ATB0,+ is a Transporter for Homocysteine (Hcy) in Retinal Cells

Abstract

Abstract: : Purpose: Hcy is an excitatory amino acid that induces apoptotic death of retinal ganglion cells in vivo (Moore et al, 2001). Elevated plasma Hcy has been reported in diabetic patients. Some reports suggest a relationship between increased Hcy and diabetic retinopathy. The mechanism by which Hcy enters retinal cells is not known. ATB0,+ is a Na+- and Cl--dependent transporter for neutral and cationic amino acids. In this study, we asked whether ATB0,+ is present in retina and if it transports Hcy. Methods: The expression of ATB0,+ was analyzed by RT-PCR and in situ hybridization (ISH) in mouse eyes. ATB0,+ protein was studied immunohistochemically in intact mouse eyes and in three retinal cells lines, ganglion (RGC-5), Müller (RMC1) and RPE (ARPE-19). The function of ATB0,+ was assessed using the cloned transporter in heterologous expression systems. Results: RT-PCR analysis of isolated mouse neural retina and RPE amplified the expected 878 bp product based on the location of the primers in ATB0,+ cDNA. ISH showed that ATB0,+ is expressed in RGCs, around the soma of select cells in the inner nuclear layer, in inner segments of photoreceptor cells and in RPE. Immunolocalization studies showed that the distribution pattern for ATB0,+ is quite similar to its gene expression pattern. ATB0,+ was detected in all three retinal cell lines. In uptake studies using transfected HRPE cells, Hcy was as effective as cysteine in competing with glycine for transport via the cloned ATB0,+ (IC50 values for Hcy and cysteine were 45 ± 7 and 64 ± 5 µM, respectively). Electrophysiological analysis using the X. leavis oocytes expressing the cloned ATB0,+ showed that Hcy induced Na+- and Cl--dependent inward currents, which were not detected in control oocytes. Conclusions: ATB0,+ is expressed in several retinal cell types, particularly ganglion, Müller and RPE. Uptake and electrophysiological studies demonstrate its ability to transport Hcy. Future studies will determine whether the expression of ATB0,+ in retinal cells is altered in diabetes and how this would influence the Hcy-induced changes in retinal function.

Keywords: excitatory neurotransmitters • oxidation/oxidative or free radical damage • diabetic retinopathy