Molecular Mechanisms For Adenosine To Inhibit Chronic Inflammatory Pain

Objective: Adenosine is one of endogenous neuromodulators,which regulates multiple pathophysiological processes by acting on adenosine receptors.It has been widely established that systematic or spinal delivery of adenosine is able to alleviate pathological pain,whereas the analgesic mechanisms remain to be elucidated.N-methyl-D-aspartate(NMDA)subtype of glutamate receptor(NMDAR)is one of the important excitatory receptors in central nervous system.The hyperfunction of NMDAR is critical for the initiation and maintenance of chronic pathological pain.Glycine receptor(GlyR)is one of the inhibitory receptors in the central nervous system and plays a critical role in the modification of nociceptive signals.To unravel the possible mechanisms underlying adenosinergic analgesia,the current study was designed to investigate the influence of adenosine on the synaptic transmission mediated by NMDAR and GlyR.Methods: The chronic inflammatory pain model was established by injecting Complete Freund's Adjuvant(CFA)subcutaneously into the plantar surfaces of hindpaws of rats or mice.Paw Withdrawal Thresholds(PWT)and Paw Withdrawal Latencies(PWL)were measured by using Von Frey monofilaments and a thermal stimulator respectively.Western blotting was performed to examine the synaptic content of NMDA receptor GluN2 B subunit.Electrophysiological patch clamp records were conducted to record the dorsal root-evoked excitatory postsynaptic currents(eEPSCs)mediated by GlyRs.Results:(1)Bath application of adenosine(1 ?mol/L)had no effect on the amplitudes of NMDAR-eEPSCs recorded in spinal cord slices prepared from intact rats.However,a higher dose of adenosine(50 ?mol/L)significantly reduced NMDAR synaptic responses.Meanwhile,adenosine(50 ?mol/L)treatment of spinal slices for 60 min reduced the decay time(?)of NMDAR-eEPSCs.(2)Incubation of spinal slices from intact rats with adenosine(50 ?mol/L)for 60 min reduced the synaptic expression of NMDA receptor GluN2 B subunit.(3)Adenosine(1 ?mol/L?10 ?mol/L?50 ?mol/L)perfusion didn't generate any effects on the amplitudes and frequencies of GlyRs-mIPSCs in spinal slices from intact rats.(4)In slices from inflamed rats,adenosine(10 ?mol/L)perfusion significantly enhanced the amplitudes of GlyR-mIPSCs,with frequencies unaltered.Pretreatment with Adenosine A1 receptor(A1R)antagonist DPCPX(100 nmol/L)for 60 min blocked the increasing effect of adenosine on GlyR-mIPSCs amplitudes,while adenosine A2 receptor(A2R)antagonist CP66713(10 ?mol/L)and adenosine A3 receptor(A3R)antagonist VUF5574(10 nmol/L)had no effects.(5)Direct activation of A1 R by CPA(200 nmol/L)enhanced GlyRs-mIPSCs amplitudes in slices from inflamed rats.(6)Postsynaptic loading of G?i/o protein inhibitor GDP-?-S(500 ?mol/L)through the recording pipettes prevented adenosine from increasing GlyRs-mIPSCs amplitudes in slices from inflamed rats.(7)Sequestination of G?? dimmer by postsynaptic induction of Gallein(100 ?mol/L)failed to block the effect of adenosine on GlyRs-mIPSCs.(8)In the presence of cAMP–dependent protein kinase(PKA)inhibitor H-89(1 ?mol/L),adenosine did not increase GlyRs-mIPSCs amplitudes in slices from inflamed rats.(9)Behavioral tests showed that intradermal injection of CFA significantly decreased PWT and PWL values,which could be reversed by intrathecal application of adenosine(10 ?g).A1 R antagonist DPCPX,but not A2 R antagonist CP66713 and A3 R antagonist VUF5574,blocked the analgesic action of adenosine.Conclusions: Adenosine inhibited NMDAR-mediated synaptic transmission by reducing the synaptic expression of GluN2 B.Adenosine had no effect on the glycinergic synaptic responses in normal rats,but enhanced glycinergic synaptic responses in inflamed rats through A1R/G?i/o/PKA signaling.