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 selec tively expressed on the surface of immune cells in neuronal and non-ne uronal tissues. Despite intensive research on its involvement in the i mmune response, the exact mechanism whereby it affects intercellular s ignaling is far from clear yet. In this study, the effect of activatio n P2Z/P2X7 receptor was investigated on the bacterial lipopolysacchari de induced nitric oxide production in RAW 264.7 macrophage call line u sing the nitrite/nitrate assay. The P2Z/P2X7 receptor agonist 3'-O-(4- benzoylbenzoyl)-adenosine 5'triphosphate increased concentration-depen dency the lipoplysaccharide(10 mu g/ml) induced nitric oxide productio n between 10 mu M and 250 mu M. ATP also increased nitric oxide produc tion in response to lipopolysaccharide, while ADP, 2-methylo-thio-aden osine 5'-triphosphate and adenosine 5'triphosphate-gamma-S was without effect. Pretreatment with oxidized adenosine triphosphate, the select ive P2Z/P2X7 receptor antagonise (300 mu M-1 mu M) strongly decreased lipopolysaccaride induced nitric oxide production. Furthermore, on mac rophages, pretreated with oxidized adenosine 5'triphosphate (300 mu M - 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 relea se of endogenous ATP from RAW 264.7 cells, measured by the luciferin-l uciferase assay. The effect of lipopolysaccharide to promote ATP relea se was concentration-dependent between 1-10 mu g/ml. In summary, our r esults show that P2Z/P2X7 receptor activation results in an increase i n nitric oxide production in response to lipopolysaccharide challenge. Since the P2Z/P2X7 receptor antagonist oxidized adenosine triphosphat e decreased lipopolysaccharide induced nitric oxide production, and li popolysaccharide was able to promote ATP release from macrophage cells , it seems likely that endogenous ATP is involved in nitric oxide form ation during endotoxin challenge. (C) 1998 Elsevier Science Ltd. All r ights reserved.