A novel approach to study the release of ATP from red blood cells during hypoxia

Background: Tissue oxygen demand requires sufficient blood flow to maintain metabolic homeostasis. Prior studies suggest that red blood cells (RBCs) release adenosine triphosphate (ATP) as a vasodilatory factor in response to a hypoxic environment and/or RBC deformation due to mechanical stress. However, existing methods to assess RBC-derived ATP provide only a static measurement of circulating ATP at one specific pressure of oxygen (PO 2 ), which limits our understanding of the mechanisms that regulate the onset and progression of RBC ATP release. The objective of this study was to develop a novel method to quantify RBC ATP release continuously and in real-time using an Oroboros Oxygraph-2k respirometer (O2k). Methods: Whole blood was collected via venipuncture from 8 young, healthy participants and RBCs were isolated and washed. RBCs were studied at 5% hematocrit in O2k chambers with 5 µM magnesium green (MgG), a fluorophore that preferentially binds to free ATP. Chambers were constantly injected with 100% nitrogen gas at 1 ml/min for 30 minutes to induce hypoxia. Over the course of the 30 minutes, PO 2 (mmHg) and extracellular [ATP] were measured using a polarographic oxygen sensor and LED-based fluorescence detection of ATP-bound MgG. Cell viability and supernatant hemoglobin (Hb) were measured in isolated RBC samples before and after exposure to hypoxia. PO 2 and fluorescence data were measured every 2 seconds during each experiment and absolute [ATP] (µM) were quantified from fluorescence data using a standard curve collected prior to each experiment. Michaelis Menten-like kinetics were applied to calculate the sensitivity of RBC ATP release to hypoxia (K m ) and maximum rate of RBC ATP release (V max ). We calculated the onset of ATP release as 2 SD above the rate of ATP release during normoxia. Results: After 30 minutes of progressively induced hypoxia, PO 2 in the chamber decreased from 121.9 ± 1.3 to 9.8 ± 0.8 mmHg. Extracellular [ATP] began being released at ~60 mmHg, consistent with oxygen tension across skeletal muscle capillaries in vivo. The peak rate of RBC ATP release during hypoxia was 5.68 ± 5.307 fold greater than during baseline normoxia. Hemoglobin measurements suggest at least some of the ATP released could be due to hemolysis, although its exact contributions are unclear at this time. Conclusions: This novel approach to measuring RBC-derived extracellular [ATP] has allowed us to identify real-time kinetics of ATP release from RBCs. More specifically, we have identified a PO 2 threshold at which extracellular ATP rapidly accumulates. This method has the potential to allow for targeted mechanistic studies into the relationship between hypoxia, Hb desaturation, and RBC ATP release but the role of hemolysis must be considered to define the true physiological relevance of this phenomenon. National Institutes on Aging (R21AG075517) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.