Characterization of cyclosporin a transport in cultured rabbit corneal epithelial cells: P-glycoprotein transport activity and binding to cyclophilin

PURPOSE. The purpose of this study was to characterize cyclosporin A (CsA) uptake and transport in cultured rabbit corneal epithelial cells (RCECs). METHODS. CsA uptake was evaluated by measuring time-dependent H-3-CsA accum ulation in confluent RCECs. Bidirectional H-3-CsA fluxes mere measured acro ss the RCEC layers grown on Transwell-COL culture plate inserts. The anti-P -gp monoclonal antibody C219 was used in western blot analysis to probe for the presence of P-gp in these cells. RESULTS. The accumulation of H-3-CsA was time and temperature dependent. St eady state was reached by 60 minutes. The initial uptake was saturable and was suppressed as a function of increases in preloading with unlabeled CsA. This uptake process was enhanced by metabolic inhibition with either 3-O-m ethylglucose, MG, or 10 mM NaN3 and 3-O-MG. The largest increase was obtain ed with 10 mM NaN3 in combination with 3-O-MG. In their presence, uptake in creased by 40%. A multidrug-resistance (MDR)-reversing agent (i.e., 500 mu M verapamil, 100 mu M vincristine, 100 mu M progesterone, 100 mu M testoste rone, 500 mu M quinidine, or 100 mu M chlorpromazine) significantly increas ed H-3-CsA accumulation. The largest increase was obtained with 500 mu M qu inidine (i.e., 36%). Conversely, verapamil and vincristine produced the lar gest inhibition of 3H-CsA efflux (i.e., 19% and 28%, respectively.). Howeve r, in the presence of 10 mu M unlabeled CsA, H-3-CsA efflux increased. H-3- CsA flux across RCEC layers showed marked directional asymmetry. The stroma l (S) to tear (T) side transcellular H-3-CsA permeability coefficient (P-tr ans) was approximately seven times higher than that in the T-to-S direction . The S-to-T P-trans was reduced by an MDR-reversing agent by up to 40%. We stern blot analysis of lysates revealed a 170-kDa membrane protein band. CONCLUSIONS. These results suggest that in RCEC the tear-side-facing membra ne has a P-gp-mediated drug efflux pump. In addition, there is suggestive e vidence for the presence of the cytosolic protein, cyclophilin. The presenc e of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.