Does Cholinergic Stimulation Affect the P2X7 Receptor-Mediated Dye Uptake in Mast Cells and Macrophages?

Abstract Background Extracellular ATP signaling through excitatory and calcium-permeable P2X receptor channels is considered as a critical player in pain generation and maintenance. P2X7 has attracted much attention over the past decade because of its prominent role in driving inflammatory processes. The role of P2X7 in mast cells in peripheral pain remains unclear.Methods P2X expression in mouse peritoneal mast cells was detected by RT-PCR. The subtypes of P2X receptors in mouse peritoneal mast cells were determined with a series of blockers by using calcium imaging and electrophysiology. The regulation of inflammatory factors mediated by different P2X subtypes were detected by ELISA and real-time PCR. The role of mast cells and P2X7 receptor in peripheral pain was explored by behavioral assays, pathological analysis and real-time PCR. Several anti-inflammatory small molecules were screened based on P2X7 in mast cells by using calcium imaging, electrophysiology and molecular docking.Results We found that ATP was significantly increased in inflammatory pain. Mouse peritoneal mast cells expressed P2X1, P2X3, P2X4 and P2X7 and could be activated by different concentrations of extracellular ATP, which could be blocked by specific ion channel antagonists. In particularly, high concentration of ATP could induce mast cells to release inflammatory mediators such as histamine, IL-1β and CCL3 through P2X7 receptor. Furthermore, peripheral pain induced by high concentration of ATP could be alleviated by P2X7 blockers or mast cell defects. Interestingly, salicylic acid and aspirin could attenuate the inward current, the release of inflammatory factors and peripheral pain induced by ATP with high concentration. Furthermore, salicylic acid and aspirin also inhibited the inward current evoked by P2X7 agonist BZATP. Molecular docking results showed that salicylic acid and aspirin had affinity to the cytoplasmic GDP-binding region of P2X7.Conclusions We concluded that P2X7 on mast cells involved in peripheral pain. Salicylic acid and aspirin could inhibit the activity of P2X7 via interacting with the GDP binding region. P2X7 receptor was a potential target for salicylic acid and aspirin analgesia.

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ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells

Journal of Leukocyte Biology ◽

10.1189/jlb.0808470 ◽

2009 ◽

Vol 85 (4) ◽

pp. 692-702 ◽

Cited By ~ 20

Author(s):

B. Elena ◽

B. Vadim ◽

O. Zane ◽

K.-N. Friedrich ◽

H. Friedrich ◽

...

Keyword(s):

Mast Cells ◽

P2x7 Receptor ◽

P2x3 Receptors ◽

Chemokine Expression

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Upregulation of P2X7 receptor signaling by interleukin-33 in mouse bone marrow-derived mast cells

Proceedings for Annual Meeting of The Japanese Pharmacological Society ◽

10.1254/jpssuppl.94.0_1-p2-27 ◽

2021 ◽

Vol 94 (0) ◽

pp. 1-P2-27

Author(s):

Kazuki Yoshida ◽

Yuu Ishida ◽

Masaaki Ito ◽

Isao Matsuoka

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

P2x7 Receptor ◽

Mouse Bone Marrow ◽

Receptor Signaling ◽

Interleukin 33

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The P2X7 receptor–pannexin connection to dye uptake and IL-1β release

Purinergic Signalling ◽

10.1007/s11302-009-9141-7 ◽

2009 ◽

Vol 5 (2) ◽

pp. 129-137 ◽

Cited By ~ 110

Author(s):

Pablo Pelegrin ◽

Annmarie Surprenant

Keyword(s):

P2x7 Receptor ◽

Dye Uptake

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Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor-expressing cells: implications for its therapeutic action

British Journal of Pharmacology ◽

10.1111/j.1476-5381.2011.01254.x ◽

2011 ◽

Vol 163 (5) ◽

pp. 912-926 ◽

Cited By ~ 56

Author(s):

C Marques-da-Silva ◽

MM Chaves ◽

NG Castro ◽

R Coutinho-Silva ◽

MZP Guimaraes

Keyword(s):

P2x7 Receptor ◽

Cationic Dye ◽

Therapeutic Action ◽

Dye Uptake

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N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

Purinergic Signalling ◽

10.1007/s11302-017-9598-8 ◽

2017 ◽

Vol 14 (1) ◽

pp. 83-90

Author(s):

Karin Dreisig ◽

Nikolaj Pagh Kristensen ◽

Maja Wallentin Dommer ◽

Niklas Rye Jørgensen ◽

Birgitte Rahbek Kornum

Keyword(s):

P2x7 Receptor ◽

Calcium Influx ◽

Dye Uptake

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8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2018.11.036 ◽

2019 ◽

Vol 27 (8) ◽

pp. 1449-1455 ◽

Cited By ~ 7

Author(s):

P.A.F. Pacheco ◽

R.M.S. Galvão ◽

A.F.M. Faria ◽

N.l. Von Ranke ◽

M.S. Rangel ◽

...

Keyword(s):

P2x7 Receptor ◽

Dye Uptake ◽

Murine Macrophages

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N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1βRelease from Murine Macrophages

Mediators of Inflammation ◽

10.1155/2016/2097219 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Ronald Sluyter ◽

Kara L. Vine

Keyword(s):

P2x7 Receptor ◽

Colorimetric Assay ◽

Dependent Manner ◽

Dye Uptake ◽

Anticancer Activities ◽

Concentration Dependent Manner ◽

Parent Molecule ◽

Murine Macrophages ◽

Ldh Release ◽

Isatin Derivatives

Extracellular adenosine 5′-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1βrelease from murine J774 macrophages. ATP-induced IL-1βand lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1βrelease in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-{4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1βrelease by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates thatN-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1βrelease from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment.

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