Functional significance of glutamate-cysteine ligase modifier for erythrocyte survival in vitro and in vivo

Abstract

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm -/-) mice. Erythrocytes from gclm -/- mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm -/- erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm -/- mice showed dramatically increased hemolysis compared with gclm +/+ controls. In addition, PHZ-treated gclm -/- mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm +/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm -/- mice and none in gclm +/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm -/- mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm +/+ mice survived, 90% of PHZ-treated gclm -/- mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm -/- erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm +/+ erythrocytes, an effect at least partially due to increased cytosolic Ca 2+ concentration. Under resting conditions, gclm -/- mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress. © 2013 Macmillan Publishers Limited All rights reserved.