RBC eNOS Regulates O2 Delivery Phenotype during Circulatory Transit Via Control of Intracellular pH

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.1074-1074
• Main Authors: Rogers, Stephen C, Safari, Zohreh, Brummet, Mary E, Bennett, Daniel, Odunaiya, Emioluwa, Eziemefe, Adaora, Nguyen, Jacqueline, Enjati, Sarah, Conelius, Olivia, Issaian, Aaron, monikA.dzieciatkow, Monika, Gamboni, Fabia, Held, Jason, D'Alessandro, Angelo, Dunaway, Luke S, Brant, Isakson, Cortese Krott, Miriam, Doctor, Allan
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-210690

BACKGROUND: As red blood cells (RBCs) traverse circulatory O2 gradients, RBC endothelial nitric oxide synthase (eNOS) migrates on and off the membrane, undergoing oscillatory activation/inactivation. Upon deoxygenation RBC eNOS is activated in a process dependent on hemoglobin (Hb) conformation, stimulation of the mechanosensitive Piezo1 channel, calcium influx and calmodulin association. Previously, we have demonstrated eNOS dependent regulation of RBC energetics, following glycolytic metabolon disassembly and key enzyme inactivation, resulting from protein s-nitrosylation. We have additional evidence of eNOS mediated s-nitrosylation of an array of RBC proteins (both cytosolic and membrane associated), including the cytoplasmic domain of Band 3 (cdB3) and carbonic anhydrase (CA). Considering the central role of these proteins in governing the Bohr effect, we hypothesized a role for eNOS in the regulation of Hb oxygen affinity (HbO2 affinity). METHODS: Murine blood (C57BL/6J, or eNOS(-/-)) was drawn into heparin vacutainers. CBC values (ABX Micros ESV60, Horiba) and HbO2 affinity were determined, via construction of oxygen dissociation curves (ODCs) at 3 fixed partial pressures of carbon dioxide (pCO2; 20, 40, 70 mmHg). Diluted blood samples (Krebs buffer) were manipulated in a thin film rotating tonometer (Meon Medical Solutions, TM8000) utilizing high precision gas blenders (Meon Medical Solutions HP-GMS, or Warner Instruments, Oxystreamer). Samples were measured by arterial blood gas (ABG) machine to determine oximetry and small metabolite/electrolyte levels (Radiometer, ABL90Flex). Intracellular pH was determined via fluorescent probe (SNARF-1), utilizing afluorescent plate reader (Biotek Neo2, USA) comprising an environmental chamber set to mimic circulatory physiologic pO2 and pCO2 gradients (i.e., the lung or maximally exercising tissue; lung = pO2 21%, pCO2 3%; tissue = pO2 1%, pCO2 14%). O2 responsive S-nitrosylation of key cytosolic and RBC membrane proteins was determined utilizing the clickable SNO-selective probe (PBZyn) and detection via LC-MS. RESULTS: No difference was observed in p50 between C57BL/6J or eNOS(-/-) groups at each pCO2. However, a significantly lower intra mouse p50 delta and area between low to high pCO2 ODC curves, was observed in eNOS-/- compared to C57BL/6J mice, demonstrating an impaired Bohr effect in the eNOS(-/-)mice. In alignment with the ODC findings, eNOS(-/-) mice demonstrated a lower RBC intracellular pH delta wh