Existence of a p-glycoprotein drug efflux pump in cultured rabbit conjunctival epithelial cells.
OBJECTIVE
To determine whether a p-glycoprotein (P-gp) drug efflux pump exists in cultured rabbit conjunctival epithelial cells (RCEs) to restrict the absorption of cyclosporin A (CSA) and other lipophilic drugs such as verapamil and dexamethasone.


METHODS
The anti-P-gp monoclonal antibody (mAb) C219 was used in western blot analysis to reveal the presence of P-gp in freshly isolated and cultured RCEs. Bidirectional transport of tritiated CSA, verapamil, and dexamethasone (0.5 or 5.0 microM) across cultured RCEs was evaluated in the absence and presence of P-gp inhibitors and an external mAb to P-gp (4E3).


RESULTS
Western blot analysis of lysates of freshly isolated and cultured RCEs with C219 mAb revealed a 170-kDa membrane protein band. At 0.5 microM CSA, the basal-to-apical (ba) apparent permeability coefficient (P(app) that is, efflux) was 9.3 times higher than that in the apical-to-basal direction (that is, influx). At 5 microM, this ratio was halved. Net CSA secretion was blocked completely at 4 degrees C. Verapamil (100 microM), progesterone (100 microM) and 4E3 mAb (5 microg/ml) increased CSA influx three times, while reducing efflux by 50% to 70%. Verapamil and progesterone inhibited CSA efflux in a concentration-dependent manner. In all cases, net secretory CSA flux was markedly reduced. The P(app) for verapamil (0.5 microM) and dexamethasone (0.5 microM) in the ba direction was 3.4 and 1.6 times, respectively, which was higher than that in the opposite direction. The 4E3 mAb reduced net verapamil secretion by 65%.


CONCLUSIONS
There may exist a P-gp-mediated drug efflux pump on the apical aspect of the rabbit conjunctiva to restrict the absorption of cyclosporin A and other lipophilic drugs.
Saha
P
P
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles 90033, USA.
Yang
J J
JJ
Lee
V H
VH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000042:Absorption; D000818:Animals; D000911:Antibodies, Monoclonal; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016572:Cyclosporine / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D016623:Ion Pumps / Q000378:metabolism*; D008297:Male; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D011817:Rabbits; D014700:Verapamil / Q000378:metabolism / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D016623:Ion Pumps; D004364:Pharmaceutical Preparations; D011374:Progesterone; D003907:Dexamethasone; D016572:Cyclosporine; D014700:Verapamil