In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs)
2-PAM
MDCK cells were seeded on transwells (24 well; PE; 0.33 cm2 area; 0.4 µm pore diameter, Corning, Corning, NY, USA) coated with collagen I (rat tail, 10 µg cm?2, Corning) at a density of 6 × 105 cells cm?2. Permeability experiments were performed 4 days after seeding. The medium was changed 2 h before the experiment and transepithelial electrical resistance (TEER) was measured before all experiments (Endohm/EVOM2). For MDCK cells, permeability measurements were performed if the TEER was ?90 ? cm2 (see Additional file 1: Table S1: TEER values for transwell experiments) [24, 25]. To confirm the integrity of the MDCK monolayers, the permeability of Lucifer yellow, 100 µM, was measured for up to 2 h. In all cases the permeability was ?1 × 10?6 cm s?1: 0.71 ± 0.34 × 10?6 cm s?1 (MDCKII), 0.38 ± 0.20 × 10?6 cm s?1 (MDCKII-MDR1), and 0.46 ± 0.21 × 10?6 cm s?1 (MDCKII-FLuc-ABCG2). The measured permeability for Lucifer yellow under the same conditions in transwells without cells, was 4.35 × 10?5 cm s?1.
All experiments with MDCK cells were performed in Hank’s balanced salt solution (HBSS) with 10 mM HEPES (Sigma) and 15 mM glucose (Sigma), pH 7.4. After incubation in media for 2 h, cell monolayers were immersed in fresh HBSS for 30 min to remove any traces of media. Then 100 µM or 10 µM 2-PAM (pralidoxime chloride, Sigma) in HBSS was pipetted into either the apical or basolateral chamber, with HBSS on the receiving side. Cell monolayers with test solutes were incubated at 37 °C with 5 % CO2 on a rocker to ensure good mixing.
For experiments with BC1-hBMECs, cells were seeded on transwells (24 well; PE; 0.33 cm2 area; 0.4 µm pore diameter, Corning) coated overnight with a 50/50 mixture collagen IV (100 ?g mL?1; Sigma) and fibronectin (50 ?g mL?1; Sigma) at a density of 1 × 106 cells mL?1. For BC1-hBMECs, permeability measurements were performed with cells that had a TEER of ?1500 ? cm2. Experiments were performed in transport buffer (distilled Millipore water with 0.12 M NaCl, 25 mM NaHCO3, 3 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 0.4 mM K2HPO4, 1 mM HEPES, and 0.1 % human platelet poor-derived serum) without the pre-incubation of media or rocking of the cells. After 2 days in EC media, 100 µM of 2-PAM in transport buffer was pipetted into the apical or basolateral well with transport buffer on the receiving side.
The concentration of 2-PAM was determined by HPLC (1260 Infinity HPLC, Agilent, Santa Clara, CA, USA) with UV Vis detection at 296 nm. Solvents were degassed by sonication for 45 min before use and all samples were run at room temperature. An isocratic flow of 44 vol.% acetonitrile (HPLC grade, Chromasolv, Sigma) and 56 vol.% ammonium acetate (0.03 M; HPLC grade, Sigma), pH 4.5, was used with a PolyCAT A column (100 × 2.1 mm, 5 µM, 300 Å, 102CT05-03, Poly LC Inc, Columbia, MD, USA) [26]. Calibration curves were constructed from standard solutions with concentrations of 0.1, 1, 10 and 100 µM. Due to the simplicity of the procedure, no internal standard was used.