Presynaptic inhibition of nociceptive neurotransmission by somatosensory neuron-secreted suppressors.

Noxious stimuli cause pain by activating cutaneous nociceptors. The Aδ- and C-fibers of dorsal root ganglion (DRG) neurons convey the nociceptive signals to the laminae I-II of spinal cord. In the dorsal horn of spinal cord, the excitatory afferent synaptic transmission is regulated by the inhibitory neurotransmitter γ-aminobutyric acid and modulators such as opioid peptides released from the spinal interneurons, and by serotonin, norepinepherine and dopamine from the descending inhibitory system. In contrast to the accumulated evidence for these central inhibitors and their neural circuits in the dorsal spinal cord, the knowledge about the endogenous suppressive mechanisms in nociceptive DRG neurons remains very limited. In this review, we summarize our recent findings of the presynaptic suppressive mechanisms in nociceptive neurons, the BNP/NPR-A/PKG/BKCa channel pathway, the FSTL1/α1Na+-K+ ATPase pathway and the activin C/ERK pathway. These endogenous suppressive systems in the mechanoheat nociceptors may also contribute differentially to the mechanisms of nerve injury-induced neuropathic pain or inflammation-induced pain.

Targeting AMPK for the Alleviation of Pathological Pain.

Chronic pain is a major clinical problem that is poorly treated with available therapeutics. Adenosine monophosphate-activated protein kinase (AMPK) has recently emerged as a novel target for the treatment of pain with the exciting potential for disease modification. AMPK activators inhibit signaling pathways that are known to promote changes in the function and phenotype of peripheral nociceptive neurons and promote chronic pain. AMPK activators also reduce the excitability of these cells suggesting that AMPK activators may be efficacious for the treatment of chronic pain disorders, like neuropathic pain, where changes in the excitability of nociceptors is thought to be an underlying cause. In agreement with this, AMPK activators have now been shown to alleviate pain in a broad variety of preclinical pain models indicating that this mechanism might be engaged for the treatment of many types of pain in the clinic. A key feature of the effect of AMPK activators in these models is that they can lead to a long-lasting reversal of pain hypersensitivity even long after treatment cessation, indicative of disease modification. Here, we review the evidence supporting AMPK as a novel pain target pointing out opportunities for further discovery that are likely to have an impact on drug discovery efforts centered around potent and specific allosteric activators of AMPK for chronic pain treatment.

Mutations in TrkA Causing Congenital Insensitivity to Pain with Anhidrosis (CIPA) Induce Misfolding, Aggregation, and Mutation-dependent Neurodegeneration by Dysfunction of the Autophagic Flux.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder characterized by insensitivity to noxious stimuli and variable intellectual disability (ID) due to mutations in the NTRK1 gene encoding the NGF receptor TrkA. To get an insight in the effect of NTRK1 mutations in the cognitive phenotype we biochemically characterized three TrkA mutations identified in children diagnosed of CIPA with variable ID. These mutations are located in different domains of the protein; L213P in the extracellular domain, Δ736 in the kinase domain, and C300stop in the extracellular domain, a new mutation causing CIPA diagnosed in a Spanish teenager. We found that TrkA mutations induce misfolding, retention in the endoplasmic reticulum (ER), and aggregation in a mutation-dependent manner. The distinct mutations are degraded with a different kinetics by different ER quality control mechanisms; although C300stop is rapidly disposed by autophagy, Δ736 degradation is sensitive to the proteasome and to autophagy inhibitors, and L213P is a long-lived protein refractory to degradation. In addition L213P enhances the formation of autophagic vesicles triggering an increase in the autophagic flux with deleterious consequences. Mouse cortical neurons expressing L213P showed the accumulation of LC3-GFP positive puncta and dystrophic neurites. Our data suggest that TrkA misfolding and aggregation induced by some CIPA mutations disrupt the autophagy homeostasis causing neurodegeneration. We propose that distinct disease-causing mutations of TrkA generate different levels of cell toxicity, which may provide an explanation of the variable intellectual disability observed in CIPA patients.

Protein kinase C gamma interneurons in the rat medullary dorsal horn: distribution and synaptic inputs to these neurons, and subcellular localization of the enzyme.

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia.

Balancing GRK2 and EPAC1 levels prevents and relieves chronic pain.

