assay was initiated by adding ECF buffer with [
3H]L-ornithine (16.7 nM) and [
14C]D-mannitol (18.2 nM) to the donor side and ECF buffer to the receiver side. In the study of the apical-to-basal (A-to-B) and basal-to-apical (B-to-A) transport, a 20-μL aliquot of the receiver-side buffer was sampled at designated times, and then replaced with an equal volume of fresh buffer. In the study of the apical-to-cell (A-to-C) and basal-to-cell (B-to-C) uptake, the assay was terminated at the designated time, and the cells were lysed with 1N NaOH and neutralized with 1N HCl. The radioactivity was measured using a liquid scintillation counter (LSC-7400), and
Equation 3 and
Equation 4 were used to calculate the compound permeated and cell-to-medium (cell/medium) ratio, respectively.