Characterization of cyclosporin A transport in cultured rabbit corneal epithelial cells: P-glycoprotein transport activity and binding to cyclophilin.
OBJECTIVE
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 3H-CsA accumulation in confluent RCECs. Bidirectional 3H-CsA fluxes were measured across 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 3H-CsA was time and temperature dependent. Steady 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-methylglucose, MG, or 10 mM NaN3 and 3-O-MG. The largest increase was obtained with 10 mM NaN3 in combination with 3-O-MG. In their presence, uptake increased by 40%. A multidrug-resistance (MDR)-reversing agent (i.e., 500 microM verapamil, 100 microM vincristine, 100 microM progesterone, 100 microM testosterone, 500 microM quinidine, or 100 microM chlorpromazine) significantly increased 3H-CsA accumulation. The largest increase was obtained with 500 microM quinidine (i.e., 36%). Conversely, verapamil and vincristine produced the largest inhibition of 3H-CsA efflux (i.e., 19% and 28%, respectively). However, in the presence of 10 microM unlabeled CsA, 3H-CsA efflux increased. 3H-CsA flux across RCEC layers showed marked directional asymmetry. The stromal (S) to tear (T) side transcellular 3H-CsA permeability coefficient (Ptrans) was approximately seven times higher than that in the T-to-S direction. The S-to-T Ptrans was reduced by an MDR-reversing agent by up to 40%. Western blot analysis of lysates revealed a 170-kDa membrane protein band.


CONCLUSIONS
These results suggest that in RCEC the tear-side-facing membrane has a P-gp-mediated drug efflux pump. In addition, there is suggestive evidence for the presence of the cytosolic protein, cyclophilin. The presence of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.
Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmology Research Division, Ikoma-chi, Japan.
K
K
Kawazu
K
K
Yamada
M
M
Nakamura
A
A
Ota
D019325:3-O-Methylglucose / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D048429:Cell Size; D002478:Cells, Cultured; D016572:Cyclosporine / Q000493:pharmacokinetics*; D004338:Drug Combinations; D018432:Drug Resistance, Multiple; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D010539:Permeability; D011817:Rabbits; D019810:Sodium Azide / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004338:Drug Combinations; D019325:3-O-Methylglucose; D016572:Cyclosporine; D019810:Sodium Azide; D019696:Peptidylprolyl Isomerase