Blood-to-lens transport of reduced glutathione in an in situ perfused guinea-pig eye.
Transport mechanisms of reduced glutathione (GSH) in intact eye are poorly understood. In this study, an in situ vascular eye perfusion (VEP) model was used to characterize the transport kinetics of circulating GSH into the aqueous humor and lens in guinea pigs. Radiolabeled [35S]GSH or [3H]GSH and [14C]sucrose (an extracellular space marker) were exposed to the blood-aqueous barrier up to 10 min, and uptake of tracers by the aqueous humor and lens was determined in the presence of different concentrations of unlabeled peptide as GSH, a gamma-glutamyl compound as a derivative of GSH (GSH monoethyl ester), and an inhibitor of gamma-glutamyl transpeptidase (GGT) activity. Plasma-aqueous and aqueous-lens compartmental unidirectional transport constant, K(in), and the initial rapid volume of distribution, Vi, were estimated by multiple-time-point graphic analysis. Our results indicated that both labeled GSH and sucrose entered the aqueous humor slowly at comparable rates with respective blood-aqueous K(in) values of 1.34 +/- 0.12 and 1.25 +/- 0.08 min-1 x 10(3). In contrast to blood-aqueous transport, GSH uptake by the lens was rapid, and the respective aqueous-lens K(in) values for labeled GSH and sucrose were 79.3 +/- 4.1 and 3.5 +/- 0.7 min-1 x 10(3). Over 94% of plasma-derived GSH remained in its original molecular form of GSH in the lens, during the 10 min perfusion both with and without the GGT inhibitor, serine borate. The amount of [35S]GSH in lens anterior epithelium (dpm mg-1) was more than three times that of aqueous humor (dpm microliter-1) within only 10 min of VEP. There was also significant accumulation of [35S]GSH in the interior cortex, as indicated by 10-min cortex/aqueous ratio of 0.65. A specific GGT independent GSH transport system was demonstrated in the lens in situ, with a Km of 26 +/- 3 microM, and Vmax of 34 +/- 3 pmol min-1 g-1 of whole lens tissue water. The lenticular influx of GSH was inhibited by GSH monoethyl ester with an affinity that was half that for GSH. It is concluded that: (a) uptake of plasma-derived GSH into the aqueous humor is by simple diffusion, and (b) cellular uptake of GSH by the lens is carrier-mediated via mechanism that is separate from the transpeptidation metabolic pathway.
Zlokovic
B V
BV
Department of Neurological Surgery, Childrens Hospital Los Angeles, CA.
Mackic
J B
JB
McComb
J G
JG
Kaplowitz
N
N
Weiss
M H
MH
Kannan
R
R
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D001693:Biological Transport, Active; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005978:Glutathione / Q000097:blood*; D006168:Guinea Pigs; D007908:Lens, Crystalline / Q000378:metabolism*; D008297:Male; D008433:Mathematics; D013462:Sulfur Radioisotopes; D013997:Time Factors
D002250:Carbon Radioisotopes; D013462:Sulfur Radioisotopes; D005978:Glutathione