Roles Of Spinal NMDA Receptor NR1 Subunit Phosphorylation On Nociception-induced Pain

ObjectiveNeuropathic pain arising from peripheral nerve injury is a clinical disorder characterized by a combination of spontaneous pain,hyperalgesia and allodynia,and remains a significant clinical problem as it is often poorly relieved by conventional analgesics.Although central sensitization resulting from neuronal plasticity in the spinal cord has been considered to play a crucial role,the mechanisms of neuropathic pain are not fully understood.Orphanin FQ was discovered as the endogenous ligand for the opioid receptor-like-1(ORL-1) receptor.ORL-1 receptor and its mRNA are densely distributed in the spinal dorsal horn.In addition,nociceptin transcripts and the precursor protein for nociceptin are highly expressed in the dorsal horn of the spinal cord and medulla,particularly in the superficial superficial dorsal horn of the spinal cord.In accordance with these localizations,OFQ is shown to be involved in nociception at the spinal level,but the results obtained by intrathecal administration of N/OFQ are contradictory,depending on the testing paradigms,animal species,and doses.OFQ at low doses(picogram range) induced allodynia and thermal hyperalgesia in conscious mice and facilitated the flexor reflex.By contrast,N/OFQ at high doses, produced analgesia,antihyperalgesic and antiallodynic effects.Fast excitatory and inhibitory neurotransmission in the spinal cord,as well as in other regions of the brain, is mediated by the amino acids L-glutamate and glycine and.Similar to behavioral studies,electrophysiological studies demonstrated that N/OFQ inhibited excitatory glutamatergic neurotransmission,without affecting glycine or GABA receptormediated synaptic responses,by decreasing the synaptic release of glutamate from synaptic terminals.However,the mechanisms of low dose of OFQ pain facilitation are not understood.The cellular mechanisms responsible for the spinal actions of N/OFQ largely remain to be clarified.Considerable evidence has demonstrated that activation of the N-methyl-D-aspartate(NMDA) subtype of glutamate receptors and subsequent nitric oxide(NO) production are key events in pain perception and central hyperexcitability in the spinal cord.In fact,a previous study demonstrated that the analgesic effect of an ORL receptor antagonist on neuropathic pain was mediated by inhibition of neuronal NO synthase(nNOS),the enzyme required for NO production.Study also showed that the NMDA receptor antagonist D-AP5 blocked the nociceptin-induced allodynia. Collectively,these results support the current interpretation that OFQ ligands may directly or indirectly modulate NMDA receptor complex functions.The function and localization of NMDA receptors is also modulated by post-translational modifications including phosphorylation.Phosphorylation of the NR1 subunit(pNR1) at protein kinase C(PKC)-dependent sites(serine 896) has been demonstrated to play a key role in enhancement of NMDA receptor activity related to pain transmission in the spinal cord.The present study was designed to characterize nociceptive response induced by OFQ and to investigate effects of pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine(Chel),an inhibitor of PKC,and nocistatin,a functional antagonist of OFQ on OFQ-induced spontaneous pain and hyperalgesia were investigated.In addition,we examined whether thisOFQ mediated PKC dependent phosphorylation of NR1at serine 896 sites plays an important role in sensory function using formaldehyde -induced pain model.Part One Effect of intrathecal administration of orphanin FQ on protein kinase C dependent phosphorylation of the spinal cord NMDA receptor subunit NR1 in ratsMaterials and MethodsAll animal procedures were conducted after approval of protocol by the Chinese Medical University Animal Care Committee.Male SD rats were kept under standard conditions with unrestricted access to food and water.1.Studies on Spontaneous Pain.Immediately following the intrathecal injection of orphanin FQ(OFQ),each rat was placed into the transparent cage,and the time spent for induced spontaneous behavioral pain responses was measured at 5-min intervals for 20 min.2.Studies on Hyperalgesia.Warm water tail-flick test was used to assess the nociceptive response.The thermal nociceptive stimulus was 50℃water,with the latency to tail-flick or withdrawal taken as the endpoint.The response time of the rats to the heat was measured at 15 min,the point of the maximal hyperalgesic effect obtained with 50 pg of OFQ.3.The effects of pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine,an inhibitor of PKC,and nocistatin,a functional antagonist of orphanin FQ on OFQ-induced spontaneous pain and hyperalgesia were investigated.4.The effects of intrathecal administration of OFQ on protein kinase C alpha(PKCα) dependent phosphorylation of the NMDA receptor subunit NR1(phosphorylated at serine 896) as a marker of NMDA receptor sensitization was analyzed by western blot methods.Results1.Orphanin FQ induced nociceptive behavior.The intrathecal administration of OFQ(50 pg) resulted in a characteristic behavioral response consisting of vigorous biting and licking with occasional crutching,which peaked at 10-15 min and had disappeared by 20 min post injection.2.There was no significant difference in the tail flick latency period between rat that had not received an intrathecal injection and those treated with intrathecal saline(at 15 min).Intrathecal administration of OFQ(50 