Molecular Identification of Membrane Potential Driven Organic Cation Transporters in the Rabbit Conjunctival Epithelium

Abstract

Abstract: : Purpose: We have identified the expression of a proton-dependent organic cation transporter (OCTN2) in the rabbit conjunctival epithelium before. Functional evidence suggested the involvement of a membrane potential-dependent organic cation transporter (OCT) in the penetration of organic cations across the conjunctiva. Here, we investigated the molecular identity of the OCT in the rabbit conjunctival epithelial cells (RCEC). Methods: Total RNA was extracted from freshly isolated RCEC and cultured RCEC using TRIZOLâ reagent. RT-PCR of OCT1, OCT2 and OCT3 gene transcript was conducted using the following primers: rbOCT1s, 5'-CCG CTG ACC TCA AGA TG-3'and rbOCT1as, 5'-CTG CCA GAC CTC CAT CA-3'; rbOCT2s 5'-GGA AGC ACA CCT GCA TCT TG-3' and rbOCT2as 5'-GAG ATT CCT GAT GAA CGT GG-3'; rbOCT3s 5'-CTG GGT GGT CCC TGA GTC TCC CCG-3' and rbOCT3as 5'-GCG TCG TCC AAG GCG CTC AAT GGT-3'. After visualized on a 1.5% agarose gel stained with ethidium bromide, the sample band was purified using QIAquickTM Gel Extraction Kit and was sequenced by Genemed Synthesis, Inc. Poly(A)+RNA was purified using Oligotex mRNA Mini Kit. mRNA samples (5 mg/lane) were fractionated and probed with HRP-labeled 335bp rbOCT3 cDNA fragment. Western blot was conducted to detect OCT3 using polyclonal rabbit anti-rOCT3 antibody (Alpha Diagnostic International) in crude membrane proteins prepared from freshly isolated RCEC and cultured RCEC. Results: No band representing OCT1 or OCT2 was amplified by as high as 35 cycles of RT-PCR in freshly isolated RCEC and cultured RCEC, whereas a 335 bp OCT3 fragment was identified. The sequence of this fragment showed 99% identity with the 216 bp rabbit OCT3 cDNA fragment in NCBI Genbank, and 85% to 88% identity with the corresponding sequences of human, rat and mouse OCT3 cDNA. Northern blot showed that OCT3-specific transcript, 3.4 kb in size, is present in freshly isolated RCEC and cultured RCEC. Western Blot identified OCT3, 65 KD in size, in membrane proteins prepared from freshly isolated RCEC and cultured RCEC. Conclusions: OCT3, not OCT1 or OCT2, is expressed in rabbit conjunctival epithelial cells. OCT3 might be involved in the organic cation transport mechanism reported in the rabbit conjunctival epithelium.

Keywords: conjunctiva • molecular biology • anterior segment