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

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.

Pannexin-1 channel opening is critical for COVID-19 pathogenesis

“…Other purinergic receptors, such as P2Y 2 , P2Y 6 , and P2X 7 , could also contribute to pathological effects in pulmonary cell types. P2Y 2 , activated by ATP but not ADP (Abbracchio and Ceruti, 2006), regulates endothelial inflammation, most notably promoting the adhesion and chemotaxis of inflammatory cells (Alberto et al, 2016;Burnstock, 2017;Buscher et al, 2006;Gabl et al, 2015;Liu et al, 2016;Marcet et al, 2004;Muhleder et al, 2020;Sophocleous et al, 2020;van Heusden et al, 2020). P2Y 12 has also been demonstrated to contribute to endothelial cell pathology (Sidiropoulou et al, 2021;Vemulapalli et al, 2020).…”

Anti-Inflammatory Effects of Novel P2X4 Receptor Antagonists, NC-2600 and NP-1815-PX, in a Murine Model of Colitis

“…In order to corroborate the results obtained from in vivo experiments, we designed a set of in vitro experiments to evaluate the effect of the P2X4 antagonists on modulating the canonical and noncanonical pathways involved in NLRP3 activation. Therefore, we performed experiments in LPS-primed and PMA-differentiated THP-1 cells treated with ATP, an established model to investigate the functions of purinergic receptors in monocyte/macrophage cells [37][38][39]. THP-1 cells were incubated with LPS, a wellrecognized activator of the rst step of NLRP3 signaling as well as a hallmark of altered intestinal permeability, a condition typically observed in the murine model of colitis and IBD patients [40,41].…”

The P2X4 Receptor: Cellular and Molecular Characteristics of a Promising Neuroinflammatory Target

“…P2X4 receptors are also expressed on mast cells [ 34 ], where they are involved in mast cell degranulation [ 35 ], as well as on other myeloid cells including monocytes [ 36 , 37 , 38 ], tissue-resident macrophages [ 3 , 39 ], and eosinophils [ 40 ]. P2X4 receptors are also commonly observed on cultured mammalian monocyte and macrophage cells and differentiated cell lines [ 39 , 41 , 42 , 43 , 44 , 45 , 46 ]. P2X4 receptors expressed on microglia have also been well-documented, and together with macrophages, have known roles in signalling pathways that mediate neuroinflammatory responses and chronic pain [ 3 , 4 , 24 , 47 , 48 , 49 ].…”