May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Transfection of the Human Sodium Dependentvitamin C Transporter (hSCVT2) Corrects the Impaired Uptake in Rodent Lens
Author Affiliations & Notes
  • M.E. M. Obrenovich
    Pathology,
    CASE, Cleveland, OH
  • X.X. Fan
    Pathology,
    CASE, Cleveland, OH
  • S.M. Jarvis
    Research School of Biosciences, University of Kent, Research School of Biosciences, University of Kent, United Kingdom
  • L. Reneker
    Department of Ophthalmology, University of Missouri, Columbia, MO
  • J.R. Reddan
    Biological Sciences, Oakland University, Rochester, MI
  • V.M. Monnier
    Pathology,
    Biochemistry,
    CASE, Cleveland, OH
  • Footnotes
    Commercial Relationships  M.E.M. Obrenovich, None; X.X. Fan, None; S.M. Jarvis, None; L. Reneker, None; J.R. Reddan, None; V.M. Monnier, None.
  • Footnotes
    Support  NEI/NIH EY 07099, T32 EY 07157 and P30EY11373
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1860. doi:
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      M.E. M. Obrenovich, X.X. Fan, S.M. Jarvis, L. Reneker, J.R. Reddan, V.M. Monnier; Transfection of the Human Sodium Dependentvitamin C Transporter (hSCVT2) Corrects the Impaired Uptake in Rodent Lens . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1860.

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

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Abstract

Abstract: : Purpose: Age–related pigmentation and crosslinking of human lens crystallins has been linked to the Maillard reaction resulting from glycation with reducing sugars or reactions with ascorbic acid (ASA). Searching for appropriate models of ascorbylation using 19F–ASA we uncovered profound differences in ASA uptake and homeostasis in mouse vs. to human lens epithelium (LE). Confirmation of the defect was crucial before developing a transgenic (Tg) mouse model of ASA pigmentation. Methods: Confluent human HLE B–3, rabbit LE N/N 1003A and mouse LE 17EM15 and 21EM15 were incubated with either 14C–ASA or 14C–DehydroASA (DHA) and F–ASA or F–DHA for up to 2 hours. Cells were washed, lysed and analyzed by 750 MHz NMR spectroscopy or liquid scintillation spectrometry for ASA transport. Cells and whole mouse lens explants were transfected with the human SVCT2 transporter (hSVCT2) using a CMV or α crystallin promoter and a Δ chick enhancer h SVCT2 construct (EN–hSVCT2). Results: F–ASA and F–DHA, were both taken up by SVCT2 in human and mouse cells. Moreover, up to ten fold less F–ASA or F–DHA or 14C–ASA uptake was found in mouse 17EM15 (p<0.0001) and 21EM15 cells (p<0.001) compared to human cell lines. The Na+–dependent component as percent of total uptake was ∼36%, 63% and 52% in HLE B–3, 17EM15 and 21EM15 cells respectively. SVCT2 activity was reduced by about 1/2, in HLE B–3 and 21EM15 cells, when cultured in high glucose. Plasmids containing a construct for the full–length human gene were transiently and stably overexpressed in CHO and mouse cells or mouse lens explants. The hSVCT2 construct, not mock transfection, rescued impaired ASA uptake in both mouse cells and whole lens explants. Conclusions: Our data confirm the defect in ASA uptake in the mouse lens, which is reversible with the hSVCT2 construct. In silico research revealed human and mouse share >80% SVCT2 gene homology but deletions and several mutations in the mouse may result in functional mouse impairments. Conversely, suppression of SVCT2 activity may be evolutionary and linked to the nocturnal life cycle, explaining why lens ASA concnetration is ∼ zero in rodents and 1–3 mM in humans. Currently, a Tg mouse was generated and expresses the EN–hSVCT2 transporter in the lens, which will serve as a model of lens pigmentation and crosslinking by ascorbylation.

Keywords: aging • receptors: pharmacology/physiology • metabolism 
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