No abstract

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(؊/؊) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(؊/؊) mice were 9 -16% of that in Gclm(؉/؉) littermates. Cysteine levels in Gclm(؊/؊) mice were 9, 35, and 40% of that in Gclm(؉/؉) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an ϳ2-fold increase in K m for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(؊/؊) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H 2 O 2 . We conclude that the Gclm(؊/؊) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.Glutathione (GSH) is the most abundant non-protein thiol found in most aerobic organisms (1, 2). GSH is an antioxidant and, by way of direct scavenging and enzymatic reduction reactions, participates in cellular protection against both endogenous and exogenous electrophiles. GSH also regulates the cellular redox balance by maintaining the essential thiol status of proteins (3). Moreover, GSH serves as a storage depot for cysteine and as a cofactor for the enzymic reduction of ribonucleotides (4, 5). Because of one or more of these functions, GSH has been shown to be involved in decisions concerning the fate of the cell, including cellular proliferation, differentiation, and apoptosis (6, 7).GSH is synthesized from its precursor amino acids in two sequential enzymatic reactions. Glutamate-cysteine ligase (GCL) 1 catalyzes the formation of ␥-glutamylcysteine (␥-GC) from glutamate and cysteine. Glutathione synthetase then couples glycine to ␥-GC to form GSH. The reaction catalyzed by GCL is rate-limiting in GSH biosynthesis, and the product GSH is a feedback inhibitor of GCL activity. Higher eukaryotes contain GCL as a heterodimer comprised of a 72.8-kDa catalytic subunit (GCLC) and a 30.8-kDa modifier subunit (GCLM), which are encoded by genes on different chromosomes (8 -10). Studies with both native and recombinant GCL protein demonstrate that the catalytic subunit is necessary and sufficient for ␥-GC biosynthesis (11,12). Targeted disruption of the Gclc gene in mice demonstrated that GCLC and most likely GSH are essential for embryonic development but not survival of cells in culture (13,14).Much of our understanding about the function of the...

Multidrug resistance-associated proteins (Mrps) are adenosine triphosphate-dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubinglucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2-related factor-2 (Nrf2)

ABSTRACT:The multidrug resistance-associated proteins ( , -3, -4, -6, and -7 mRNA expression. Mrp3 was also induced by two other CAR activators phenobarbital and diallyl sulfide, two PXR ligands, pregnenalone-16␣-carbonitrile and spironolactone, and the PPAR␣ ligands clofibrate, ciprofibrate, and diethylhexylphthalate. The Nrf2 activators (butylated hydroxyanisole, oltipraz, and ethoxyquin) induced Mrp2-6. In conclusion, a variety of mechanisms are suggested for Mrp3 induction, including AhR, CAR, PXR, PPAR␣, and Nrf2, whereas on a whole, a predominant role for AhR and Nrf2 in hepatic induction of the Mrp family was observed. Thus, these specific transcription factors are implicated in regulation of both drug metabolism and efflux transport.