Adenosine A 2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-α by equine monocytes

Abstract

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A 1, A 2A, A 2B and A 3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A 2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-α (TNF-α), and to assess the selectively of these agonists for equine adenosine A 2A over the A 1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A 1 and A 2A receptor subtypes yielded a rank order of affinity for the equine A 2A receptor of ATL307 > ATL309 ≈ ATL310 ≈ ATL313 > ATL202 ≈ ATL361 ≈ ATL376 > ATL372 > CGS21680 > NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-α production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A 2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-α occurred via stimulation of A 2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A 3 receptor agonist, reduced LPS-induced TNF-α production, this effect of IB-MECA was inhibited by the A 2A selective antagonist ZM241385 but not by the A 3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A 2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A 2A receptor antagonist ZM241385 or the adenosine A 2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-α production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-α by equine monocytes primarily via activation of adenosine A 2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A 2A receptors may be suitable for development as anti-inflammatory drugs in horses.