Chronic pain is a major clinical problem, yet the mechanisms underlying the transition from acute to chronic pain remain poorly understood. In mice, reduced expression of GPCR kinase 2 (GRK2) in nociceptors promotes cAMP signaling to the guanine nucleotide exchange factor EPAC1 and prolongs the PGE2-induced increase in pain sensitivity (hyperalgesia). Here we hypothesized that reduction of GRK2 or increased EPAC1 in dorsal root ganglion (DRG) neurons would promote the transition to chronic pain. We used 2 mouse models of hyperalgesic priming in which the transition from acute to chronic PGE2-induced hyperalgesia occurs. Hyperalgesic priming with carrageenan induced a sustained decrease in nociceptor GRK2, whereas priming with the PKCε agonist ΨεRACK increased DRG EPAC1. When either GRK2 was increased in vivo by viral-based gene transfer or EPAC1 was decreased in vivo, as was the case for mice heterozygous for Epac1 or mice treated with Epac1 antisense oligodeoxynucleotides, chronic PGE2-induced hyperalgesia development was prevented in the 2 priming models. Using the CFA model of chronic inflammatory pain, we found that increasing GRK2 or decreasing EPAC1 inhibited chronic hyperalgesia. Our data suggest that therapies targeted at balancing nociceptor GRK2 and EPAC1 levels have promise for the prevention and treatment of chronic pain.

Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors.

OBJECTIVE: To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG). METHODS: Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO. RESULTS: Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization. INTERPRETATION: The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

Extracellular signal-regulated kinase activation in the chronic constriction injury model of neuropathic pain in anaesthetized rats.

BACKGROUND: The role of extracellular signal-regulated kinases (ERKs) in nociception has been explored in the last years. While in spinal cord their activation is frequently correlated with pain or acute noxious stimuli, supraspinally, this association is not so evident and remains unclear. This study aims to evaluate ERK1/2 activation in the spinal cord and brainstem nuclei upon neuropathy and/or an additional mechanical stimulus. METHODS: Acute noxious mechanical stimulation was applied in the left hindpaw of anaesthetized SHAM-operated and chronic constriction injured (CCI, neuropathic pain model) rats. Other SHAM or CCI rats did not receive any stimulus. Immunohistochemistry against the phosphorylated isoforms of ERK1/2 (pERK1/2) was performed in lumbar spinal cord and brainstem sections to assess ERK1/2 activation. RESULTS: In the spinal cord, stimulation promoted an increase in pERK1/2 expression in the superficial dorsal horn of SHAM rats. No significant effects were caused by CCI alone. At supraspinal level, changes in ERK1/2 activation induced by CCI were observed in A5, locus coeruleus (LC), raphe obscurus (ROb), raphe magnus, dorsal raphe (DRN), lateral reticular and paragigantocellularis nucleus. CCI increased pERK1/2 expression in all these nuclei, with exception of LC, where a significant decrease was verified. Mechanical noxious stimulation of CCI rats decreased pERK1/2 expression in ROb and DRN, but no further changes were detected in either SHAM- or CCI-stimulated animals. CONCLUSION: ERK1/2 are differentially activated in the spinal cord and in selected brainstem nuclei implicated in nociception, in response to an acute noxious stimulus and/or to a neuropathic pain condition.

Regulation of the Na,K-ATPase gamma-subunit FXYD2 by Runx1 and Ret signaling in normal and injured non-peptidergic nociceptive sensory neurons.

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons which relay nociceptive, thermoceptive, mechanoceptive and proprioceptive information from peripheral tissues toward the central nervous system. These neurons establish constant communication with their targets which insures correct maturation and functioning of the somato-sensory nervous system. Interfering with this two-way communication leads to cellular, electrophysiological and molecular modifications that can eventually cause neuropathic conditions. In this study we reveal that FXYD2, which encodes the gamma-subunit of the Na,K-ATPase reported so far to be mainly expressed in the kidney, is induced in the mouse DRGs at postnatal stages where it is restricted specifically to the TrkB-expressing mechanoceptive and Ret-positive/IB4-binding non-peptidergic nociceptive neurons. In non-peptidergic nociceptors, we show that the transcription factor Runx1 controls FXYD2 expression during the maturation of the somato-sensory system, partly through regulation of the tyrosine kinase receptor Ret. Moreover, Ret signaling maintains FXYD2 expression in adults as demonstrated by the axotomy-induced down-regulation of the gene that can be reverted by in vivo delivery of GDNF family ligands. Altogether, these results establish FXYD2 as a specific marker of defined sensory neuron subtypes and a new target of the Ret signaling pathway during normal maturation of the non-peptidergic nociceptive neurons and after sciatic nerve injury.

Bone marrow expression of poly(ADP-ribose) polymerase underlies diabetic neuropathy via hematopoietic-neuronal cell fusion.

