Hypoxia limits antioxidant capacity in red blood cells by altering glycolytic pathway dominance

Abstract

The erythrocyte membrane is a newly appreciated platform for thiol-based circulatory signaling, and it requires robust free thiol maintenance. We sought to define physiological constraints on erythrocyte antioxidant defense. Hemoglobin (Hb) conformation gates glycolytic flux through the hexose monophosphate pathway (HMP), the sole source of nicotinamide adenine dinucleotide phosphate (NADPH) in erythrocytes. We hypothesized elevated intraerythrocytic deoxyHb would limit resilience to oxidative stress. Human erythrocytes were subjected to controlled oxidant (superoxide) loading following independent manipulation of oxygen tension, Hb conformation, and glycolytic pathway dominance. Sufficiency of antioxidant defense was determined by serial quantification of GSH, NADPH, NADH redox couples. Hypoxic erythrocytes demonstrated greater loss of reduction potential [Delta GSH E(hc) (mV): 123.4+/-9.7 vs. 57.2+/-11.1] and reduced membrane thiol (47.7+/-5.7 vs. 20.1+/-4.3%) (hypoxia vs. normoxia, respectively; P<0.01), a finding mimicked in normoxic erythrocytes after HMP blockade. Rebalancing HMP flux during hypoxia restored resilience to oxidative stress at all stages of the system. Cell-free studies assured oxidative loading was not altered by oxygen tension, heme ligation, or the inhibitors employed. These data indicate that Hb conformation controls coupled glucose and thiol metabolism in erythrocytes, and implicate hypoxemia in the pathobiology of erythrocyte-based vascular signaling.

Figure 1.

Relation between oxygen tension and reducing capacity in RBCs. A simplified scheme of glucose metabolism in the RBC (A), which proceeds through either the Embden-Meyerhof pathway (EMP, orange arrows), or the hexose monophosphate pathway (HMP, blue arrows). Both share glucose-6-phosphate (G6P) as an initial substrate. HMP is the sole source of NADPH in RBCs and generates fructose-6-phosphate (F6P) or glyceraldehyde-3-phosphate (G3P), which rejoin the EMP prior to glyceraldehyde-3-phosphate dehydrogenase (G3PD), a key regulatory point in the EMP. The EMP generates NADH (utilized by metHb reductase), as well as ATP (to drive ion pumps) and 2,3-DPG (to modulate hemoglobin p50) (neither ATP or 2,3 DPG shown). Relative flux through the EMP and the HMP is modulated by O₂-linked transitions in Hb conformation due to competitive binding for the cytoplasmic domain of Band 3 (cdB3) between deoxyHb and key EMP enzymes (PFK, Aldo, G3PD, PK, and LDH). In fully oxygenated RBCs (B), these EMP enzymes are inactivated by sequestration on cdB3, resulting in glucose being channelled through the HMP, maximizing NADPH (and thus GSH) recycling capacity. In deoxygenated RBCs (C), EMP enzymes are dispersed by deoxyHb binding to Band 3, creating competition for G6P as a substrate and thereby constraining recycling capacity for NADPH and GSH and limiting resilience to attack by ROS. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) are the principal endogenous ROS encountered by RBCs. Both ROS may be generated internally (not shown); however, only H₂O₂ can cross the membrane directly. O₂⁻ enters RBCs through Band 3 (anion exchange protein 1, or AE-1). H₂O₂ and O₂⁻ are ultimately reduced to water by catalase or glutathione peroxidase (GPx). CAT, catalase; GSH, glutathione; GR, glutathione reductase; NADPH, nicotinamide adenine dinucleotide phosphate; PFK, phosphofructokinase; Aldo, aldolase; PK, pyruvate kinase; LDH, lactate dehydrogenase.

Figure 2.

Partitioning of RBC membrane proteins into intrinsic and extrinsic fractions. Membrane proteins from oxygenated and deoxygenated RBC lysates (total) were processed into intrinsic (membrane spanning) and extrinsic (skeletal, anchoring, or membrane-associated) proteins. Aliquots from membrane fractions containing equal amounts of protein were separated by SDS-PAGE and either visualized by Coomassie blue staining (A), or transferred to a nitrocellulose membrane for immunoblotting (B). Note appropriate partitioning of proteins known to be associated with each fraction. Relative to signal from the unfractionated (total) preparation, partitioning led to enhancement of individual proteins within their respective fractions, precluding quantitative comparisons.

Figure 3.

Conservation of reduced RBC membrane thiol in ghost and intact RBCs preparations exposed to graded oxidative stress. Percentage of reduced membrane thiol remaining (indexed to membrane protein) following graded oxidative stress (45-min exposure). Protein fractions in ghosts prepared from oxygenated RBCs (A) and deoxygenated RBCs (B) were compared to fractions from intact oxygenated RBCs (C) and intact deoxygenated RBCs (D). Data are plotted as means ± se; n = 5–11. *P < 0.05, **P < 0.01 for intact deoxygenated RBC protein fractions vs. corresponding intact oxygenated RBCs. ^ƒP < 0.05 for intact deoxygenated RBC protein fractions vs. corresponding ghosts from deoxygenated RBCs.

Figure 4.

Serial glutathione redox state of deoxygenated and oxygenated RBCs exposed to graded oxidative stress. Measurement of total glutathione (GSH+GSSG) and GSSG alone and calculation of GSH (from the difference) in oxygenated or deoxygenated washed RBCs exposed to HX/XO. Glutathione-related parameters, including half-cell reduction potential E_hc (A, B), total GSH + GSSG (C, D), GSH alone (E, F), and GSSG alone (G, H) are shown for oxygenated RBCs (top panels) and deoxygenated RBCs (bottom panels). Data are presented as means ± se; n = 4–6. *P < 0.05, **P < 0.01 for deoxygenated RBCs vs. corresponding oxygenated RBC sample. Note that P < 0.01 for all 0.4 and 0.8 U/ml XO deoxygenated RBC E_hc values from 5 min onward, vs. corresponding oxygenated RBC E_hc (not presented graphically).

