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

Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca2+ and was blocked by Ca2+ chelator BAPTA. IL-6 release and Ca2+ elevation occurred rapidly, consistent with vesicular IL-6 staining in unstimulated cells. P2X7R stimulation did not trigger IL-1β release in these unprimed cells. P2X7R-mediated IL-6 release was enhanced in RPE cells from the ABCA4−/− mouse model of retinal degeneration. In summary, P2X7R stimulation triggers rapid Ca2+-dependent IL-6 release across the apical membrane of RPE cells.

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Probenecid Blocks Human P2X7 Receptor-Induced Dye Uptake via a Pannexin-1 Independent Mechanism

PLoS ONE ◽

10.1371/journal.pone.0093058 ◽

2014 ◽

Vol 9 (3) ◽

pp. e93058 ◽

Cited By ~ 46

Author(s):

Archana Bhaskaracharya ◽

Phuong Dao-Ung ◽

Iman Jalilian ◽

Mari Spildrejorde ◽

Kristen K. Skarratt ◽

...

Keyword(s):

P2x7 Receptor ◽

Dye Uptake ◽

Pannexin 1 ◽

Independent Mechanism

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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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Antiapoptotic Effects of Estrogen in Normal and Cancer Human Cervical Epithelial Cells

Endocrinology ◽

10.1210/en.2004-0807 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5568-5579 ◽

Cited By ~ 36

Author(s):

Qifang Wang ◽

Xin Li ◽

Liqin Wang ◽

Ying-Hong Feng ◽

Robin Zeng ◽

...

Keyword(s):

Epithelial Cells ◽

P2x7 Receptor ◽

Mitochondrial Permeability Transition ◽

Calcium Influx ◽

Cycle Phase ◽

Caspase 9 ◽

Antiapoptotic Effect ◽

17Β Estradiol ◽

Cervical Epithelial Cells ◽

Cervical Cells

Abstract The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca2+-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nm 17β-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2′,3′-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16–1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17β-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca2+-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

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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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