Diabetic neuropathy is the most common diabetic complication. The pathogenetic pathways include oxidative stress, advanced glycation end product (AGE) formation, protein kinase C, and NF-κB activation, as well as increased polyol flux. These metabolic perturbations affect neurons, Schwann cells, and vasa nervorum, which are held to be the primary cell types involved. We hypothesize that diabetes induces the appearance of abnormal bone marrow-derived cells (BMDCs) that fuse with neurons in the dorsal root ganglia (DRG) of mice, leading to diabetic neuropathy. Neuronal poly(ADP-ribose) polymerase-1 (PARP-1) activation in diabetes is known to generate free radical and oxidant-induced injury and poly(ADP-ribose) polymer formation, resulting in neuronal death and dysfunction, culminating in neuropathy. We further hypothesize that BM-specific PARP expression plays a determining role in disease pathogenesis. Here we show that bone marrow transplantation (BMT) of PARP-knockout (PARPKO) cells to wild-type mice protects against, whereas BMT of wild-type cells to PARPKO mice, which are normally "neuropathy-resistant," confers susceptibility to, diabetic neuropathy. The pathogenetic process involving hyperglycemia, BMDCs, and BMDC-neuron fusion can be recapitulated in vitro. Incubation in high, but not low, glucose confers fusogenicity to BMDCs, which are characterized by proinsulin (PI) and TNF-α coexpression; coincubation of isolated DRG neurons with PI-BMDCs in high glucose leads to spontaneous fusion between the 2 cell types, while the presence of a PARP inhibitor or use of PARPKO BMDCs in the incubation protects against BMDC-neuron fusion. These complementary in vivo and in vitro experiments indicate that BMDC-PARP expression promotes diabetic neuropathy via BMDC-neuron fusion.

Early-life stress produces muscle hyperalgesia and nociceptor sensitization in the adult rat.

Chronic pain in adults has been associated with early-life stress. To examine the pronociceptive effect of early-life stress, we evaluated cutaneous and muscle nociception and activity in muscle nociceptors in an animal model of neonatal stress, limited bedding, in the rat. In this neonatal limited bedding (NLB) model, litters are exposed to limited bedding between postnatal days 2 and 9, and controls to standard bedding. In adult NLB-treated rats, mechanical nociceptive threshold in skeletal muscle was significantly lower (~22%) than in controls. Furthermore, administration of prostaglandin E(2) in skin as well as muscle produced markedly prolonged hyperalgesia, an effect prevented by spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cε (PKCε), a second messenger in nociceptors that has been implicated in the induction and maintenance of chronic pain. In electrophysiological studies, mechanical threshold of muscle nociceptors was reduced by ~31% and conduction velocity significantly increased (~28%). These findings indicate that neonatal stress induces a persistent hyperalgesia and nociceptor sensitization manifest in the adult and that the second messenger PKCε may be a target against which therapies might be directed to treat a chronic pain syndrome that is associated with early-life traumatic stress.

Up-regulation of low-threshold tetrodotoxin-resistant Na+ current via activation of a cyclic AMP/protein kinase A pathway in nociceptor-like rat dorsal root ganglion cells.

The effects of forskolin on low-threshold tetrodotoxin-resistant (TTX-r) Na(+) currents was studied in small diameter (average ≈ 25 µm) dorsal root ganglion (DRG) cells. All DRG cells included in the study were categorized as type-2 or non-type-2 based on the expression of a low-threshold A-current. In all type-2 and some non-type-2 DRG cells held at -80 mV, the adenylyl cyclase (AC) activator forskolin (10 µM) up-regulated TTX-r Na(+) currents evoked with steps to -55 mV through -35 mV (low-threshold current). Up-regulation of low-threshold current by forskolin was mimicked by the protein kinase A (PKA) agonist Sp-cAMPs and the inflammatory mediator serotonin, and blocked by the PKA antagonist Rp-cAMPs. Forskolin-induced up-regulation of low-threshold current evoked from a holding potential of -60 mV was blocked by 40 ms steps to 0 mV, which presumably induced a long lasting inactivation of the low-threshold channels. Reducing to 3 ms the duration of steps to 0 mV, significantly increased the number of DRG cells where low-threshold current was up-regulated by forskolin, presumably by reducing the long-lasting inactivation of the low-threshold channels. In the same cells, high-threshold current, evoked by 40 ms or 3 ms steps to 0 mV, was consistently up-regulated by forskolin. The selective Na(V)1.8 channel blocker A-803467 markedly blocked high-threshold current but not low-threshold current. The different voltage protocols observed to activate and inactivate the low- and high-threshold currents, and the observation that A-803467 blocked high- but not low-threshold current suggests that the two currents were mediated by different channels, possibly Na(V)1.8 and Na(V)1.9, respectively. Inflammatory mediators may simultaneously up-regulate Na(V)1.8 and Na(V)1.9 channels in the same nociceptor via a AC/PKA signaling pathway, increasing nociceptor signaling strength, and lowering nociceptor threshold, respectively.

