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N Zhang, VH L Lee; Molecular Identification of Organic Cation Transporters in the Rabbit Conjunctival Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3261.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the molecular identity of the carrier mediated transport system for organic cations in the rabbit conjunctival epithelium Methods: Total RNA was extracted from freshly isolated rabbit conjunctival epithelial cells using TRIZOL® Reagent. RT-PCR was performed using degenerate primers based on highly conserved regions of all cloned organic cation transporters OCTs and OCTNs in human, mouse and rat. The PCR product was subcloned into pGEM-T® vector and sequenced. Sequence similarity was determined using the NCBI BLAST program. To confirm the possible existence of OCTN2, uptake studies were carried out in primary rabbit conjunctival epithelial cell culture (RCEC) using 1.6mCi/ml (20nM) [3H]L-carnitine, a typical substrate of OCTN2. All uptake studies were conducted in the bicarbonated Ringer’s solution (BRS) maintained at pH7.4 under 95% air/5% CO2. Results: A 472bp fragment was generated by RT-PCR. The resulting sequence showed highest homology to OCTN2 of other species (85%-86%), followed by OCTN1 (66%-73%) and OCTN3 (71%). Functional characterization showed that apical uptake of 20nM L-carnitine by RCEC over 10 min was 5 times higher than basolateral uptake. Apical uptake was almost abolished when the temperature was lowered to 40C or when Na+ was excluded from BRS on both sides of the RCEC. In addition, apical uptake was inhibited by several organic cations, including 1mM verapamil (by 90%); 2.5mM guanidine (by 20%), TEA (by 37%), and cimetidine (by 75%). Conclusion: OCTN2 is expressed in the rabbit conjunctival epithelium. (Supported in part by EY12356)
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