Endogenous Endothelial Nitric Oxide Synthase (eNOS) Modulates Oxygen (O₂) Responsive Control of Red Blood Cell (RBC) Energy Metabolism and Antioxidant Systems

BACKROUND: Red blood cells (RBCs) confront substantial oxidative loads, which may exceed antioxidant capacity during O₂ delivery constraint, injuring proteins/lipids critical to blood flow control and O₂ delivery homeostasis. As such, RBCs exhibit O₂ responsive glycolysis in which Embden Meyerhof pathway (EMP) or hexose monophosphate pathway (HMP) flux is apportioned to optimize reducing equivalent recycling for either thiol-based antioxidant systems (via HMP) or metHb reduction and ATP generation (via EMP). O₂ linked glycolytic routing arises from Hb conformation-based control of metabolon assembly on the Band 3 membrane protein (B3): deoxyHb competes with key EMP enzymes for B3 binding, with dominance oscillating between HMP (arteries) and EMP (veins) - aligning flux to context. However, during hypoxia, deoxyHb persistence in arterial blood masks B3 binding sites, limiting the increase in HMP dynamic range that should occur during arterial transit. As a result, desaturated RBCs lose NADPH and GSH recycling ability, preventing antioxidant scaling to ROS abundance. Hypoxia-responsive metabolism is commonly coordinated by HIF-responsive pathways (here, via erythropoietin, EPO) as well as by Nitric oxide (NO) and S-nitrosothiol (SNO) based signaling. While RBCs are known to exhibit O₂ responsive processing of plasma NO equivalents, the role of endogenous endothelial NO synthase (eNOS) in mature RBCs is newly appreciated and poorly understood. We explored the hypothesis that RBC eNOS (as a function of hypoxia a/o EPO) modulates O₂ responsive control of glycolytic flux to support antioxidant systems during stress.

METHODS: NO/SNO based regulation of glycolysis was evaluated in RBCs from healthy humans or eNOS (-/-) & wt mice (C57BL/6J) during oxygenation/deoxygenation +/- L-S-nitrosocysteine (SNOCys, 100uM) and +/- UV illumination. Additionally, EPO mediated control of glycolytic routing (via RBC eNOS) in hypoxic RBCs was evaluated (human RBCs +/- EPO, +/- selective inhibition of: PI3K/Akt (wortmannin), B3 metabolon assembly (CO, WWW781), arginase (NOHA) and eNOS (NIO). In these RBCs, dynamic range in HMP flux (+/- methylene blue stimulation) was assessed by ¹H-NMR following incubation with [2-¹³C] D-glucose. O₂ responsive metabolon assembly (for which GAPDH is a key element) on B3 (and eNOS/B3 binding) was determined by immunofluorescent imaging and proximity ligation assay (Duolink PLA). We also quantified O₂ responsive S-nitrosylation of key cytosolic and RBC membrane proteins with a clickable SNO-selective probe (PBZyn).

RESULTS: We found (1) expected O₂ responsive variation in HMP dynamic range and reducing equivalent recycling & ROS resilience, linked to (2) O₂ dependent B3 metabolon assembly with reciprocal GAPDH & eNOS binding to B3 and (3) SNO-based modulation of GAPDH binding/activity that appeared mediated by (4) eNOS, prompted by either hypoxia or EPO via PI3K/Akt. These data fit the paradigm of O₂ dependent control of RBC metabolism, arising from cyclic B3 glycolytic metabolon assembly (oxygenation) and disassembly (deoxygenation), with eNOS facilitating metabolon disassembly but blunting EMP flux and stabilizing antioxidant resilience. Importantly, UV treatment (de-nitrosylation) in deoxygenated RBCs enhanced GAPDH/cdB3 interaction, whereas L-CSNO (S-nitrosylation) reversed the reciprocal effect in oxygenated RBCs with expected modulation of HMP dynamic range. Selective inhibition of PI3/Akt/eNOS in EPO-stimulated RBCs suggest a role for this pathway in supporting HMP dynamic range and antioxidant capacity during O₂ delivery constraint. Murine WT RBCs followed the same patterns as human RBCs, with deoxygenated eNOS (-/-) RBCs retaining GAPDH/cdB3 Duolink signal, suggesting that eNOS derived NO facilitates metabolon disassembly from B3.

SUMMARY: This work further illuminates adaptive dynamics in RBC energy metabolism, suggesting hypoxia-responsive modulation by eNOS, possibly along two time-scales: acutely (with circulatory O₂ gradients) and sub-acutely (with HIF-based EPO signaling, ie sustained anemia a/o hypoxia). Of note, these data are the first demonstration of functionally significant O₂ dependent eNOS migration in human RBCs, which appears (at least) to mediate (via S-nitrosylation) glycolytic metabolon assembly/disassembly in circulating RBCs, supporting antioxidant systems during stress.

No relevant conflicts of interest to declare.