pg) shortened the response latency at 15 min after intrathecal injection,demonstrating that the experimental rats developed increased sensitivity to the thermal stimuli by intrathecal OFQ.3.Intrathecal treatment with MK-801(0.5μg),chelerythrine(20μg) or nocistatin (10μg) alone did not produce any noticeable pain behaviour for 20 min after injection, but significantly inhibited spontaneous pain and hyperalgesia induced by intrathecal injection of OFQ. 4.Expression of phosphorylation of NR1(at serine896) and PKCαin the spinal cord was found to increase as early as 30 min after intrathecal injection of either OFQ ),and reached the peak effect 60 min after injection.Collectively,these findings demonstrate that PKC dependent phosphorylation of the NMDA receptor subunit NR1 (phosphorylated at Ser896) is involved in the facilitation of nociception associated with OFQ receptor activation.Part Two Effects of intrathecal administration of orphanin FQ receptor antagonist nocistatin on intrathecal NMDA-induced pain and spinal NOS activityMaterials and MethodsThe present study was aimed at determining whether an intrathecal injected OFQ receptor antagonist,NST,can modulate NMDA-induced behavioral responses and neuronal activity.1.NMDA at a dose of(μg) was administered intrathecally 10 min after treatment with the NST or saline.Immediately following the NMDA injection,cumulative time spent scratching,biting and lifting the paw was measured.Warm water tail flick test was used to assess thermal hyperalgesia.Rats received intrathecal administration of NST or saline followed by intrathecal NMDA 10 min later.The effects of pretreatment with NST on NMDA-induced pain were also observed.2.As the subsequent activation of NMDA receptors promotes an increase in intracellular calcium resulting in the production of nitric oxide(NO) in the spinal cord. Nicotinamide adenine dinucleotide phosphatediaphorase(NADPH-d) histochemistry method was used to investigat possible role for nocistatin in the effects of NMDA-induced NOS activitation.Results1.Intrathecal administration of NMDA(1μg) induced nociceptive behaviors, including scratching,biting and lifting and significantly decreased tail-flick latency responses to thermal stimulation.Pretreatment with intrathecal NST significanty decreased NMDA-induced spontaneous pain and NMDA-induced decrease in tail tick latency.2.Intrathecal injection of NST significantly inhibited NMDA-evoked increase in NOS expression,compared with intrathecal injection of NS.Part Three Modulatory role of intrathecal administration of nocistatin on protein kinase C dependent phosphorylation of the spinal cord NMDA receptor subunit NR1 induced by formaldehydeMaterials and Methods1.The formaldehyde test was carried out.100μl of 4%formaldehyde in PBS was injected subcutaneously into the dorsal surface of the right hind paw.Immediately after the injection,each rat was returned to the observation chamber.The amount of time that animals spent licking the injected paw was monitored.2.Immunocytochemistry and histochemistry were performed to determine the effects of pretreatment with NST on formaldehyde -induced changes of Fos-like immunoreactive(Fos-LI) and nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-d) positive neurons,respectively.3,Western blot was used to determine the effects of the intrathecal injection of NST on formaldehyde-induced phosphorylation of NMDA NR1 subunit at serine 896 site(pNR1) and phosphorylated PKCα.Results1.Intrathecal pretreatment with the selective OFQ receptor antagonist,NST significantly reduced formaldehyde-induced pain behaviors.2.Formaldehyde-induced pain behaviors were associated with the expression of Fos-like immunoreactivity(Fos LI) throughout the spinal dorsal horn with highest effect seen in laminaeⅠ-Ⅱ.Formaldehyde also induced an increase in nitric oxide synthase(NOS) activity in superficial layers of the dorsal horn as revealed by NADPH diaphorase histochemistry.Pretreatment with the NST significantly inhibited formaldehyde-evoked Fos-LI,NADPH-d-positive and Fos-LI/NADPH-d double-labeled neurons in the spinal dorsal horn.Intrathecal injection of NST also reduced formaldehyde-evoked pNR1 expression at the protein kinase C-dependent site,serine-896 in spinal cord.SummaryIntrathecal injection of OFQ elicited spontaneous pain and hyperalgesia.On the other hand,the nociceptive response was inhibited by pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine,an inhibitor of PKC,or nocistatin, a functional antagonist of orphanin FQ.Western blot assays of pNR1 in the spinal cord indicated that intrathecal injection of the OFQ significantly enhanced pNR1 at the protein kinase C-dependent site,serine-896 expression in the spinal cord.In another set of experiments,NST reduced NMDA-induced pain behaviour and the nitric-oxide synthase(NOS) immunoreactivity.Finally,pretreatment with the NST significantly inhibited formaldehyde evoked pain and formaldehyde-evoked pNR1 expression at the protein kinase C-dependent site,serine-896 in spinal cord.Conclusions1.Activation of the ORL-1 receptor by orphanin FQ produces spontaneous pain and thermal hyperalgesia in conscious rats.2.PKCαand PKC dependent phosphorylation of the NMDA receptor subunit NR1(phosphorylated at serine896) is involved in the facilitation of orphanin FQ associated with OFQ receptor activation.3.Inhibition of of spinal orphanin FQ inhibites formaldehyde-induced pain via PKC dependent phosphorylation of the NMDA receptor NR 1 subunit and subsequent activation of NOS.