P2X4 Receptor-Dependent Ca2+ Influx in Model Human Monocytes and Macrophages
Janice A Layhadi1, Samuel J Fountain2
1School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK. j.layhadi@uea.ac.uk.
Abstract:
Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5'-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage.
Insights
P2X4 receptor activation significantly contributes to calcium responses in human macrophages stimulated by adenosine 5'-triphosphate (ATP). This study highlights P2X4 receptors as key players in ATP-mediated calcium signaling in macrophages.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Monocytes and macrophages express P2 receptors for adenosine 5'-triphosphate (ATP), a damage-associated molecular pattern molecule.
- ATP binding to P2 receptors triggers calcium responses via direct permeation (P2X) or intracellular store mobilization (P2Y).
Purpose of the Study:
- To compare the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages.
- To elucidate the specific roles of different P2X receptor subtypes in macrophage calcium signaling.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) and immunocytochemistry to confirm P2X receptor expression.
- Measurement of intracellular calcium changes in response to ATP using a microplate reader.
- Pharmacological inhibition of P2X receptors and endocytosis/exocytosis pathways.
- shRNA-mediated knockdown of P2X4 receptors.
Main Results:
- ATP induced concentration-dependent calcium increases in both monocytes and macrophages.
- Tg-resistant ATP-evoked calcium responses in macrophages were extracellular calcium-dependent, indicating influx.
- P2X4 antagonists (5-BDBD, PSB-12062) and P2X4 knockdown significantly attenuated these responses.
- Ivermectin (IVM) potentiated the Tg-resistant component, while dynasore inhibited its magnitude but slowed decay, suggesting endocytosis involvement.
Conclusions:
- P2X4 receptor activation significantly contributes to the ionotropic calcium response evoked by ATP in model human macrophages.
- These findings identify P2X4 receptors as critical mediators of ATP-induced calcium influx in macrophages.
- The study provides insights into the complex mechanisms of purinergic signaling in innate immune cells.
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