Sc. Lu et al., BIDIRECTIONAL GLUTATHIONE TRANSPORT BY CULTURED HUMAN RETINAL-PIGMENTEPITHELIAL-CELLS, Investigative ophthalmology & visual science, 36(12), 1995, pp. 2523-2530
Purpose. To characterize glutathione (GSH) transport by cultured human
retinal pigment epithelial (HRPE) cells. Methods. Cultured HRPE cells
were pretreated with acivicin for GSH efflux and with buthionine sulf
oximine for GSH uptake to prevent the breakdown and resynthesis of GSH
. Efflux was measured by the linear rate of accumulation of GSH in the
supernatant; uptake was measured using [S-35] GSH plus varying concen
trations of GSH. Molecular forms were verified by high-performance liq
uid chromatography. HRPE cell mRNA was probed for the presence of the
two recently cloned rat sinusoidal and canalicular GSH transporters, (
RsGshT and RcGshT), by Northern blot analysis. Results. Glutathione ef
flux was temperature dependent (undetectable at 4 degrees C), and it a
veraged 23 +/- 3.3 pmol/10(6) cells/minute or 10% of the total GSH eff
luxed per hour (total cell GSH = 13.6 +/- 1.5 nmol/10(6) cells). Efflu
x was not influenced by dithiothreitol or sulfobromophthalein-reduced
GSH adduct, agents known to affect liver sinusoidal GSH transport. Glu
tathione uptake tvas linear up to 45 minutes and was temperature depen
dent. The difference between 37 degrees C and 4 degrees C uptake value
s represented true uptake. Glutathione uptake (2 mu Ci/ml + 1 mM mass)
was Na independent and was inhibited significantly by phenol-3,6-dibr
omphthaleim disulfonate. The kinetics of GSH uptake was assessed by me
asuring uptake with S-35-GSH and 0.05 to 40 mM extracellular GSH for 3
0 minutes. Uptake was saturable with V-max = 18.7 +/- 1.7 nmol/10(6) c
ells/30 minutes, K-m = 12.1 +/- 1.9 mM, n (binding site) = 1. On North
ern blot analysis, HRPE cells express mRNA for RcGshT but not for RsGs
hT. Conclusions. The similarities in functional characteristics of GSH
transport and the presence of RcGshT-like mRNA suggest GSH transport
in HRPE cells is mediated by a RcGshT homolog. Although the transporte
r can operate bidirectionally, it is expected to be a net efflux pump
under normal physiologic conditions because the intracellular GSH conc
entration is much higher.