A novel approach to study mechanisms of age-related dysfunction in hypoxia-induced erythrocyte ATP release

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PROJECT SUMMARY: Matching blood flow and oxygen delivery to tissue oxygen demand is one of the most essential fundamental physiological processes. Recent studies show that red blood cells (RBCs) sense hypoxia and respond by releasing ATP. RBC-derived ATP causes vasodilation that improves local blood flow and oxygen delivery via binding to endothelial purinergic (P2) receptors. Our laboratory and others have demonstrated that RBC ATP release is impaired in healthy older adults, as well as patients with type II diabetes and pulmonary hypertension. Current methodology to study hypoxia-induced RBC ATP release is limited to static measures of ATP at discrete levels of oxygenation (PO2), and thus the critical barrier to understanding hypoxia-induced RBC ATP release is the inability to simultaneously measure PO2 and ATP release in real-time. Our preliminary data indicates that fluo-oximetry with magnesium green (Mg-G) can simultaneously measure ATP release and PO2 in real-time, allowing for precise quantification of variables of RBC function that include total ATP release, the PO2 required to initiate ATP release, peak rate of ATP release, and others. Although it is well established that the final conduit for regulated ATP release during hypoxia occurs via pannexin-1 channels, the mechanisms stimulating RBC ATP release in response to hypoxia remain unclear. RBC deformability has been linked with hypoxia-induced ATP release, and we have demonstrated that improving deformability of RBCs from older adults restores ATP release. Recent data implicate the mechanically activated cation channel Piezo1 in shear-mediated RBC ATP release, however the role of Piezo1 in hypoxia-induced RBC ATP release is unknown. Therefore, the overall goal of this exploratory research proposal is to establish our novel approach for monitoring real-time RBC ATP release and PO2 simultaneously, and to explore the role of Piezo1 in stimulating ATP release during hypoxia in young and older adults. In Specific Aims 1.1 and 1.2, we will use continuous, simultaneous measurement of PO2 and ATP to define parameters of RBC ATP release during progressive hypoxia. We will validate our approach by demonstrating ATP release during hypoxia is abolished via pannexin-1 channel blockade. In Specific Aims 2.1 and 2.2, we will determine whether stimulation of Piezo1 channels is requisite for hypoxia-induced RBC ATP release in young adults, and whether reduced stimulation of Piezo1 channels explains the impairment in RBC ATP release in older adults. We will also determine whether pharmacological stimulation of mechanosensitive Piezo1 channels reverses the age-related impairment in RBC ATP release. The findings from the proposed studies will establish a novel approach for studying RBC physiology during hypoxia, and will provide the first data regarding the mechanistic role of Piezo1 in hypoxia-induced RBC ATP release in young and older adults. Our results could be the impetus for future studies designed to improve circulating ATP in older adults and various patient populations suffering from exercise intolerance or tissue ischemia due to impaired local regulation of blood flow and oxygen delivery.