April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Quantification of Expression of B-Crystallin in Mitochondria in Human Retinal Pigment Epithelial Cells and Its Secretion Using Enzyme-Linked Immunosorbent Assay (ELISA)
Author Affiliations & Notes
  • M. Kitamura
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • P. G. Sreekumar
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • S. Sonoda
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • C. Spee
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • S. J. Ryan
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • R. Kannan
    Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • D. R. Hinton
    Doheny Eye Institute, Los Angeles, California
    Pathology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • Footnotes
    Commercial Relationships  M. Kitamura, None; P.G. Sreekumar, None; S. Sonoda, None; C. Spee, None; S.J. Ryan, None; R. Kannan, None; D.R. Hinton, None.
  • Footnotes
    Support  NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel Beckman foundation
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1414. doi:https://doi.org/
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      M. Kitamura, P. G. Sreekumar, S. Sonoda, C. Spee, S. J. Ryan, R. Kannan, D. R. Hinton; Quantification of Expression of B-Crystallin in Mitochondria in Human Retinal Pigment Epithelial Cells and Its Secretion Using Enzyme-Linked Immunosorbent Assay (ELISA). Invest. Ophthalmol. Vis. Sci. 2010;51(13):1414. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The aim of the present study was to a) standardize and validate an enzyme-linked immunosorbent assay (ELISA) for quantification of αB-crystallin and b) to apply the assay for measurement of αB-crystallin in mitochondria and secretion into extracellular media of human cultured fetal RPE.

Methods: : Early passage, confluent, cultured fetal human RPE cells and polarized RPE cells were used for the study. RPE cells were switched to serum free medium for 16 h and were then exposed to 150 µM tert-butylhydroperoxide (tBH) for 2-4 h. Cytosol and mitochondria were isolated using a Mitochondria/Cytosol Fractionation Kit for αB-crystallin determination by ELISA using reagents from Stressgen. In separate experiments, 24 h secretion of αB crystallin into extracellular medium was measured in polarized and non-polarized RPE under unstimulated conditions.

Results: : The ELISA standard curves were linear in the range 1.25 ng/ml-40 ng/ml (r2=0.9996). The levels of αB-crystallin (ng/mg protein) in cytosol and mitochondria of non-polarized RPE were 2522 ± 61 and 162 ± 4.7 respectively. tBH-induced oxidative stress caused an increase in mitochondrial expression of αB-crystallin while the cytosolic pool decreased. The 24 h secretion of αB-crystallin in non polarized RPE was 2.22 ± 0.55 ng/106 cells. αB-crystallin was selectively secreted to the apical side with negligible secretion to the basolateral side.

Conclusions: : Our data show that αB-crystallin is expressed in mitochondria and is regulated by oxidative stress. Using polarized RPE, we could also obtain quantitative evidence for preferential secretion of αB-crystallin to the apical surface.

Keywords: crystallins • oxidation/oxidative or free radical damage • retinal pigment epithelium 
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