Nucleotide receptor signalling and the generation of reactive oxygen species

Abstract

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology.

Fig. 1

Working model for the involvement of P2X and P2Y receptors in regulating the phagocytic NADPH oxidase complex. In the resting state, a small portion of gp91^phox (Nox2) and p22^phox are located at the plasma membrane (although priming with factors such as LPS or chemoattractants can recruit additional gp91^phox and p22^phox to the membrane and promote the assembly of the NADPH complex (see text)). The oxidase function of the gp91^phox/p22^phox is dormant until it is complexed with the cytosolic components p67^phox, p47^phox, p40^phox, and Rac1/2. Stimulus-induced phosphorylation of p47^phox results in a conformational change that allows it to be recruited to the membrane and additional phosphorylation of the p47^phox/p40^phox/p67^phox complex allows for its structural reorganization and assembly with gp91^phox/p22^phox at the membrane. Also, stimulus-induced recruitment of activated (GTP-loaded) Rac2 to the membrane facilitates the assembly of the functional NADPH oxidase complex. With respect to phox protein phosphorylation, several kinases are postulated to be important, including PKC isoforms and p38 MAP kinase. Also, Ca⁺⁺ fluxes can promote NADPH oxidase assembly, in part via Ca⁺⁺-dependent activation of kinases (e.g., PKC isoforms). Moreover, Rac activation may also contribute to phox protein phosphorylation via initiating p38 MAP kinase activation. Final assembly/activation of the NADPH complex requires additional phosphorylation steps. In terms of P2X and P2Y receptor-associated activation of NADPH oxidase activity, it is hypothesized that increases in intracellular Ca⁺⁺ induced by P2X agonists result in the activation of protein kinases, such as PKC isoforms, that are essential for the phosphorylation/activation of NADPH oxidase subunits including p47^phox. Furthermore, activation of P2X₇ has been linked to the stimulation of p38 MAP kinase, which would also be expected to facilitate the phosphorylation/activation of NADPH oxidase subunits. In the case of P2Y receptors, many of these receptors can regulate certain phospholipase C (PLC) isoforms with the subsequent conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to IP₃ and diacylglycerol (DAG), which in turn would lead to the elevation of cytoplasmic free Ca⁺⁺ and the activation of PKC isoforms, respectively. These events would also be expected to promote the phosphorylation/activation of NADPH oxidase subunits. Furthermore, certain P2Y receptors have also been reported to lead to Rac activation, which in turn would be predicted to facilitate the phosphorylation, activation and assembly of NADPH oxidase