Net glutathione secretion across primary cultured rabbit conjunctival epithelial cell layers.
OBJECTIVE
Metabolism and transport of glutathione (GSH), the endogenous thiol antioxidant, in conjunctival tissue to date are poorly understood. The purpose of the present study was to define transport characteristics of GSH in primary cultured rabbit conjunctival epithelial cells (RCECs).


METHODS
RCECs were grown on membrane filters to exhibit tight barrier properties (transepithelial electrical resistance, TEER, approximately 1 k(Omega)/cm(2)). Uptake, efflux, and transepithelial transport of GSH were determined in the presence or absence of extracellular Na(+) under conditions of inhibition of GSH biosynthesis and degradation. Uptake was determined at 15 minutes after instillation of (3)H-GSH to the apical or basolateral bathing fluid. GSH efflux was estimated from the time course of release of prebiosynthesized (35)S-GSH. Transepithelial transport was assessed by instillation of (3)H-GSH in either the apical or basolateral bathing fluid, followed by sampling from respective contralateral sides.


RESULTS
Apical uptake and efflux showed Na(+) dependency up to 65%. GSH uptake in the initial 15 minutes was linear in the presence of 1 mM GSH (labeled and unlabeled) in Na(+)-containing buffer. The uptake rate was higher from the apical fluid than from the basolateral fluid. A Hill analysis of the Na(+)-dependent process yielded a coupling ratio for Na(+) to GSH of 1.25:1. The efflux rate of GSH into the apical fluid was marginally dependent on the apical presence of Na(+) and was significantly greater than that in the basolateral fluid. Basolateral efflux of GSH was primarily Na(+) independent, whereas basolateral uptake almost exclusively was Na(+) dependent. Depolarizing the RCEC membrane potential decreased GSH efflux into either apical or basolateral fluids (5 pmol/min small middle dot 10(6) cells). Hyperpolarization significantly increased the rate of GSH efflux into the apical fluid (120 pmol/min small middle dot 10(6) cells), whereas the basolateral efflux was not affected. Apparent permeability of GSH across RCEC layers was approximately eight times higher in the basolateral-to-apical (secretion) direction than the opposite (absorption) direction.


CONCLUSIONS
GSH is transported across RCEC membranes by both Na(+)-dependent and -independent processes. Analysis of the Na(+)-dependent uptake process gave an approximate 1:1 coupling ratio for Na(+)-GSH cotransport. The Na(+)-independent component is highly sensitive to cell membrane potential. Net secretion of GSH into the apical fluid may play a role in the protection of conjunctival tissue and tear film from oxidant insults.
Gukasyan
Hovhannes J
HJ
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90033, USA.
Lee
Vincent H L
VH
Kim
Kwang-Jin
KJ
Kannan
Ram
R
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000378:metabolism*; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D012965:Sodium Chloride; D005723:gamma-Glutamyltransferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione