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MD Gandhi, AK Mitra; Evidence of a Neutral Amino Acid Transporter in ARPE - 19 Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3268.
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Purpose: To determine the presence of neutral amino acid transporter, peptide transporter PepT1 and membrane bound p-glycoprotein in ARPE – 19 cells. Method: ARPE-19 cells were used as in-vitro model for the experiments. Cells were cultured between passage 11-50 as confluent monolayers in DMEM/DME-F12. Uptake and transport studies were performed using the appropriate substrates. For transport studies, the cells were grown on collagen-coated membranes and experiments performed on confluent monolayers 37°C. Mannitol was used as a marker for paracellular permeability and monolayer integrity. 3H cyclosporine was used for transport studies that was carried out in apical to basolateral as well as basolateral to apical directions. Transport studies were carried out using side by side diffusion chambers. Samples were withdrawn from the receiver chambers at predetermined time intervals, between 5 min-2 hrs, and analyzed using liquid Scintillation counter. Transport studies were performed in triplicates at 37°C within 17-27 days of plating cells. Uptake studies were also performed using 12 well cluster plates at 37°C. Results: Uptake studies showed that there was no significant difference in uptake of PepT1 substrate glycyl sarcosine in both presence, and absence of PepT1 inhibitor. Further studies carried out indicated a probable lack of functional expression of the PepT1 transporter in this cell line. Preincubation with p-glycoprotein inhibitor Quinidine did not appreciably change the cyclosporine transport characteristics across the ARPE – 19 cells. These studies showed no significant levels of expression of p-glycoprotein in this cell line. Uptake studies using radiolabeled large neutral amino acids showed the possible presence of a neutral amino acid transporter. Inhibition studies showed an increased specificity of this transporter for L – amino acids compared to the corresponding D isomers. Conclusions: Lack of expression of P-glycoprotein is observed in ARPE-19 cells. The experimental data shows no evidence of PepT1 expression. This neutral amino acid transporter can be exploited for drug targeting in the eye using amino acid prodrugs of antiviral agents.
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