Reduced Erythrocyte Equlibrative Nucleoside Transporter Expression Underlies Increased Circulating Adenosine Levels and Contributes to Pathophysiology of Sickle Cell Disease

Abstract 3232

Sickle cell disease (SCD) is a severe genetic disorder with a high morbidity and mortality. Understanding the molecular basis responsible for sickling, a central pathogenesis of SCD, is critical for developing new therapeutic strategies. Using a nonbiased high throughput metabolomic screen, coupled with genetic and pharmacological approaches, recent studies demonstrated that excessive adenosine signaling through the A_2B adenosine receptor triggers sickling by induction of 2,3-bisphosphoglycerate (2,3-BPG), an erythroid specific metabolite that induces O₂ release from hemoglobin. Adenosine is a signaling nucleoside that elicits many physiological effects by engaging membrane receptors. Notably, equlibrative nucleoside transporters (ENTs) on erythrocytes have been long speculated to regulate extracellular adenosine concentrations under hypoxic conditions. Thus, we hypothesize that ENT is likely a key molecule responsible for elevated circulating adenosine levels and contributing to pathophysiology of SCD. To test this hypothesis, we first conducted western blot analysis to compare expression profiles of ENTs on normal and sickle erythrocytes. We found that ENT1 is the major ENT expressed on both mouse and human erythrocytes. Unexpectedly, ENT1 levels were significantly reduced in sickle erythrocytes compared to normal erythrocytes in both humans and mice, suggesting that ENT1 may contribute to increased adenosine levels seen in SCD. Next, we performed pharmacological studies to determine the exact role of ENT in normal and sickle erythrocytes. We found that treatment with dipyridamole or an ENT1 specific inhibitor (NBMPR) enhanced adenosine-induced elevation of 2,3-BPG in cultured mouse RBCs. Using Hemox Analyzer, we found that co-treatment of adenosine with either dipyridamole or NBMPR resulted in a further right shift of oxygen equilibrium curve (OEC) and further increase in P₅₀ compared to the cells treated with adenosine alone. Similar to our pharmacological studies, we found that genetic deletion of ENT1 further enhanced adenosine-induced 2,3-BPG production in cultured erythrocytes, additional right shift of OEC and increased P₅₀. Extending mouse studies to human, we demonstrate that co-treatment of adenosine with either dipyridamole or NBMPR further enhanced the adenosine alone-mediated 2,3-BPG induction in cultured erythrocytes isolated from normal individuals and SCD patients. Finally, we found that dipyridamole treatment significantly enhanced hypoxia-mediated 2,3-BPG production, right shift of OEC and substantial sickling in cultured erythrocytes isolated from SCD patients. Overall, our studies demonstrate that 1) ENT1 is a major transporter expressed by RBCs and that inhibition or deletion of ENT1 results in enhanced adenosine-mediated 2,3-BPG induction and deoxygenation in normal RBCs; 2) Decreased ENT1 expression in sickle erythrocytes is responsible for elevated circulating adenosine and thereby contributes to sickling by promoting 2,3-BPG production and triggering deoxygenation. Therefore, our findings reveal a previously unrecognized role of ENT1 in erythrocyte physiology, add a new insight to the pathophysiology of SCD and open up new therapies for the disease.