Vitamin C transport in human lens epithelial cells: Evidence for the presence of SVCT2

R. Kannan, A. Stolz, Q. Ji, P. D. Prasad, V. Ganapathy

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Vitamin C [ascorbic acid (AA)] is an important antioxidant present in mM amounts in the aqueous humor. Recently, two specific transporters for vitamin C (SVCT1, SVCT2) have been cloned in the rat and the human. The aim of the present study was to characterize vitamin C transport in an immortalized human lens epithelial cell line (HLE-B3). AA uptake was linear for 120 min in experiments conducted with 14C AA + 40 μM unlabelled AA. Uptake was measured at varying AA concentrations (0.04-1 mM) in Na+-containing and Na+-free buffers for 30 min at 37°C. Effect of potential inhibitors of AA transport was also examined. Presence (or absence) of SVCT1 and SVCT2 was studied by RT-PCR of HLE-B3 poly (A)+ RNA using gene specific primers. Uptake studies revealed that AA uptake was highly Na+-dependent and exhibited saturation. Na+-dependent 14C-AA uptake was strongly inhibited (85-90 %) by 10 mM unlabelled AA, Incubation of HLE-B3 cells with cAMP (0.1 mM), cytocholasin B (0.1 mM) and phorbol dibutyrate (1 μM) resulted in partial inhibition (36-51%) of AA uptake. Under similar conditions, D-glucose (10 mM) and staurosporine (0.1 μM) had no effect. RT-PCR showed the presence of SVCT2 while SVCT1 could not be amplified. Exposure to the chemical oxidant tertbutylhydroperoxide (TBH) up-regulated SVCT2 gene expression in HLE-B3 cells. Our data suggest that Na+-dependent transport of AA in normal lens epithelium is most likely mediated by SVCT2 rather than by SVCT1. This transport system may be subject to regulation by oxidant stress and by various second messenger signals.

Original languageEnglish (US)
Pages (from-to)159-165
Number of pages7
JournalExperimental eye research
Volume73
Issue number2
DOIs
StatePublished - Jan 1 2001

Fingerprint

Lenses
Ascorbic Acid
Epithelial Cells
Oxidants
Polymerase Chain Reaction
Staurosporine
Aqueous Humor
Second Messenger Systems
Buffers
Epithelium
Antioxidants
Gene Expression
Glucose

Keywords

  • Ascorbic acid
  • Human lens epithelial cells
  • Membrane transport
  • Oxidant stress
  • SVCT1
  • SVCT2

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Vitamin C transport in human lens epithelial cells : Evidence for the presence of SVCT2. / Kannan, R.; Stolz, A.; Ji, Q.; Prasad, P. D.; Ganapathy, V.

In: Experimental eye research, Vol. 73, No. 2, 01.01.2001, p. 159-165.

Research output: Contribution to journalArticle

Kannan, R. ; Stolz, A. ; Ji, Q. ; Prasad, P. D. ; Ganapathy, V. / Vitamin C transport in human lens epithelial cells : Evidence for the presence of SVCT2. In: Experimental eye research. 2001 ; Vol. 73, No. 2. pp. 159-165.
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abstract = "Vitamin C [ascorbic acid (AA)] is an important antioxidant present in mM amounts in the aqueous humor. Recently, two specific transporters for vitamin C (SVCT1, SVCT2) have been cloned in the rat and the human. The aim of the present study was to characterize vitamin C transport in an immortalized human lens epithelial cell line (HLE-B3). AA uptake was linear for 120 min in experiments conducted with 14C AA + 40 μM unlabelled AA. Uptake was measured at varying AA concentrations (0.04-1 mM) in Na+-containing and Na+-free buffers for 30 min at 37°C. Effect of potential inhibitors of AA transport was also examined. Presence (or absence) of SVCT1 and SVCT2 was studied by RT-PCR of HLE-B3 poly (A)+ RNA using gene specific primers. Uptake studies revealed that AA uptake was highly Na+-dependent and exhibited saturation. Na+-dependent 14C-AA uptake was strongly inhibited (85-90 {\%}) by 10 mM unlabelled AA, Incubation of HLE-B3 cells with cAMP (0.1 mM), cytocholasin B (0.1 mM) and phorbol dibutyrate (1 μM) resulted in partial inhibition (36-51{\%}) of AA uptake. Under similar conditions, D-glucose (10 mM) and staurosporine (0.1 μM) had no effect. RT-PCR showed the presence of SVCT2 while SVCT1 could not be amplified. Exposure to the chemical oxidant tertbutylhydroperoxide (TBH) up-regulated SVCT2 gene expression in HLE-B3 cells. Our data suggest that Na+-dependent transport of AA in normal lens epithelium is most likely mediated by SVCT2 rather than by SVCT1. This transport system may be subject to regulation by oxidant stress and by various second messenger signals.",
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