April 2011
Volume 52, Issue 14
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ARVO Annual Meeting Abstract  |   April 2011
Cyclic Stretch Increased Intracellular Succinate Concentration In Cultured Retinal Pigment Epithelial Cells Through A Calcium Dependent Pathway
Author Affiliations & Notes
  • Hirofumi Kinoshita
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Kiyoshi Suzuma
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Toshihide Maki
    Joint Research Center, Nagasaki University, Nagasaki, Japan
  • Makiko Matsumoto
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Mao Kusano
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Masafumi Uematsu
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Takashi Kitaoka
    Ophthalmology & Vis Sci, Nagasaki Univ Grad Sch of BioMed Sci, Nagasaki, Japan
  • Footnotes
    Commercial Relationships  Hirofumi Kinoshita, None; Kiyoshi Suzuma, None; Toshihide Maki, None; Makiko Matsumoto, None; Mao Kusano, None; Masafumi Uematsu, None; Takashi Kitaoka, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1796. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • retinal neovascularization • age-related macular degeneration 
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