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P2y2 and p2x4 receptors mediate ca2+ mobilization in dh82 canine macrophage cells

Journal Article


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Abstract


  • © 2020, MDPI AG. All rights reserved. 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.

Publication Date


  • 2020

Citation


  • Sophocleous, R., Miles, N., Ooi, L. & Sluyter, R. (2020). P2y2 and p2x4 receptors mediate ca2+ mobilization in dh82 canine macrophage cells. International Journal of Molecular Sciences, 21 (22), 1-22.

Scopus Eid


  • 2-s2.0-85096239317

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2812&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1778

Number Of Pages


  • 21

Start Page


  • 1

End Page


  • 22

Volume


  • 21

Issue


  • 22

Place Of Publication


  • Switzerland

Abstract


  • © 2020, MDPI AG. All rights reserved. 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.

Publication Date


  • 2020

Citation


  • Sophocleous, R., Miles, N., Ooi, L. & Sluyter, R. (2020). P2y2 and p2x4 receptors mediate ca2+ mobilization in dh82 canine macrophage cells. International Journal of Molecular Sciences, 21 (22), 1-22.

Scopus Eid


  • 2-s2.0-85096239317

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2812&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1778

Number Of Pages


  • 21

Start Page


  • 1

End Page


  • 22

Volume


  • 21

Issue


  • 22

Place Of Publication


  • Switzerland