Endotoxemia, a common occurrence in horses with colic, is caused by the movement of lipopolysaccharides (LPS) from enteric gram negative bacteria into the bloodstream.  LPS then binds to LPS binding protein, which then facilitates its interaction with CD14 receptors on monocytes and neutrophils.  This leads to a cascade of events resulting in the release of pro-inflammatory cytokines, including Tumor Necrosis Factor-a (TNF-a). 
 
Recent studies have demonstrated that adenosine analogs have an inhibitory effect on TNF-a production by human and rodent mononuclear cells¹.  However, the mechanisms responsible for this inhibitory effect have not been elucidated.  In this study, we investigated the effects of CGS 21680, an adenosine A2a receptor agonist, and IB-MECA, an adenonsine A3 receptor agonist, on the production of TNF-a and thromboxane A2 in equine monocytes.  We also measured intracellular concentrations of cAMP of equine monocytes and Mono-Mac-6 cells stimulated with CGS 21680, IB-MECA, and 2-Chloroadenosine (2 ClAdo), a non-selective adenosine analog, to determine whether the effects of these agonists could be attributed to changes in adenylyl cyclase activity.  The results of previous studies indicate that CGS 21680 increases adenylyl cyclase activity, whereas IB-MECA has the opposite effect. 

A bioassay was used to quantify TNF-a production by equine monocytes exposed to LPS and varying concentrations of CGS or IB-MECA.  TxB2, the breakdown product of thromboxane A2, was measured in supernatants of equine monocytes exposed to LPS using an ELISA kit.  Intracellular cAMP accumulation was measured in [3H]adenine-loaded cells after exposure to medium containing either forskolin (a direct stimulator of adenylyl cylcase), CGS 21680, IB-MECA, or 2-ClAdo (alone or in the presence of LPS).  We demonstrated that CGS does increase adenylyl cyclase activity.  In addition, both CGS and IB-MECA decreased LPS-induced TNF-a production in a concentration dependent manner.
 
If the remainder of the results of these experiments are as predicted, further research will need to be performed to determine how adenosine analogs can be used in the treatment of endotoxic horses.
*Department of Large Animal Medicine and Surgery; **Department of Physiology and Pharmacology

¹Eigler A, Greten TF, Sinha B, Haslberger C, Sullivan GW, Endres S. "Endogenous adenosine curtails lipopolysaccharide
-stimulated tumour necrosis factor synthesis." Scandinavian Journal of Immunology 1997; 45:132-139.