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

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.