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.

Figure 1

Erythrocytes isolated from gclm^−/− mice show low glutathione levels and are prone to cell death. (a) Cytosolic glutathione concentration in untreated erythrocytes isolated from resting gclm^+/+ and gclm^−/− mice. Data are the mean±S.E.M. of n=10 mice/group. For all Figures, white bar=gclm^+/+; black bar=gclm^−/−. ***P<0.001 (Student's t-test). (b) Representative photographs of erythrocytes that were isolated from gclm^+/+ and gclm^−/− mice, and incubated at 37 °C for 6–12 h (some variability in the extent of gclm^−/− erythrocyte lysis is seen at this time point) in the absence (upper row) or presence (lower row) of 250 μM Trolox (antioxidant)

Figure 2

Massive hemolysis in gclm^−/− mice subjected to oxidative stress. (a) Concentration of free Hb in serum from gclm^+/+ and gclm^−/− mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=4 mice/group. For all Figures, white bars=gclm^+/+; black bars=gclm^−/−. *P<0.05; **P<0.01 (Student's t-test). (b) Lactate dehydrogenase activity in serum from gclm^+/+ and gclm^−/− mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=4 mice/group. (c) Weight loss of gclm^+/+ and gclm^−/− mice following 24 h of PHZ (0.07 mg/g b.w.) treatment. Data were expressed relative to each mouse's starting weight. Results shown are the mean±S.E.M. of n=4 mice/group

Figure 3

PS-exposing erythrocytes accumulate in the spleens of gclm^−/− mice upon exposure to oxidative stress. (a) H&E-stained sections (upper panels) and Prussian Blue-stained (to detect iron) sections (lower panels) of splenic tissue collected from gclm^+/+ (left) and gclm^−/− (right) mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Results are representative of four mice/group. For all Figures, white bar=gclm^+/+; black bar=gclm^−/−. ***P<0.001 (Student's t-test). (b) Percentage of PS-externalizing cells among total erythrocytes isolated from spleens of gclm^+/+ and gclm^−/− mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=4 mice/group

Figure 4

Hemosiderin in the kidneys and reduced kidney function of gclm^−/− mice treated with PHZ. (a) H&E-stained sections (upper panels) and Prussian Blue-stained (to detect iron) sections (lower panels) of renal tissue collected from gclm^+/+ (left) and gclm^−/− (right) mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Results are representative of four mice/group. For all Figures, white bars=gclm^+/+; black bars=gclm^−/−. *P<0.05 (Student's t-test). (b) Concentrations of creatinine (left panels) and urea (right panels) in serum collected from gclm^+/+ and gclm^−/− mice at 24 h post-PHZ (0.07 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=4 mice/group

Figure 5

Survival of gclm^−/− mice is dramatically reduced following treatment with PHZ. Survival curves of gclm^+/+ and gclm^−/− mice (n=12/group) following PHZ (0.07 mg/g b.w.) treatment

Figure 6

Hemolysis is triggered in GCLM-deficient mice also upon treatment with low-dose PHZ. (a) Concentration of free Hb in serum from gclm^+/+ and gclm^−/− mice before (left bars) and at 24 h post-PHZ (right bars; 0.014 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=3–4 mice/group. For all Figures, white bars=gclm^+/+; black bars=gclm^−/−. *** or ^###P<0.001; Student's t-test). (b) GSH/GSSG ratio (upper panel), concentration of GSH (lower left panel) and concentration of GSSG (lower right panel) in erythrocytes from gclm^+/+ and gclm^−/− mice before (left bars) and at 24 h post-PHZ (right bars; 0.014 mg/g b.w.) treatment. Data are the mean±S.E.M. of n=4 mice/group

Figure 7

Ex vivo, gclm^−/− erythrocytes die of Ca²⁺-induced cell death. (a) Percentage of total erythrocytes from gclm^+/+ and gclm^−/− mice that externalized PS. Erythrocytes were stained to detect PS immediately upon blood collection. Data are the mean±S.E.M. of n=5 mice/group. For all Figures, white bars=gclm^+/+; black bars=gclm^−/−. * or ^#P<0.05; ** or ^##P<0.01; *** or ^###P<0.001 (Student's t-test). (b) Percentage of total erythrocytes from gclm^+/+ and gclm^−/− mice that externalized PS after incubation at 37 °C for 30 min in vitro in the absence of treatment (left), or in the presence of 0.3 mM TBOOH (right). Data are the mean±S.E.M. of n=8 mice/group (analysis of variance). (c) Representative original histogram displaying Annexin V fluorescence of the erythrocytes in (b, right); that is, gclm^+/+ and gclm^−/− erythrocytes that were incubated at 37 °C for 30 min with 0.3 mM TBOOH. (d) Cytosolic Ca²⁺ concentration as determined by Fluo3 fluorescence of erythrocytes that were isolated from gclm^+/+ and gclm^−/− mice and incubated at 37 °C for 30 min without treatment. Data are expressed in arbitrary units and are the mean±S.E.M. of n=3 mice/group. (e) Cytosolic Ca²⁺ concentration as determined by Fluo3 fluorescence of erythrocytes that were isolated from gclm^+/+ and gclm^−/− mice and incubated at 37 °C for 4–8 without treatment (left bars) or with 250 μM Trolox (right bars). Data are normalized fluorescence intensities expressed in relative units and are the mean±S.E.M. of n=10 experiments with erythrocytes from eight mice/group (analysis of variance). (f) Percentage of total erythrocytes from gclm^+/+ and gclm^−/− mice that externalized PS after incubation at 37 °C for 4–8 h in vitro in the presence (+Ca²⁺) or absence (−Ca²⁺) of extracellular Ca²⁺ without (−Trolox) or with 250 μM Trolox (+Trolox). Data are the mean±S.E.M. of n=10 experiments with erythrocytes from eight mice/group (analysis of variance)

Figure 8

Gclm^−/− mice exhibit reticulocytosis but show an otherwise unaltered blood count. (a) Percentage of total blood cells that were reticulocytes in resting gclm^+/+ and gclm^−/− mice. Data are the mean±S.E.M. of n=8 mice/group. For all Figures, white bars=gclm^+/+; black bars=gclm^−/−. *P<0.05 (Student's t-test). (b) Standard hematological parameters in resting gclm^+/+ and gclm^−/− mice. Data are the mean±S.E.M. and are expressed as indicated (n=3–4 mice/group)