Vitamin C [ascorbic acid (AA)] is an important antioxidant present in m M 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 microM unlabelled AA. Uptake was measured at varying AA concentrations (0.04-1 m M) in Na+-containing and Na+-free buffers for 30 min at 37 degrees 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 m M unlabelled AA. Incubation of HLE-B3 cells with cAMP (0.1 m M), cytocholasin B (0.1 m M) and phorbol dibutyrate (1 microM) resulted in partial inhibition (36-51%) of AA uptake. Under similar conditions, D -glucose (10 m M) and staurosporine (0.1 microM) had no effect. RT-PCR showed the presence of SVCT2 while SVCT1 could not be amplified. Exposure to the chemical oxidant tert-butylhydroperoxide (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.
Copyright 2001 Academic Press.