Potentiation by ATP of lipopolysaccharide-stimulated nitric oxide production in cultured astrocytes.

The functional changes of astrocytes are deeply involved in neurodegenerating processes of various CNS diseases. ATP is released during various neuronal damages such as brain ischemia and may control astrocyte functions. We examined the effect of ATP on the production of nitric oxide in the cultured astrocytes from rat embryo. The astrocytes were stimulated by lipopolysaccharide instead of pathological activation in vivo. Nitric oxide production was evaluated by the fluorometric assay of nitrite accumulated in the medium. The expression of inducible nitric oxide synthase was analyzed by Western blotting. Nitric oxide production induced by 1 ng/ml lipopolysaccharide was enhanced by ATP with maximal enhancement of three- to four-fold; a half-effective concentration was about 0.3 mM. In the absence of ATP, half-effective concentration of lipopolysaccharide on nitric oxide production was about 3 ng/ml; however, half-effective concentration shifted to 0.3 ng/ml in the presence of 1.5-mM ATP. Several other P2 receptor agonists (uridine triphosphate, ADP, adenosine monophosphate, 2'- and 3'-O - (4-benzoylbenzoyl)-ATP, and 2-methylthioATP) showed a similar enhancing effect, and an antagonist, ATP-2',3'-dialdehyde, showed an inhibiting effect. Western blotting analysis revealed that the extent of lipopolysaccharide-induced expression of nitric oxide synthase increased several-fold by the addition of ATP; half-effective concentration was about 0.5 mM. These results suggest that the extracellular ATP plays an important role as a transmitter and regulates astrocyte functions via a certain P2 receptor and that such a change in astrocyte function is involved in either protection or aggravation in neurodegenerative processes.