Abstract
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.
Highlights
The activation of purinergic receptors by nucleotides such as adenosine 5 -triphosphate (ATP) and uridine 5 -triphosphate (UTP) is crucial for a number of inflammatory processes, including those in the central nervous system (CNS) such as chronic pain [1,2,3,4] and remyelination of nerves following injury to the CNS [5,6]
Studies have demonstrated that DH82 cells express a number of macrophage markers, such as CD11c and CD18 [21], and can secrete tumour necrosis factor (TNF)-α and interleukin (IL)-6 similar to that observed in lipopolysaccharide (LPS)-stimulated canine monocytes [22]
Through investigation of canine P2 receptor expression and functional characterisation of these receptors, this study has identified the P2Y2 receptor and, to a lesser extent, the P2X4 receptor, as the primary functional P2 receptors in DH82 cells, which are responsible for nucleotide-mediated Ca2+ mobilization
Summary
The activation of purinergic receptors by nucleotides such as adenosine 5 -triphosphate (ATP) and uridine 5 -triphosphate (UTP) is crucial for a number of inflammatory processes, including those in the central nervous system (CNS) such as chronic pain [1,2,3,4] and remyelination of nerves following injury to the CNS [5,6]. P2 receptors, such as the P2X4 and P2Y2 receptors, are commonly expressed on human and rodent macrophages and macrophage cell lines [8,9,10,11,12,13,14], and have demonstrated roles in signalling pathways that control chronic pain and inflammation in humans or rodents [2,8,15,16,17,18]. Human and rodent macrophage or myeloid cell lines, such as THP-1 and RAW264.7 cells, are well-established models for studying endogenous P2 receptors commonly expressed on human and rodent macrophages [31,32,33,34,35,36,37,38]. Through investigation of canine P2 receptor expression and functional characterisation of these receptors, this study has identified the P2Y2 receptor and, to a lesser extent, the P2X4 receptor, as the primary functional P2 receptors in DH82 cells, which are responsible for nucleotide-mediated Ca2+ mobilization
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