Glutamate-evoked release of adenosine and regulation of peripheral nociception

Glutamate (which facilitates peripheral nociception) releases adenosine (which inhibits peripheral nociception via adenosine A₁ receptors) when injected locally into the rat hindpaw. The present study determined whether this locally released adenosine could modulate spontaneous pain behaviors produced by a local injection of 1.5% formalin, by determining the effect of 8-cyclopentyl-theophylline (CPT; selective adenosine A₁ receptor antagonist) on flinching produced by formalin/glutamate combinations. Experiments were performed following a prior conditioning injection of 2.5% formalin into the contralateral hindpaw 3-4 days earlier. CPT augmented flinching behaviors produced by 1.5% formalin/1 μmol glutamate, but had no effect on behaviors produced by formalin or glutamate alone. CPT also augmented flinches generated by formalin/α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) and formalin/kainic acid, but not by formalin/N-methyl-D-aspartate (NMDA) combinations. The conditioning leads to a clearer expression of the peripheral inhibitory effect of adenosine (inhibitory effect of an inhibitor of adenosine kinase on flinching also was observed), rather than an increased release of adenosine (no enhanced release observed by microdialysis). Microglia appear to be involved in the conditioning, as microglia are activated in the dorsal spinal cord 3 days following injection of 2.5% formalin, and augmentation of formalin/glutamate-induced flinching by CPT is inhibited by the glial metabolic inhibitor fluorocitrate. The augmentation of flinching by CPT is also eliminated following a spinal pretreatment with MK-801 (NMDA receptor antagonist), cyclohexyladenosine (adenosine A₁ receptor agonist), N^G-nitro-L-arginine methyl ester HCl (nitric oxide synthetase inhibitor), and chelerythrine (protein kinase C inhibitor). The conditioning pretreatment with 2.5% formalin does not lead to a generalized chemical or thermal hypersensitivity in the contralateral hindpaw. This study demonstrates that prior exposure to 2.5% formalin in the contralateral hindpaw reveals an inhibitory effect of adenosine on peripheral nociception in the presence of glutamate; this conditioning involves microglia and other mechanisms involved in central sensitization.