Figure 5.

Effect of oxygen tension, heme occupancy, and presence of inhibitors on oxidation of GSH by HX/XO. Cell-free system to isolate effect of oxygen tension, heme occupancy, and presence of inhibitors on oxidant production from HX/XO, as measured by GSH conversion to GSSG. HX (1.5 mM) and XO (0.2 U/ml) alone, or purified Hb tetramers (500 μM) with 2,3-DPG (500 μM), HX (1.5 mM) and XO (0.2 U/ml) were incubated with GSH (500 μM), in Krebs buffer (pH 7.4, 37°C) under oxygenated or deoxygenated conditions (A). In addition, experiments were performed under oxygenated (B) and deoxygenated (C) conditions with GSH (500 μM), HX (1.5 mM), and XO (0.2 U/ml) in the presence of either DIDS (500 μM), DHEA (5 μM), or KA (15 μM). GSSG/GSH redox ratios were calculated from the measurement of total and oxidized glutathione . In the presence of HX/XO, buffer oxygen content (pO₂∼500 or 10 Torr) did not significantly influence the rate of GSH oxidation to GSSG (A). Hb, in the presence of HX/XO, significantly accelerated the oxidation of GSH to GSSG (P<0.05); this effect was less pronounced following Hb deoxygenation (HbSO₂ 88 or 24%) (A). DIDS, DHEA, or KA with HX/XO did not affect the rate of GSH oxidation to GSSG under either high- or low-oxygen conditions (B, C). Data are plotted as means ± se; n = 3–6. *P < 0.05 vs. corresponding low oxygen tension sample.

Figure 6.

Serial NADPH and NADH redox ratios from oxygenated and deoxygenated RBCs exposed to graded oxidative stress. NADPH/NADP (A, B) and NAD/NADH (C, D) redox ratios calculated from the measurement of total and reduced pyridine nucleotides. Change in the (NADPH/NADP_Total) × 100 ratio over time during steady oxidant production reflects recycling efficiency for the NADPH-reducing systems in RBCs. Data are presented from oxygenated (A, C; left panel) and deoxygenated (B, D; right panel) RBCs. Data are plotted as means ± se; n = 3–6. *P < 0.05, **P < 0.01 for deoxygenated RBC samples vs. corresponding oxygenated sample.

Figure 7.

Reducing equivalent recycling: effect of Hb conformation and the inhibition of superoxide entry into RBCs. Paired NADPH and GSH redox ratios from oxygenated and deoxygenated RBCs exposed to HX/XO (1.5 mM and 0.8 U/ml, respectively), plotted with data from deoxygenated RBCs pretreated with Band 3 inhibitor DIDS (500 μM, 1 h, 37°C; to block superoxide entry into RBCs) (A, B), or deoxygenated RBCs pretreated with 100% CO (5 min, RT, forming >97% carboxyHb; to lock Hb conformation in R state and prevent release of EMP enzymes from Band 3; see Fig. 1) (C, D). Glutathione redox ratio is calculated as (GSSG/GSH_Total) × 100. NADPH redox ratio is calculated as (NADPH/NADP_Total) × 100. Recycling capacity in deoxygenated RBCs was preserved by preventing superoxide entry into deoxygenated RBCs (DIDS), or by preventing hypoxia-induced transition in Hb conformation (CO treatment). Data are plotted as means ± se; n = 3–4. *P < 0.05, **P < 0.01 for CO or DIDS values vs. deoxygenated values.

Figure 8.

Reducing equivalent recycling: effect of glycolytic pathway dominance. Paired NADPH and GSH redox ratios from oxygenated and deoxygenated RBCs exposed to HX/XO (1.5 mM and 0.8 U/ml, respectively), plotted with data from glucose depleted oxygenated RBC (A, B); oxygenated RBCs pretreated with the G6PD inhibitor DHEA (5 μM, 15 min, 37°C; to block the HMP) (C, D), or deoxygenated RBCs pretreated with the G3PD inhibitor KA (15 μM, 15 min, 37°C; to block the EMP) (E, F). Glutathione redox ratio is calculated as (GSSG/GSH_Total) × 100. NADPH redox ratio is calculated as (NADPH/NADP_Total) × 100. Reducing equivalent recycling capacity was lost in oxygenated RBCs following glucose depletion (A, B) and HMP blockade at G6PD (C, D); reducing equivalent recycling capacity was restored in hypoxic RBCs following EMP blockade (E, F). Data are plotted as means ± se; n = 3–4. *P < 0.05, **P < 0.01 for glucose-depleted or DHEA values vs. oxygenated values or KA values vs. deoxygenated values.

Figure 9.

Conservation of reduced RBC membrane thiol in RBCs confronted with oxidative stress. Percentage of reduced membrane thiol remaining (indexed to membrane protein) in RBCs exposed to HX/XO (1.5 mM and 0.8 U/ml, respectively) for 45 min. Following either oxygenation or deoxygenation in the tonometer, washed RBCs (in Krebs) were removed, portioned into aliquots, and incubated with HX/XO in a shaking heater block (37°C). As observed for measures of recycling capacity for reducing equivalents (Figs. 7 and 8), oxygenated RBCs lost the ability to defend membrane thiols following HMP blockade with DHEA, while hypoxic RBCs were rescued by stabilizing Hb conformation (CO), preventing superoxide entry into RBCs (DIDS), or following EMP blockade (KA). Data are plotted as means ± se; n = 4–8. *P < 0.05.