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P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium(+) uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium(+) uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium(+) uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.

Contains fulltext : 89873.pdf (Publisher’s version ) (Closed access) 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. 01 september 2010

The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

P2X7, a damage-associated molecular pattern receptor and adenosine 5'-triphosphate (ATP)-gated cation channel, plays an important role in the activation of the NALP3 inflammasome and subsequent release of interleukin (IL)-1β from human monocytes; however its role in monocytes from other species including the dog remains poorly defined. This study investigated the role of P2X7 in canine monocytes, including its role in IL-1β release. A fixed-time flow cytometric assay demonstrated that activation of P2X7 by extracellular ATP induces the uptake of the organic cation, YO-PRO-1(2+), into peripheral blood monocytes from various dog breeds, a process impaired by the specific P2X7 antagonist, A438079. Moreover, in five different breeds, relative P2X7 function in monocytes was about half that of peripheral blood T cells but similar to that of peripheral blood B cells. Reverse transcription-PCR demonstrated the presence of P2X7, NALP3, caspase-1 and IL-1β in LPS-primed canine monocytes. Immunoblotting confirmed the presence of P2X7 in LPS-primed canine monocytes. Finally, extracellular ATP induced YO-PRO-1(2+) uptake into and IL-1β release from these cells, with both processes impaired by A438079. These results demonstrate that P2X7 activation induces the uptake of organic cations into and the release of IL-1β from canine monocytes. These findings indicate that P2X7 may play an important role in IL-1β-dependent processes in dogs.

Epithelial cells are important in inflammation and immunity. In this study, we examined if Madin-Darby canine kidney (MDCK) epithelial cells express functional P2X7 receptors, which bind the damage-associated molecular pattern extracellular adenosine 5'-triphosphate (ATP). Reverse transcription (RT)-PCR and immunoblotting revealed the expression of P2X7 in MDCK cells. A flow cytometric assay demonstrated that ATP or 2'(3')-O-(4-benzoylbenzoyl)ATP induced ethidium(+) uptake into MDCK cells, and that this process was impaired by the P2X7 antagonists KN-62 and A438079. RT-PCR also demonstrated the presence of Toll-like receptor 4, NALP3, caspase-1, interleukin-1β and interleukin-18 in MDCK cells, as well as in positive control LPS-primed canine monocytes. In conclusion, the MDCK epithelial cell line expresses functional P2X7, as well as Toll-like receptor 4 and molecules associated with the NALP3 inflammasome. This cell line may help elucidate the role of these molecules in kidney epithelial cells and renal disorders in dogs and humans.

Background The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. Methods Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. Results RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~ 116 μM, and this uptake was reduced in the presence of extracellular Ca2+ and Mg2+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. Conclusions P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. General significance RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.