Measurement of OCT2 Transporter Uptake Activity in HEK293 and MDCKII Cells

Transport studies were conducted in OCT2-expressing MDCKII and HEK293 cells with [¹⁴C]-metformin with or without 1 mM cimetidine (OCT2 inhibitor). For transport assays, OCT2-expressing MDCKII and HEK293 cells were grown in 24-well poly-d-lysine coated plates, at a density of 75,000 cells per well with 1 ml of high glucose Dulbecco’s modified Eagles’ medium (changed daily). On day 3, cells were washed with 1 ml/well Dulbecco’s phosphate-buffered saline buffer and incubated at 37°C with KRH buffer (5.6 mM glucose, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM CaCl₂, 1.2 mM MgSO₄, and 25 mM HEPES) containing 5.5 µM [¹⁴C]-metformin with or without 1 mM cimetidine (OCT2 inhibitor). After 2 minutes (when uptake is linear), the uptake (in triplicate) was terminated by washing the cells three times with ice-cold KRH buffer (1 ml each). Then, the cells were lysed with 0.5 ml 1 M NaOH at 37°C for 2 hours and neutralized with 0.5 ml 1 M HCl. Thirty microliters of this lysate solution was used for total protein estimation using the BCA method, and 200 µl was used to analyze total radioactivity by Tri-Carb Liquid Scintillation Counters (PerkinElmer, Waltham (MA)). OCT2-mediated uptake was determined by the difference in the metformin uptake in the presence/absence of 1 mM cimetidine. The in vitro OCT2-mediated clearance of metformin was calculated as the ratio of OCT2-mediated uptake and metformin concentration in the media.