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Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
P2X currents in peritoneal macrophages of wild type and P2X4 -/- mice
Bert Brône1, Diederik Moechars, Roger Marrannes
1Gobal Preclinical Development, Johnson and Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B2340 Beerse, Belgium. bbrone@prdbe.jnj.com
Abstract:
In this study the ATP-induced (P2X) currents in isolated peritoneal macrophages of wild type (WT) and P2X(4) knockout (P2X(4)(-/-)) mice were studied by means of whole-cell patch clamp in order to (1) survey the P2X currents of native macrophages and (2) to investigate the expression of P2X(4)-like currents in the WT versus P2X(4)(-/-) mice. Three types of currents were observed in the isolated macrophages: (1) in approximately 10% of both WT and P2X(4)(-/-) macrophages a fast activating and inactivating P2X1-like current was recorded with low concentrations (0.1-1 microM) of ATP; (2) 85% of wild type and 100% of P2X(4)(-/-) macrophages exhibited a non-desensitizing P2X(7)-like current activated at high concentrations of ATP (10mM). The identity of the P2X(7) current was confirmed using the specific blocker A-740003; (3) 88.6% of the WT but none of the P2X(4)(-/-) macrophages showed a small P2X(4)-like current that desensitized slowly upon ATP application at intermediate concentrations (3-300 microM). Several observations indicated that the slowly desensitizing current in WT macrophages was P2X(4). The EC50 value of 5.3 microM ATP was as expected for P2X(4) and the current induced by 3-300 microM ATP was absent in P2X(4)(-/-) mice. Upon application of 3 microM ivermectin, a P2X(4)-selective modulator, the amplitude of this current was increased and the desensitization was inhibited in WT cells. In addition, this current was facilitated by 10 microM Zn(2+) but inhibited by Cu(2+) (in contrast to P2X(2)). We conclude that the P2X(4) and P2X(7) currents are functionally expressed in recruited peritoneal macrophages of WT mice and that the P2X(4)-like current is absent in P2X(4)(-/-) mice.
Insights
This study investigated ATP-induced currents in mouse macrophages, finding functional P2X4 and P2X7 channels in wild-type mice. P2X4-like currents were absent in P2X4 knockout mice, confirming channel specificity.
Area of Science:
- Immunology
- Cell Physiology
- Molecular Biology
Background:
- Adenosine triphosphate (ATP) acts as an extracellular signaling molecule.
- Purinergic receptors, specifically P2X receptors, are ion channels activated by ATP.
- Macrophages play crucial roles in immune responses and express various P2X receptor subtypes.
Purpose of the Study:
- To characterize ATP-induced (P2X) currents in isolated peritoneal macrophages from wild-type (WT) and P2X(4) knockout (P2X(4)(-/-)) mice.
- To investigate the functional expression and specific contribution of P2X(4) receptors in macrophages.
- To survey the profile of native P2X currents in macrophages.
Main Methods:
- Whole-cell patch-clamp electrophysiology was employed to record P2X currents.
- Macrophages were isolated from peritoneal cavities of WT and P2X(4)(-/-) mice.
- Pharmacological tools, including a P2X(7) blocker (A-740003) and a P2X(4) modulator (ivermectin), were used.
Main Results:
- Three distinct P2X currents were identified: P2X1-like (fast activating/inactivating), P2X(7)-like (non-desensitizing), and P2X(4)-like (slowly desensitizing).
- The P2X(4)-like current was present in 88.6% of WT macrophages but completely absent in P2X(4)(-/-) macrophages.
- P2X(7)-like currents were observed in both WT (85%) and P2X(4)(-/-) (100%) macrophages, confirmed by A-740003.
- Ivermectin modulated the P2X(4)-like current amplitude and desensitization in WT cells.
Conclusions:
- Peritoneal macrophages from WT mice functionally express P2X(4) and P2X(7) receptor channels.
- The P2X(4)-like current identified is specifically mediated by the P2X(4) receptor, as evidenced by its absence in P2X(4)(-/-) mice.
- These findings contribute to understanding purinergic signaling in macrophage function.
