May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Molecular Evidence and Functional Expression of P-Glycoprotein (MDR1) in Human and Rabbit Cornea and Corneal Epithelial Cell Line
Author Affiliations & Notes
  • S. Dey
    School of Pharmacy, Univ of Missouri Kansas City, Kansas City, MO, United States
  • J. Patel
    School of Pharmacy, Univ of Missouri Kansas City, Kansas City, MO, United States
  • B.S. Anand
    School of Pharmacy, Univ of Missouri Kansas City, Kansas City, MO, United States
  • B. Jain-Vakkalagadda
    School of Pharmacy, Univ of Missouri Kansas City, Kansas City, MO, United States
  • P.R. Kaliki
    School of Pharmacy, Univ of Missouri Kansas City, Kansas City, MO, United States
  • V. Ganapathy
    Biochemistry & Molecular Biology, Medical College of Georgia, Augusta, GA, United States
  • D. Pal
    Biochemistry & Molecular Biology, Medical College of Georgia, Augusta, GA, United States
  • A.K. Mitra
    Biochemistry & Molecular Biology, Medical College of Georgia, Augusta, GA, United States
  • Footnotes
    Commercial Relationships  S. Dey, None; J. Patel, None; B.S. Anand, None; B. Jain-Vakkalagadda, None; P.R. Kaliki, None; V. Ganapathy, None; D. Pal, None; A.K. Mitra, None.
  • Footnotes
    Support  EY09171-08, EY10659-07
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1376. doi:
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      S. Dey, J. Patel, B.S. Anand, B. Jain-Vakkalagadda, P.R. Kaliki, V. Ganapathy, D. Pal, A.K. Mitra; Molecular Evidence and Functional Expression of P-Glycoprotein (MDR1) in Human and Rabbit Cornea and Corneal Epithelial Cell Line . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1376.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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