Molecular Evidence and Functional Expression of P-Glycoprotein (MDR1) in Human and Rabbit Cornea and Corneal Epithelial Cell Line

Abstract

Abstract: : Purpose: Efflux pump like P-glycoprotein (P-gp) is believed to be a major barrier to drug delivery. The purpose of this work was to determine if cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump. Methods: Cultured primary rabbit corneal epithelial cells (rPCEC) and a corneal cell line (SIRC) 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 done using appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing and protein translation were performed to confirm P-gp. Results: Permeabilites of [³H] CsA across SIRC were found 1.74 X 10^-6cm/s in apical to-basolateral and 5.1 X 10^-6cm/s in basolateral-to-apical direction. Uptake of Rho-123 across both SIRC and rPCEC was time and temperature dependent. Rho-123 uptake in SIRC was 14.4 pmoles/mg protein and and in the presence of CsA (10 microM) was 70.8 pmoles/mg protein in 3 hrs. Uptake in rPCEC had the highest uptake in 3 hours. CsA was found to be a very potent inhibitor (IC₅₀=2 mM) of Rho-123 uptake. Testosterone was not very potent but it did increase uptake 2.2 fold at 200 microM concentration. A series of P-gp inhibitors were checked to confirm their inhibitory potential. CsA (10 microM) showed the highest inhibition (2.49 fold increase in Rho-123 uptake) whereas testosterone (100 microM) showed almost no inhibition (1.07 fold increase). Quinidine, verapamil, progesterone and all other inhibitors showed significant P-gp inhibition (1.43 - 2.49 fold increase in Rho-123 uptake). Sodium azide, a metabolic poison, inhibited P-gp mediated Rho-123 efflux. Rho-123 uptake was increased 2.7 fold in the presence of 1 mM NaN₃. 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 translated from the fragment product indicated 89% homology with human MDR1. Conclusions:Functional and molecular characterization proves the existence of P-gp in human cornea, rabbit cornea and cell lines. This knowledge of existence of P-gp will help in better ocular drug delivery strategies.

Keywords: cornea: epithelium • ion transporters • pharmacology