P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells.
Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium+ uptake into MEL cells in a concentration-dependent fashion and with an EC(50) of approximately 154 microM. The most potent P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium+ uptake. ATP-induced ethidium+ and YO-PRO-1(2+) uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.
Constantinescu
Patrick
P
School of Biological Sciences, University of Wollongong, Wollongong, Australia.
Wang
Bin
B
Kovacevic
Kati
K
Jalilian
Iman
I
Bosman
Giel J C G M
GJ
Wiley
James S
JS
Sluyter
Ronald
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D004996:Ethidium / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D010718:Phosphatidylserines / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7
C546469:P2rx7 protein, mouse; D010718:Phosphatidylserines; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D004996:Ethidium