A novel approach to real-time monitoring of erythrocyte ATP release as a function of hypoxia
Date
2020
Authors
Smith, Meghan E., author
Dinenno, Frank, advisor
Lark, Daniel, committee member
Amberg, Gregory, committee member
Journal Title
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Abstract
Background: Matching blood flow to tissue oxygen demand is essential for maintaining metabolic homeostasis and sustaining human life. Recent studies suggest that red blood cells (RBCs) play a role in local vasodilatory signaling by releasing ATP in response to hypoxia. RBC ATP release and overall function are impaired with age and disease. Until now, luciferin/luciferase bioluminescence is the only method described to quantify ATP release from RBCs. Here, we describe a novel approach where ATP release is measured as a function of hypoxia continuously and in real time using an Oroboros Oxygraph O2K respirometer. Purpose: This report describes the development and application of this new approach. Methods: We obtained blood samples from 10 healthy, young adults (18-35y) via venipuncture. Washed RBCs were diluted to 5% hematocrit and added to the glass chamber of a calibrated Oxyfluorimeter along with 5μM Mg-G. Nitrogen gas was constantly injected into the chamber at 1 ml/min to decrease PO2. An LED-based fluorescence detection device monitored Mg-G fluorescence, which was used to calculate extracellular [ATP]. Results: When 5% HCT RBCs were exposed to 30 minutes of hypoxia, Mg-G fluorescence (V) continuously increased. During 30 minutes of progressive hypoxia, PO2 in the chamber decreased from 121.9 ± 1.3 to 9.8 ± 0.8 mmHg, and D extracellular [ATP] from normoxia (μM) increases from 0 to 6,985.0 ± 793.6 μM. Extracellular [ATP] accumulates markedly when PO2 in the chamber reaches 50.60 ± 1.52 mmHg. Conclusion: Using this novel method, we identified a PO2 threshold at which extracellular ATP accumulates rapidly, which is consistent with the range of PO2 that elicits Hb desaturation in RBCs. This approach may allow for detailed mechanistic studies into the relationship between hypoxia, Hb desaturation, and RBC ATP release.