Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines.
OBJECTIVE
Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump.


METHODS
Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp.


RESULTS
Permeability of [(3)H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10(-6) cm/s in the apical-to-basolateral and 5.1 x 10(-6) cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 micro M) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1.


CONCLUSIONS
Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.
Dey
Surajit
S
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Patel
Jignesh
J
Anand
Banmeet S
BS
Jain-Vakkalagadda
Blisse
B
Kaliki
Preetham
P
Pal
Dhananjay
D
Ganapathy
Vadivel
V
Mitra
Ashim K
AK
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D003315:Cornea / Q000378:metabolism*; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020112:Rhodamine 123 / Q000037:antagonists & inhibitors / Q000378:metabolism; D013739:Testosterone / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D020112:Rhodamine 123; D013739:Testosterone; D016572:Cyclosporine