👤 Penny Roon

To analyze the mechanisms of folate uptake in retinal Müller cells. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Müller cells, and rMC-1 cells for the three known folate transport proteins folate receptor alpha (FRalpha), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Laser scanning confocal microscopy (LSCM) and immunoelectron microscopy were used to determine the subcellular location of FRalpha and PCFT in primary Müller cells. The pH dependence of the uptake of [(3)H]-methyltetrahydrofolate ([(3)H]-MTF) was assayed in Müller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. FRalpha and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Müller cells and rMC-1 cells. LSCM localized these proteins to the plasma membrane, nuclear membrane, and perinuclear region. Immunoelectron microscopic studies revealed the colocalization of FRalpha and PCFT on the plasma membrane and nuclear membrane and within endosomal structures. Müller cell uptake of [(3)H]-MTF was robust at pH 5.0 to 6.0, consistent with PCFT activity, but also at neutral pH, reflecting RFC function. RFC was expressed in mouse Müller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. FRalpha and PCFT are expressed in retinal Müller cells and colocalize in the endosomal compartment, suggesting that the two proteins may work coordinately to mediate folate uptake. The unexpected finding of RFC expression and activity in cultured Müller cells may reflect the upregulation of this protein under proliferative conditions. Show less

GPR109A has been identified as a G-protein-coupled receptor for niacin. beta-hydroxybutyrate (beta-HB) is a physiologic ligand for the receptor. beta-HB, the predominate ketone body in circulation, is an important energy source for neurons, including retinal neurons, under various physiologic and pathologic conditions. The identification of GPR109A as the receptor for beta-HB suggests additional, hitherto unknown, functions for this metabolite. The circulating levels of beta-HB increase in diabetes. Since retinopathy is a serious complication associated with diabetes, we investigated GPR109A expression in retina and in different retinal cell types to determine if the receptor may have a role in the pathophysiology of diabetic retinopathy. RT-PCR, fluorescent in situ hybridization, and immunofluorescent techniques were used to analyze GPR109A expression in mouse retina and in three transformed retinal cell lines: ARPE-19 (RPE), RGC-5 (ganglion), and rMC-1 (Müller). Activation of GPR109A by niacin and beta-HB was demonstrated in ARPE-19 cells by cAMP assay. Studies conducted using mouse retinal tissues demonstrated that GPR109A is expressed in retina with its expression restricted to RPE, where it differentially polarizes to the basolateral membrane. These results were confirmed with cell lines, which demonstrated GPR109A expression in ARPE-19, but not in rMC-1 and RGC-5 cells. Primary cultures of mouse RPE also showed robust expression of GPR109A. cAMP assay demonstrated that GPR109A expressed in RPE is functional. These data represent the first report on GPR109A expression in retina. The exclusive expression of GPR109A in RPE basolateral membrane, which has access to beta-HB in blood, may have biologic importance in diabetic retinopathy. Show less

Monocarboxylates are primary energy substrates in the retina. Recently, the authors identified two sodium-coupled monocarboxylate transporters (SMCTs), SMCT1 (a high-affinity transporter) and SMCT2 (a low-affinity transporter). Expression of SMCT1 and SMCT2 has been studied in several tissues; however, little is known about their expression in retina. In the present study, the authors asked whether SMCT1 and SMCT2 are also expressed in retina and, if so, in which particular retinal cell types. SMCT1 and SMCT2 expression was analyzed in intact mouse retina and cultured retinal cells (ganglion, Müller, RPE) by RT-PCR, in situ hybridization, and immunofluorescence. Uptake assays were performed to demonstrate SMCT1 (RGC-5 and ARPE-19 cells) and SMCT2 (rMC-1 cells) expression at the functional level. SMCT1 mRNA and protein were detected in the ganglion cell layer, inner nuclear layer, inner/outer plexiform layers, photoreceptor inner segments, and RPE. In RPE, the expression of SMCT1 was restricted to the basolateral membrane. SMCT2 mRNA and protein were detected only in neural retina, with a pattern of protein localization consistent with labeling of Müller cells. In vitro studies confirmed the cell type-specific expression of SMCT1 and SMCT2. Uptake assays demonstrated Na(+)-coupled monocarboxylate transport in RGC-5, ARPE-19, and rMC-1 cells. These data provide the first evidence for the expression of SMCT1 and SMCT2 in the retina and for the cell-type specific distribution of these transporters within the retina. These studies suggest that SMCT1 and SMCT2 play a differential role in monocarboxylate transport in the retina in a cell type-specific manner. Show less