Adenosine kinase inhibitor attenuates the expression of inducible nitric oxide synthase in glial cells

Abstract

The present study demonstrates the anti-inflammatory effect of adenosine kinase inhibitor (ADKI) in glial cells. Treatment of glial cells with IC51, an ADKI, stimulated the extracellular adenosine release and reduced the LPS/IFNγ-mediated production of NO, and induction of iNOS and TNF-α gene expression. The recovery of IC51-mediated inhibition of iNOS expression by adenosine transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), and the inhibition of LPS/IFNγ-induced iNOS gene expression by exogenous adenosine indicate a role for adenosine release in IC51-mediated iNOS expression. The rescue of IC51-mediated inhibition of iNOS expression by adenosine receptor antagonist for A_2A, 8-(3-chlorostyryl)caffeine (CSC) and alloxazine for A_2B, further supports a role for interaction of adenosine and its receptors in anti-inflammatory activity. The IC51-mediated induction of cAMP levels, downstream target of A_2A and A_2B, and inhibition of LPS/IFNγ-induced expression of iNOS by forskolin, a cAMP activator, document a role for cAMP mediated pathway in anti-inflammatory activity of IC51. Taken together, these studies document that IC51-mediated inhibition of iNOS expression is through activation of adenosine receptors, which activates A_2A and A_2B resulting in increased cAMP levels following LPS/IFNγ stimulation. Moreover, the lack of effect of IC51 or adenosine on NFκB DNA binding activity and its transactivity indicates that the inhibition of iNOS expression mediated by IC51 may be through an NFκB independent pathway.

Introduction

Nitric oxide (NO) is a short-lived signaling molecule that mediates a wide range of biological effects, such as vasorelaxation (Palmer et al., 1987), neurotransmission (Garthwaite, 1991), and microbial and tumor cell killing (Nathan, 1992). Inducible nitric oxide synthase (iNOS), which is present in macrophages and astrocytes, is regulated at the transcriptional level in response to the stimuli of cytokines and lipopolysaccharide (LPS) (Jaffrey and Snyder, 1995). NO is important in the pathophysiology of several neuroinflammatory diseases, such as demyelinating disorders, ischemia and traumatic injuries (Bo et al., 1994, Cross et al., 1994).

Adenosine is a key homeostatic inhibitory neuromodulator that contributes to endogenous antinociceptive and anti-inflammatory response following tissue damage (Fredholm et al., 2001). Adenosine kinase (ADK; EC 2.7.1.20) is a key adenosine-catabolizing enzyme, which phosphorylates adenosine to adenosine monophosphate (AMP). Inhibition of ADK causes an increase of adenosine level in extracellular space by facilitated diffusion nucleoside transports (Dunwiddie and Masino, 2001). Adenosine is known to exert its effects via four major receptors, A₁, A_2A, A_2B, and A₃, which have been classified according to sequences and biochemical and pharmacological properties. A₁ and A₃ receptors are coupled to G_i protein to inhibit adenylate cyclase, whereas both type A₂ receptors are coupled to G_s protein to activate adenylate cyclase (Fredholm et al., 2001, Stiles, 1992). Unlike A₁ receptor, which are widely expressed in the brain, the expression A_2B and A₃ receptors in the CNS is rather low. A_2A adenosine receptors are mainly expressed in the striatum, with lower expression levels in cortex and hippocampus (Dunwiddie and Masino, 2001).

Recently, several studies reported that ADK inhibitors (ADKIs) have several effects, such as anti-inflammation; inhibiting TNF-α release, edema formation, neutrophil adhesion, and leukocyte accumulation in rheumatoid arthritis model, inflammatory pain model or sepsis model (Boyle et al., 2001, Cronstein et al., 1995, Firestein et al., 1994, Firestein et al., 1995, Poon and Sawynok, 1999, Rosengren et al., 1995), antinociception; inhibiting thermal hyperalgesia and tactile allodynia in pain models (Jarvis et al., 2002, Zhu et al., 2001), anticonvulsion; inhibiting the maximum electric shock-induced seizure (Ugarkar et al., 2000a, Ugarkar et al., 2000b, Wiesner et al., 1999), and neuroprotection; reducing infarct volume in temporary middle cerebral artery (MCA) occlusion model (Miller et al., 1996, Tatlisumak et al., 1998). In this study we examined the effect of IC51, an ADKI or adenosine on the iNOS expression and NO production in LPS/IFNγ treated C6 cells or astrocytes. The inhibition of expression of iNOS and TNF-α by IC51 is mediated via the increase of adenosine, the activation of A₂ receptor, and the increase of cAMP by an NFκB independent pathways.