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Brandi S Betts-Obregon, Federico Gonzalez-Fernandez, Andrew S Mendiola, Andrew T C Tsin; Interphotoreceptor retinoid-binding protein promotes delivery of all-trans retinol into rat Müller Cells in culture.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3472. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Interphotoreceptor Retinoid-Binding Protein (IRBP), a major component of the interphotoreceptor matrix, is thought to thought to function in the cone visual cycle. However, the mechanism that IRBP facilitates cone - Müller cell retinoid trafficking is unknown. IRBP binds to the pericellular matrix of the Müller cell villi. We hypothesize that IRBP facilitates the delivery of all-trans retinol (ATOL) into rMC-1 cells in culture.
IRBP was purified from bovine retina by a combination of ion exchange, ConA affinity, and size exclusion chromatography. The concentration of the purified IRBP was confirmed by amino acid analysis. Rat Müller cells (rMC-1) were seeded into 6-well plates at 2.5 x 105 cells per mL per well. BSA or apo-bovine IRBP (bIRBP) was pre-incubated with ATOL before the ATOL/protein mixture was introduced into the culture medium at a final concentration of 10µM ATOL and 2µM BSA or bIRBP. rMC-1 homogenates and culture media were collected at 0, 12 and 24 hours, followed by retinol extraction and HPLC analysis.
After incubation with ATOL and BSA, ATOL in the cell media decreased from 2200 pmol/mL at 0 hour to 1700 pmol/mL at 12 hours, and then to 1132 pmol/mL at 24 hours (n=6). After incubation with ATOL and bIRBP, ATOL decreased from 2200 pmol/mL at 0 hours to 1200 pmol/mL at 12 hours of incubation, and then to 704 pmol/mL at 24 hours (n=9). Homogenates from Müller cells without the addition of ATOL to cell media or with the addition of ATOL without BSA or bIRBP (i.e. ATOL in ethanol dispersed directly into the cell media at a final concentration of 10 µM) did not yield ATOL. However, homogenates from Müller cells incubated with ATOL and BSA for 12 hours yielded 1788 pmol/mg protein, and this level increased to 3452 pmol/mg at 24 hours (n=6). After incubation of ATOL and bIRBP for 12 hours, cell homogenate yielded 2100 pmol/mg protein, and this level increased to 6900 pmol/mg protein at 24 hours (n=9).
Incubation of ATOL and BSA or bIRBP resulted in a time-dependent increase of ATOL delivery into rat Müller cells in culture. In comparison to BSA, bIRBP facilitated a 2-fold higher accumulation of ATOL within a 24 hour incubation period. This may be attributable to the higher binding specificity and protective effect of retinol by bIRBP, and/or ability of IRBP to interact with the Müller cell.
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