MOLECULAR CHARACTERIZATION OF BRAIN GSH TRANSPORTERS

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DESCRIPTION (Investigator's Abstract): Although glutathione (GSH) is critical in defense against oxidative stress and is ubiquitous in aerobic cells, the brain may utilize GSH for other special functions such as storage and transfer of cysteine and modulation of neuroexcitatory transmission. We have been interested in the translocation of intact GSH across plasma membranes. Two GSH transporters have been cloned in rat liver, one of which, RcGshT, is also found in whole brain, isolated capillaries, and cultured astrocytes, and is phenobarbital inducible in brain as well as liver. In addition, we showed that GSH can be extracted at the BBB and GSH transport is mediated by sodium-dependent mechanism distinct from RcGshT (sodium-independent). Xenopus laevis oocytes expressing guinea-pig or bovine capillary mRNA exhibit bidirectional GSH transport which could be resolved by size fractionation (bovine) into at least 3 different GSH transporters--one being RcGshT (bovine homologue) and two novel ones (one of which is highly sodium dependent). Our first specific aim is to express, clone and characterize rat brain capillary GSH transporters using the Xenopus laevis oocyte expression system. We will i) perform size fractionation of mRNA and microinjection into oocytes to determine multiplicity of transporters and compare their ion requirements, ii) study contribution of RcGshT and gamma-glutamyl transpeptidase, GGT, to expression of GSH transport in the relevant size fractions using antisense oligonucleotides, iii) clone and sequence GSH transporters distinct from RcGshT, iv) study GSH uptake and efflux in oocytes expressing three cloned GSH transporters: kinetics, ion requirements, molecular forms and inhibitor specificity, v) perform Northern blot analysis of RNA from various organs probed with newly cloned GSH transporters and, vi) perform Northern and Western blots of capillaries vs capillary-depleted brain from controls and phenobarbital-treated rats. In our second aim, we will study transport of GSH in cultured astrocytes and brain endothelial cells and characterize the transporters: we will i) determine whether GSH uptake and efflux in astrocytes and endothelial cells is saturable and its specificity and ion requirements, and ii) determine the presence or absence of cloned GSH transporter mRNAs and gene products in astrocytes and endothelial cells. These studies will lead to a better understanding of the homeostasis and transport of GSH in the brain in normal physiology and pathophysiology and will be critical in the development of potential therapeutic strategies of GSH delivery in GSH-deficient neurological states.