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Wanwisa Promsote, Sudha Ananth, Rajalakshmi Veeranan-Karmegam, Nevin Lambert, Chi-Chao Chan, Vadivel Ganapathy, Pamela M. Martin; The Cysteine Prodrug L-2-Oxothiazolidine-4-Carboxylic Acid (OTC) Elicits Potent Antioxidant and Anti-inflammatory Effects in RPE: Relevance to Treatment of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6560.
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L-2-oxothiazolidine-4-carboxylic acid (OTC), a pro-drug of cysteine, has been shown to be effective at increasing endogenous glutathione levels in a number of experimental model systems as well as in human patients. Transport of OTC across the plasma membrane occurs via the Na+-coupled monocarboxylate transporter SLC5A8 (SMCT1), a discovery made recently by our group. Interestingly, most SLC5A8 substrates have proven to also be ligands for the anti-inflammatory receptor GPR109A. Whether OTC is a GPR109A ligand, or has any effect on the cellular response to inflammation, has not been studied. Therefore in the present study we asked whether OTC: (1) is a ligand for GPR109A, (2) promotes anti-inflammatory signaling in RPE, and (3) reverses/prevents development/progression of AMD-like retinal complications in the Ccl2/Cx3cr1 double knockout (DKO) mouse model of the disease.
BRET, cAMP, and radioligand binding assays were used to study GPR109A-OTC interaction. ARPE-19 and primary RPE cells isolated from Gpr109a+/+ and Gpr109a-/- mouse eyes were treated with 10 ng/ml TNF-α in the presence or absence of various concentrations of OTC followed by analysis of IL-6 and Ccl2 expression by qPCR and ELISA. Niacin, a well-characterized GPR109A ligand, served as a positive control. DKO and corresponding age-/gender-matched controls were treated with various concentrations OTC either in drinking water or via oral gavage (0-4 months). Regular drinking water or PBS served as negative controls. Fundoscopic imaging was performed 1-2X/month. Additionally, mice were sacrificed at various timepoints and eyes harvested for preparation of RNA, protein, retinal cryosections and/or JB-4 plastic sections for use in qPCR, western blotting, immunofluorescence, and morphological analyses.
OTC is a ligand for GPR109A and suppresses TNF-α-induced IL-6 and Ccl2 expression/secretion in ARPE-19 and Gpr109a+/+ primary RPE. A similar effect was observed also in Gpr109a-/- primary RPE. In vivo experiments involving DKO animals showed OTC to be a very promising compound in terms of preventing or slowing progression of AMD-like retinal lesions.
OTC is a ligand for GPR109A and potently suppresses anti-inflammatory responses in RPE, an effect not solely dependent upon its interaction with GPR109A. The fact that OTC, a compound already proven safe-for-use in humans, has dual anti-oxidant and anti-inflammatory properties in RPE suggests that the compound may be useful as a therapeutic agent for treatment of retinal diseases such as AMD in which oxidative stress and inflammation are critically involved.
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