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Hirofumi Kinoshita, Kiyoshi Suzuma, Toshihide Maki, Makiko Matsumoto, Mao Kusano, Masafumi Uematsu, Takashi Kitaoka; Cyclic Stretch Increased Intracellular Succinate Concentration In Cultured Retinal Pigment Epithelial Cells Through A Calcium Dependent Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1796.
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Recently it is suggested that posterior vitreomacular adhesion is a potential risk factor for exudative age-related macular degeneration (AMD) and the succinate receptor GPR91 in neurons has a major role in retinal angiogenesis. We evaluated the expression of succinate in retinal pigment epithelial (RPE) cells undergoing clinically relevant cyclic stretch to reveal role of succinate and mechanical stress in AMD.
Succinate extracted from ARPE19 cells, a human RPE cell line, in culture undergoing mechanical stretch were evaluated. Cells were seeded on six-well flexible-bottom plates coated with collagen1 containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin G (100IU/ml), streptomycin (100µg/ml). When ARPE19 cells were cultured to near confluency, the cells were incubated for 24 hours in 5%CO2 at 37°C with serum free DMEM. The cells were subjected to a 5, 10 or 15% cyclic stretch maintained for 1-9 hours using a computer-controlled vacuum stretch apparatus (Flexcer Cell Strain Unit; Flexcel). The medium was decanted and cells lysed directly in the culture plates with TNE buffer. Succinate concentration was assessed by enzymatic analysis of succinate and selected ion monitoring mode of liquid chromatography-mass spectrometry, and normalized with total protein quantity. Moreover succinate concentration after treatment with pharmacological inhibitors were evaluated to speculate the pathway of succinate activity in cells.
10% cyclic stretch maximally increased succinate(mg) / total protein(g) after 2 hours (30.1±1.3 to 46.5±9.6 P<0.05 Tukey test). BAPTA/AM, an intracellular calcium chelator, inhibited stretch-induced succinate increase in ARPE19 cells significantly by 86.9%, however GFX (PKC inhibitor), LY294002 (PI3 kinase inhibitor), Genistein (tyrosine kinase inhibitor), or PD98059 (MEK1 inhibitor) had no effect.
Cyclic stretch increased intracellular succinate concentration in cultured RPE cells through a calcium dependent pathway. Our data suggest that succinate has a role in pathology of AMD, and may be a target for medical treatment.
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