NOS-2, the inducible enzyme isoform in the nitric oxide-generating pathway, is a key regulator of inflammatory processes. In the brain, increased expression of NOS-2 is associated with lesions in demyelinating and neurodegenerative processes (multiple sclerosis, Alzheimer’s). In addition, nitric oxide has been implicated in neural damage mechanisms during hypoxia/ischaemia and viral infection. Purine compounds can modulate NOS-2 induction. It has been claimed that ATP down-regulates the induction of NOS-2 expression elicited by IL-1β and λ-interferon. The inhibitory effect was revealed by pre-treatment with ATP for 30-120 min before induction of NOS-2 with IL-1β and IFNλ. However, ATP is very susceptible to hydrolysis by (a) serum esterases or (b) cellular ecto-nucleotidases. The action on NOS-2 induction may be mediated by the degradation products. We have investigated the modulatory role of ATP and adenosine on the expression of NOS-2 in cultures of rat cerebellar astrocytes stimulated with IL-1β (0.5 ng ml^-1) and IFNλ (50 U ml^-1). Animals (7-day-old rats) were maintained according to international standards and humanely killed by decapitation. The induction of the enzyme was determined after 48 h by measuring nitrite production with the Griess reaction. ATP concentrations were measured by luminescence using luciferase assay.

We have measured the lifetime of ATP in culture medium. There is a rapid hydrolysis of ATP added to DMEM + 10 % FCS medium. ATP was degraded at a rate of 2.5 % min^-1. The t_1/2 was 40 ± 2 min (mean ± S.D., n = 3). The hydrolysis was even faster when ATP (100 µM) was added to astrocyte cultures. The concentration of ATP dropped to levels below 10 nM within the treatment period (< 30 min). The EC₅₀ values for ATP acting on P2Y and P2X receptors are typically higher (> 1 µM). In astrocytes, the nitrite production after a 48 h treatment with IL-1β and IFNλ was partially inhibited by ATP. However, the P2 antagonist PPADS (25 µM) and Reactive Blue (25 µM) did not prevent the action of ATP. Furthermore, the addition to the extracellular medium of the enzyme apyrase, to assure the complete hydrolysis of added ATP, failed to block the effect of ATP. Thus, the ATP effect may be mediated by a metabolite, rather than by the nucleotide itself. The end product of extracellular ATP hydrolysis is adenosine. Exogenous adenosine did inhibit NOS-2 induction by IL-1β and IFNλ as ATP did. In addition, the antagonist DPCPX (10 nM-1 µM) completely reversed the action of adenosine and, also, of ATP, indicating that the action of both compounds is mediated by adenosine receptors. The treatment with DPCPX resulted in an overshoot effect, suggesting that there is a tonic inhibition by adenosine in astrocyte cultures. These results indicate that the reported action of ATP in NOS-2 expression is mediated by adenosine receptors, activated by adenosine generated from ATP hydrolysis. This process should be checked whenever the effects of ATP treatments of more than a few minutes are investigated.

This work was supported by a research grant from Comunidad Autónoma Canaria, CAC PI2001/106. D.L.V. is supported by a fellowship from Cabildo Insular de Gran Canaria.