P2X4 Receptor-Dependent Ca2+ Influx in Model Human Monocytes and Macrophages

Layhadi, Janice A.; Fountain, Samuel J.

doi:10.3390/ijms18112261

Cited by 38 publications

(30 citation statements)

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“…Consistent with these data, 5‐BDBD has been reported as a moderately potent inhibitor of human and rodent recombinant P2X4 receptors, with limited effectiveness at submicromolar concentrations (Abdelrahman et al, 2017; Ase et al, 2015; Balazs et al, 2013; Coddou, Sandoval, Hevia, & Stojilkovic, 2019). The data presented here are also in agreement with recent studies demonstrating inhibition of endogenous P2X4 receptor activity in human monocyte‐derived macrophages (Layhadi & Fountain, 2017) and human CD4 + T cells (Ledderose et al, 2018). Furthermore, the current study reported 5‐BDBD to be ineffective at inhibiting Ca 2+ responses induced by saturating concentrations of ATP.…”

Section: Discussionsupporting

confidence: 92%

Pharmacological and genetic characterisation of the canine P2X4 receptor

Sophocleous

Berg

Finol‐Urdaneta

et al. 2020

British J Pharmacology

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Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors.Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca 2 + and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key Results:No P2RX4 missense variants were identified in any canine samples.Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2 0 (3 0 )-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors.Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptorrelated disease onset and management in dogs and humans.Abbreviations: 5-BDBD, 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one; BX430, 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea; BzATP, 2 0 (3 0 )-O-(4-benzoylbenzoyl)-ATP; ECS, extracellular Ca 2+ solution; EmGFP, Emerald GFP; SR, seal resistance; TNP-ATP, 2 0 ,3 0 -O-(2,4,6-trinitrophenyl)-ATP.

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Section: Discussionsupporting

confidence: 92%

Pharmacological and genetic characterisation of the canine P2X4 receptor

Sophocleous

Berg

Finol‐Urdaneta

et al. 2020

British J Pharmacology

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“…The agonist profile of ATP on DH82 cells demonstrated pharmacological similarities to the recombinant canine P2X4 receptor [41], as well as studies of endogenous P2X4 receptors in a human macrophage cell model [34]. BzATP induced partial Ca 2+ responses in DH82 cells with significantly lower potency compared to ATP, consistent with the recent report that BzATP is a partial agonist of recombinant canine P2X4 receptors [41].…”

Section: Discussionsupporting

confidence: 82%

“…To date, the DH82 canine macrophage cell line has primarily been utilized as a model of viral and protozoan infection [23][24][25], such as for the study of canine distemper and its oncolytic potential [55][56][57]. Although studies have described the expression of functional P2 receptors in human or rodent macrophages and macrophage cell lines [8,10,11,34], studies directly investigating P2 receptors in canine macrophages have been absent. The current study described, for the first time, the expression and function of P2 receptors in DH82 cells, demonstrating a primary role for cell surface P2Y 2 receptors in nucleotide-mediated Ca 2+ mobilization through PLC/IP 3 signal transduction.…”

Section: Discussionmentioning

confidence: 99%

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

Sophocleous

Miles

Ooi

et al. 2020

IJMS

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Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.

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“…1A). This finding is in accordance with previous studies reporting functional evidence for these receptors in monocyte-derived human macrophages (44)(45)(46)(47)(48).…”

Section: Human Macrophages Express Multiple P2x Receptorssupporting

confidence: 93%

ATP-Gated P2X7 Receptors Require Chloride Channels To Promote Inflammation in Human Macrophages

Janks

Sprague

Egan

2019

The Journal of Immunology

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Immune cells of myeloid origin show robust expression of ATP-gated P2X7 receptors, two-transmembrane ion channels permeable to Na + , K + , and Ca 2+. Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1b and IL-18. In this study, we show that ATP generates facilitating cationic currents in monocyte-derived human macrophages and permeabilizes the plasma membrane to polyatomic cationic dyes. We find that antagonists of PLA 2 and Cl 2 channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1b release. Our data demonstrate significant differences in the actions of ATP in murine and human macrophages and suggest that PLA 2 and Cl 2 channels mediate innate immunity downstream of P2X7 receptors in human macrophages.

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P2X4 Receptor-Dependent Ca2+ Influx in Model Human Monocytes and Macrophages

Cited by 38 publications

References 34 publications

Pharmacological and genetic characterisation of the canine P2X4 receptor

Pharmacological and genetic characterisation of the canine P2X4 receptor

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

ATP-Gated P2X7 Receptors Require Chloride Channels To Promote Inflammation in Human Macrophages

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