Master of Science - Research
School of Biological Sciences
Wang, Bin, The role of P2X7 in red blood cell biology, Master of Science - Research thesis, School of Biological Sciences, University of Wollongong, 2011. https://ro.uow.edu.au/theses/3607
Red blood cells (RBCs) are essential for human health, and defects in RBC development and death give rise to various disorders including anaemia and erythrocytosis. Mature RBCs or erythrocytes contain high amounts of adenosine 5’- triphosphate (ATP) which can be release under various physiological and pathophysiological conditions. Once released, ATP triggers the activation of purinergic P2 receptors in an autocrine or paracrine fashion. The P2X7 receptor is a ligand-gated ion channel belonging to the P2X receptor family. P2X7 activation induces a number of downstream effects including reactive oxygen species (ROS) formation and cell death. P2X7 is predominantly expressed on haematopoietic cells including RBCs. However, the physiological and pathophysiological roles of P2X7 on RBCs remain poorly defined. Recent works from our laboratory have identified the presence of P2X7 on an immature RBC model, murine erythroleukemia (MEL) cells. Activation of P2X7 in this cell line induces ethidium+ uptake, phosphatidylserine exposure, and cell death. This study aims to 1) to determine if activation of P2X7 induces ROS formation in MEL cells; 2) to determine the mechanism(s) of P2X7-induced death of MEL cells.
RT-PCR confirmed the presence of P2X7 in MEL cells. Cytofluorometric cation uptake assay confirmed the function of P2X7 in MEL cells. ATP induced the uptake of ethidium+ and Yo-Pro-12+ in MEL cells, and this uptake was blocked by the P2X7 antagonist, A-438079. A cytofluorometric assay using the ROS sensitive probe 2’, 7’-dichlorodihydrofluorescein diacetate demonstrated that ATP induced ROS formation in MEL cells in a time- and concentration-dependent fashion with an EC50 of ~150 μM. The most potent P2X7 agonist 3’-O-(4-benzoyl)benzoyl-adenosine 5’- triphosphate, but not adenosine 5’-diphosphate or uridine 5’-triphosphate, also induced ROS formation. Moreover, ATP-induced ROS formation was impaired by the P2X7 antagonist, A-438079. ATP-induced ROS formation was impaired by the broad spectrum ROS inhibitors, N-acetyl-l-cysteine (NAC) and diphenyleneiodonium (DPI), and the mitochondrial complex I inhibitor, rotenone, but not by the NADPH oxidase inhibitor, apocynin. None of these compounds impaired ATP-induced ethidium+ uptake. Finally, ATP-induced ROS formation was not dependent on extracellular Ca2+ influx or intracellular K+ efflux.
The colourmetric MTT assay indirectly confirmed that ATP impaired MEL cell growth. Moreover, cytofluorometric measurements of Annexin-V binding and 7- aminoactinomycin staining directly confirmed that ATP induced death of MEL cell. A-438079 impaired ATP-induced cell death in both of these assays. Flow cytometry also showed that MEL cells pre-treated with ATP were phagocytosed by J774A.1 macrophage and that this process was impaired by incubation of MEL cells with A- 438079 prior to ATP treatment. ATP-induced death of MEL cells was impaired by the broad spectrum caspase inhibitor, Z-VAD-FMK and mitogen-activated protein kinase (MAPK) p38 inhibitors, SB202190 and SB203580. None of these compounds impaired ATP-induced ethidium+ uptake. Moreover, a cytofluorometric assay and phospho-immunoblotting demonstrated that ATP induced caspase and p38 MAPK activation in MEL cells respectively, and that these processes were impaired by A- 438079. In contrast, NAC did not impair the ATP-induced death of MEL cells.
In conclusion, activation of P2X7 induces ROS formation via a mitochondrial pathway that is independent of extracellular Ca2+ influx and K+ efflux. Moreover, P2X7 induces death of MEL cells, which can result in their phagocytosis by J774A.1 macrophages. Finally, P2X7 induced MEL cell death is mediated by apoptotic pathways involving p38 MAPK and caspases, but not ROS formation. This study supports a role of P2X7 in RBC development and homeostasis.