title: Molecular expression and functional activity of efflux and influx transporters in hypoxia induced retinal pigment epithelial cells.
abstract: A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
authors.forename: Ramya Krishna
authors.initials: RK
authors.lastname: Vadlapatla
authors.forename: Aswani Dutt
authors.initials: AD
authors.lastname: Vadlapudi
authors.forename: V K Chaithanya
authors.initials: VK
authors.lastname: Ponnaluri
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Mridul
authors.initials: M
authors.lastname: Mukherji
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D060433:Administration, Ophthalmic; D001692:Biological Transport; D015153:Blotting, Western; D015687:Cell Hypoxia; D002460:Cell Line; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000008:administration & dosage / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D026901:Membrane Transport Proteins; D004364:Pharmaceutical Preparations; D012333:RNA, Messenger
keywords: Efflux and influx transporters; Hypoxia; Molecular expression and functional activity; Retinal pigment epithelial cells