Multidrug resistance protein 1 (MRP1) in rabbit conjunctival epithelial cells: its effect on drug efflux and its regulation by adenoviral infection.
OBJECTIVE
To evaluate the expression, localization, function, and regulation of multidrug resistance protein (MRP1) in rabbit conjunctival epithelial cells (RCEC).


METHODS
MRP1 gene expression in RCEC was determined by reverse transcription-polymerase chain reaction (RT-PCR), and MRP1 protein expression and its localization were determined by Western blot analysis and immunofluorescence using an anti-MRP1 monoclonal antibody, MRPr1. The effect of MRP1 on the transport and uptake of fluorescein was evaluated in RCEC grown on Transwell filters. Moreover, the effect of adenovirus type 5 (Ad5)-infected RCEC, and cytokines (Interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha (TNFalpha)) on MRP1 expression and leukotriene C4 (LTC4) uptake were investigated.


RESULTS
A 652 bp RT-PCR product from rabbit conjunctiva showed a 87% homology to human MRP1. Immunostaining with MRPr1 revealed a predominant basolateral localization of MRP1 in RCEC. Uptake of fluorescein, a MRP1 substrate, was increased (203-290%) in the presence of uricosuric drug probenecid at 100 microM, anti-inflammatory drug indomethacin at 10 microM and diclofenac, flurbiprofen, and ofloxacin at 1 mM, and by ATP depletion, but not influenced by the depletion of GSH, and the presence of antiviral cidofovir and anti-inflammatory drug cromolyn and prednisolone. Apical-to-basolateral facilitated transport of LTC4 was abolished in the presence of probenecid. Western blot analysis with MRPr1 revealed a distinct band at approximately 190 kDa for freshly isolated and cultured RCEC. Both Ad5 and cytokines (IL-1, IL-6, and TNF-alpha) up-regulated MRP1 expression, thereby reducing LTC4 uptake.


CONCLUSIONS
MRP1 appears to be primarily localized in the basolateral membrane of RCEC and function in the efflux of certain organic anions and inflammatory factors out of cells from the basolateral membrane. The upregulation in the expression of MRP1 by Ad5-infection and cytokines suggests a role of MRP1 in the transport of inflammatory factors during ocular inflammation. Supported by NIH grants EY12578, EY10421, and EY12356.
Yang
Johnny J
JJ
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA. johnny.yang@mpi.com
Ann
David K
DK
Kannan
Ram
R
Lee
Vincent H L
VH
D000256:Adenoviridae / Q000254:growth & development; D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D018938:Caco-2 Cells; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D019793:Fluorescein / Q000378:metabolism / Q000493:pharmacokinetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D017997:Leukotriene C4 / Q000378:metabolism / Q000493:pharmacokinetics; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D000894:Anti-Inflammatory Agents, Non-Steroidal; D019328:Buthionine Sulfoximine; D016207:Cytokines; D019793:Fluorescein; D007213:Indomethacin; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; C421109:multidrug resistance-associated protein 1