P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells
Patrick Constantinescu1, Bin Wang, Kati Kovacevic
1School of Biological Sciences, University of Wollongong, Wollongong, Australia.
Abstract:
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.
Insights
Murine erythroleukemia cells express the P2X7 receptor, a purinergic receptor. This receptor activation by extracellular ATP leads to cell death and microparticle release, impacting red blood cell function.
Area of Science:
- Cell Biology
- Immunology
- Pharmacology
Background:
- Extracellular ATP is known to affect cell function via purinergic receptors.
- Murine erythroleukemia (MEL) cells exhibit responses to ATP characteristic of the P2X7 receptor, but its presence was unconfirmed.
- The role of P2X7 receptor in MEL cells remained unclear.
Purpose of the Study:
- To investigate the expression and function of the P2X7 receptor in murine erythroleukemia cells.
- To determine if P2X7 receptor activation impacts MEL cell viability and behavior.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence staining were used to detect P2X7 expression.
- Flow cytometry was employed to measure ethidium+ and YO-PRO-1(2+) uptake, Annexin-V binding, and microparticle release.
- Cell viability was assessed using colorimetric assays.
Main Results:
- P2X7 receptor expression was confirmed in MEL cells via molecular and cellular techniques.
- ATP-induced ethidium+ uptake was observed in a concentration-dependent manner, specifically mediated by P2X7.
- ATP exposure impaired cell growth, induced cell death, phosphatidylserine exposure, and microparticle release, all inhibited by a P2X7 antagonist.
Conclusions:
- Murine erythroleukemia cells express functional P2X7 receptors.
- Activation of P2X7 receptors by extracellular ATP plays a significant role in inducing cell death and microparticle release in MEL cells.
- These findings suggest a potential in vivo role for P2X7 receptor activation in red blood cell death and microparticle shedding.
