Activation of the A2A adenosine receptor inhibits nitric oxide production in glial cells

Abstract

Selective adenosine receptor agonists and antagonists have marked effects on the outcome of cerebral ischemia, and adenosine receptors are expressed on astrocytes. In this study we examined the effects of various adenosine receptor agonists on the production of nitric oxide and the induction of iNOS in astrocytes activated by LPS/IFN-gamma and TNF-alpha/IL-1beta and on the production of TNF-alpha. Treatment of the cells with the A2A receptor agonist CGS 21680 inhibited both NO production and iNOS expression induced by stimulation with either LPS/IFN-gamma or TNF-alpha/IL-1beta, whereas the A1 and A3 receptor agonists, CPA and Cl-IB-MECA, respectively, did not have significant inhibitory effects. The inhibitory effect of the A2A receptor agonist was antagonized by the specific A2A receptor antagonist CSC. The A2A agonist also exerted a small inhibitory effect on the production of TNF-alpha. Similar inhibitory effects on the production of NO were obtained by cyclic AMP-elevating reagents, such as forskolin and dibutyryl cyclic AMP. Our findings suggest that activation of the A2A receptor inhibits NO production and iNOS expression likely via increased cAMP.

Fig. 1

Effects of adenosine receptor agonists and cAMP-elevating agents on NO production in C6 glioma cells. C6 cells were treated with LPS/IFN-γ and different concentrations of COS 21680 in the absence (broken line) and presence (solid line) of the A_2A antagonist CSC at a concentration of 10 μM, **P < 0.001, *P < 0.01. A: C6 cells were treated with either LPS/IFN-γ (open bars) or TNF-α/IL-lβ (shaded bars) for 24 h in the absence (labeled medium) or presence of A₁ (CPA, 1 μM), A_2A (CGS 21680, 10 μM), or A₃ (Cl-IB-MECA, 1 μM) agonists, **P < 0.001, *P < 0.003 (B), or in the presence of cAMP-elevating agents, forskolin (10 μM) and db-cAMP (100 μM), *P < 0.004 (C). Supernatants were collected for the determination of ${\text{NO}}_2^{-}$. Results represent the means±S.D. of three separate experiments.

Fig. 2

Effect of the A_2A receptor agonist CGS 21680 and db-cAMP on iNOS induction. Cells were stimulated with either LPS/IFN-γ (A) or with TNF-α/IL-lβ (B) for 24 h in the absence and presence of CGS 21680 (10 μM), CSC (10 μM) or db-cAMP (100 μM). Whole cell lysates were resolved by SDS-PAGE and proteins were immunoblotted with anti-iNOS antibody. The results are of one representative experiment out of three.

Fig. 3

Effect of adenosine agonists selective for A₁ (CPA, 1 μM), A_2A (CGS 21680, 10 μM), or A₃ (Cl-IB-MECA, 1 μM) receptors and db-cAMP (100 μM) on TNF-α secretion in LPS/IFN-γ-treated cells. C6 glioma cells were treated with LPS/IFN-γ in the presence and absence of different adenosine agonists and db-cAMP, and TNF-α levels were determined following 48 h using an ELISA kit. The results represent means ±S.D. from three separate experiments. *P < 0.002, **P < 0.01.