Nicotinamide adenine dinucleotide phosphate/neuronal nitric oxide synthase-positive neurons in the trigeminal subnucleus caudalis involved in tooth pulp nociception.

Sensory information on facial structures, including teeth pulp, periodontium, and gingiva, is relayed in the trigeminal complex. Tooth pulp inflammation constitutes a common clinical problem, and this peripheral injury can induce neuroplastic changes in trigeminal nociceptive neurons. There is considerable evidence that the trigeminal subnucleus caudalis (Vc) is the principal relay for trigeminal nociceptive information as well as modulation of the painful stimuli. Glutamatergic primary afferents innervating the tooth pulp project to the most superficial laminae of the Vc. N-methyl-D-aspartate receptor stimulation leads to the activation of the enzyme nitric oxide synthase (NOS), which synthesizes the free radical nitric oxide (NO). This enzyme is expressed mainly in lamina II interneurons, and in a small number of cells in lamina I as well as in deep laminae projection neurons of Vc. In the present study, we analyzed the temporal changes in neuronal NOS (nNOS) in Vc local circuitries after unilateral intermediate molar pulp injury. Our results demonstrate that a peripheral dental pulp injury leads to neuroplastic changes in the relative amount and activity of nNOS enzyme. Moreover, after a period of time, the nitrergic system shifts to the initial values, independently of the persistence of inflammation in the pulp tissues.

ERK1/2 signaling pathway in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons.

Sensitization of esophageal nociceptive afferents by inflammatory mediators plays an important role in esophageal inflammatory nociception. Our previous studies demonstrated that esophageal mast cell activation increases the excitability of esophageal nodose C-fibers. But the intracellular mechanism of this sensitization process is still less clear. We hypothesize that extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway plays an important role in mast cell activation-induced sensitization of esophageal nodose C-fiber neurons. Mast cell activation and in vivo esophageal distension-induced phosphorylations of ERK1/2 were studied by immuno-staining and Western blot in esophageal nodose neurons. Extracellular recordings were performed from nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Nerve excitabilities were compared by action potentials evoked by esophageal distensions before and after mast cell activations with/without pretreatment of mitogen-activated protein kinases (MAPK)/ERK kinase inhibitor U0126. The expressions of phospho-ERK1/2 (p-ERK1/2) in the same nodose ganglia were then studied by Western blot. Mast cell activation enhances in vivo esophageal distension-induced phosphorylation of ERK1/2 in nodose neurons. This can be prevented by pretreatment with mast cell stabilizer cromolyn. In ex vivo esophageal-vagal preparations, both mast cell activation and proteinase-activated receptor 2 (PAR2)-activating peptide perfusion increases esophageal distension-induced mechano-excitability of esophageal nodose C-fibers and phosphorylation of ERK1/2 in nodose neurons. Pretreatment with MAPK/ERK kinase inhibitor U0126 prevents these potentiation effects. Collectively, our data demonstrated that mast cell activation enhances esophageal distension-induced mechano-excitability and phosphorylation of ERK1/2 in esophageal nodose C-fiber neurons. This reveals a new intracellular pathway of esophageal peripheral sensitization and inflammatory nociception.

A role for extracellular signal-regulated kinases 1 and 2 in the maintenance of persistent mechanical hyperalgesia in ovariectomized mice.

Mitogen-activated protein kinases (MAPKs) are important signaling factors in many cellular processes including cell proliferation and survival during development and synaptic plasticity induced by acute nociception in the adult. There is extensive evidence for the involvement of members of the MAPK family, the extracellular signal-regulated kinases 1 and 2 (ERKs 1/2), in the development of acute inflammatory somatic and visceral pain, but their role in the maintenance of chronic pain states is unknown. We have previously shown that ovariectomy of adult mice (OVX) generates a persistent and estrogen-dependent abdominal hyperalgesic state that lasts for several months and is not related to a persistent nociceptive afferent input. Here we have used OVX mice to study a possible role of ERK 1/2 in the spinal processing of this form of chronic abdominal hyperalgesia. Eight weeks after OVX the mice showed a robust abdominal hyperalgesia and a significant increase in the activation of ERK1/2 in the lumbosacral spinal cord. This enhanced activation was not seen in control and sham-operated mice or in regions of the cord other than lumbosacral in OVX mice. Also, the increased activation of ERK 1/2 observed in OVX mice matched the time course of the hyperalgesic state as no activation was observed at week 1 after OVX when the hyperalgesic state had not yet developed. Administration of slow-release pellets containing 17ß-estradiol at week 5 post OVX reversed both the development of the hyperalgesia and the enhanced activation of ERK 1/2, suggesting that this activation, like the hyperalgesic state, was estrogen-dependent. Intrathecal injections of the ERK 1/2 inhibitor U0126 successfully rescued the mice from the abdominal hyperalgesia for up to 24 h after the injection and also reversed the enhanced expression of ERK 1/2. Our study shows, for the first time, activation of ERK 1/2 in the spinal cord matching the time course of an estrogen-dependent chronic hyperalgesic state.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1ß maturation.

