ATP released by LPS increases nitric oxideproduction in raw 264.7 macrophage cell line viaP2Z⧹P2X7 receptors

Abstract

P2Z⧹P2X7 receptor is a particular type of purinoceptor, which is selectively expressedon the surface of immune cells in neuronal and non-neuronal tissues. Despite intensive researchon its involvement in the immune response, the exact mechanism whereby it affects intercellularsignaling is far from clear yet. In this study, the effect of activation P2Z⧹P2X7 receptor wasinvestigated on the bacterial lipopolysaccharide induced nitric oxide production in RAW 264.7macrophage call line using the nitrite:nitrate assay. The P2Z⧹P2X7 receptor agonist 3'-O-(4-benzoylbenzoyl)-adenosine 5'triphosphateincreased concentration-dependency the lipo- plysaccharide (10 μg⧹ml) induced nitricoxide production between 10 μM and 250 μM. ATP also increased nitric oxideproduction in response to lipopolysaccharide, while ADP, 2-methylo-thio-adenosine5'-triphosphate and adenosine 5'triphosphate- γ-S was without effect. Pretreatment withoxidized adenosine triphosphate, the selective P2Z⧹P2X7 receptor antagonise (300 μM-1 μM) strongly decreased lipopolysaccaride induced nitric oxide production.Furthermore, on macrophages, pretreated with oxidized adenosine 5'- triphosphate (300 μM -1 μM), 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate and ATP did notaffect lipopolysaccharide induced nitric oxide production. 15 min lipopolysaccharide treatmentinduced a transient and reversible release of endogenous ATP from RAW 264.7 cells, measuredby the luciferin luciferase assay. The effect of lipopolysaccharide to promote ATP release wasconcentration-dependent between 1-10 μg⧹ml. In summary, our results show that P2Z⧹P2X7 receptor activation results in an increase innitric oxide production in response to lipopolysaccharide challenge. Since the P2Z⧹P2X7receptor antagonist oxidized adenosine triphosphate decreased lipopolysaccharide induced nitricoxide production, and lipopolysaccharide was able to promote ATP release from macrophagecells, it seems likely that endogenous ATP is involved in nitric oxide formation during endotoxinchallenge.