ATP released by LPS increases nitric oxide production in raw 264.7 macrophage cell line via P2Z/P2X7 receptors

P2Z/P2X7 receptor is a particular type of purinoceptor, which is selectively expressed on the surface of immune cells in neuronal and non-neuronal tissues. Despite intensive research on its involvement in the immune response, the exact mechanism whereby it affects intercellular signaling is far from clear yet. In this study, the effect of activation P2Z/P2X7 receptor was investigated on the bacterial lipopolysaccharide induced nitric oxide production in RAW 264.7 macrophage call line using the nitrite/nitrate assay. The P2Z/P2X7 receptor agonist 3'-O-(4-benzoylbenzoyl)-adenosine 5'triphosphate increased concentration-dependency the lipoplysaccharide (10 microg/ml) induced nitric oxide production between 10 microM and 250 microM. ATP also increased nitric oxide production in response to lipopolysaccharide, while ADP, 2-methylo-thio-adenosine 5'-triphosphate and adenosine 5'triphosphate-gamma-S was without effect. Pretreatment with oxidized adenosine triphosphate, the selective P2Z/P2X7 receptor antagonise (300 microM-1 microM) strongly decreased lipopolysaccaride induced nitric oxide production. Furthermore, on macrophages, pretreated with oxidized adenosine 5'-triphosphate (300 microM-1 mM), 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate and ATP did not affect lipopolysaccharide induced nitric oxide production. 15 min lipopolysaccharide treatment induced a transient and reversible release of endogenous ATP from RAW 264.7 cells, measured by the luciferin-luciferase assay. The effect of lipopolysaccharide to promote ATP release was concentration-dependent between 1-10 microg/ml. In summary, our results show that P2Z/P2X7 receptor activation results in an increase in nitric oxide production in response to lipopolysaccharide challenge. Since the P2Z/P2X7 receptor antagonist oxidized adenosine triphosphate decreased lipopolysaccharide induced nitric oxide production, and lipopolysaccharide was able to promote ATP release from macrophage cells, it seems likely that endogenous ATP is involved in nitric oxide formation during endotoxin challenge.