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Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-glycoprotein Transport Activity and Binding to Cyclophilin

Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-glycoprotein Transport Activity and Binding to Cyclophilin

K Kawazu, K Yamada, M Nakamura, A Ota

Invest Ophthalmol Vis Sci. 1999 Jul;40(8):1738-44.

PMID: 10393043

Abstract:

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 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42416934	Anti-phospho-NFAT3 (pSer168/170) antibody produced in rabbit			Price

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