BACKGROUND: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1ß and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). RESULTS: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1ß and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1ß and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1ß was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. CONCLUSION: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1ß maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Phosphorylation of ERK in the spinal dorsal horn following pancreatic pronociceptive stimuli with proteinase-activated receptor-2 agonists and hydrogen sulfide in rats: evidence for involvement of distinct mechanisms.

Noxious stimuli cause prompt phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following somatic and colonic nociception. Here we asked whether two distinct pronociceptive stimuli with proteinase-activated receptor-2 (PAR2) agonists and hydrogen sulfide (H(2)S) in the pancreas cause phosphorylation of ERK in the spinal dorsal horn and also examined involvement of their possible downstream signaling molecules, transient receptor potential vanilloid-1 (TRPV1) and T-type Ca(2+) channels, respectively. Capsaicin (a TRPV1 agonist), trypsin (an endogenous PAR2 agonist), SLIGRL-NH(2) (a PAR2-activating peptide), and NaHS (an H(2)S donor) were infused into the pancreatic duct in anesthetized rats, and phosphorylated ERK in the spinal cord was detected by immunohistochemistry. Intraductal administration of capsaicin and trypsin caused prompt phosphorylation of ERK in the superficial layers of T9, but not T5 or T12, spinal dorsal horn. SLIGRL-NH(2) and NaHS, administered in the same manner, also produced ERK phosphorylation in the corresponding spinal regions. Mibefradil, a T-type Ca(2+) channel blocker, abolished the phosphorylation of ERK caused by intraductal NaHS but not SLIGRL-NH(2). In contrast, capsazepine, an inhibitor of TRPV1, suppressed the phosphorylation of ERK caused by intraductal SLIGRL-NH(2) but not NaHS. Our data thus demonstrate that pancreatic pronociceptive stimuli with PAR2 agonists and H(2)S cause ERK phosphorylation in the spinal dorsal horn, through activation of TRPV1 and T-type Ca(2+) channels, respectively, and that those two pronociceptive pathways are independent of each other.

Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones.

Protease-activated receptors (PAR1-4) are activated by proteases released by cell damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia. Previous studies have shown that PAR2 receptors enhance activation of TRPV1 but the role of other PARs is less clear. In this paper we investigate the expression and function of the PAR1, 3 and 4 thrombin-activated receptors in sensory neurones. Immunocytochemistry and in situ hybridization show that PAR1 and PAR4 are expressed in 10 - 15% of neurons, distributed across all size classes. Thrombin or a specific PAR1 or PAR4 activating peptide (PAR1/4-AP) caused functional effects characteristic of activation of the PLCß/PKC pathway: intracellular calcium release, sensitisation of TRPV1, and translocation of the epsilon isoform of PKC (PKCε) to the neuronal cell membrane. Sensitisation of TRPV1 was significantly reduced by PKC inhibitors. Neurons responding to thrombin or PAR1-AP were either small nociceptive neurones of the peptidergic subclass, or larger neurones which expressed markers for myelinated fibres. Sequential application of PAR1-AP and PAR4-AP showed that PAR4 is expressed in a subset of the PAR1-expressing neurons. Calcium responses to PAR2-AP were by contrast seen in a distinct population of small IB4+ nociceptive neurones. PAR3 appears to be non-functional in sensory neurones. In a skin-nerve preparation the release of the neuropeptide CGRP by heat was potentiated by PAR1-AP. Culture with nerve growth factor (NGF) increased the proportion of thrombin-responsive neurons in the IB4- population, while glial-derived neurotropic factor (GDNF) and neurturin upregulated the proportion of thrombin-responsive neurons in the IB4+ population. We conclude that PAR1 and PAR4 are functionally expressed in large myelinated fibre neurons, and are also expressed in small nociceptors of the peptidergic subclass, where they are able to potentiate TRPV1 activity.

Purinergic receptors are involved in tooth-pulp evoked nocifensive behavior and brainstem neuronal activity.

BACKGROUND: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,ß-methyleneATP (α,ß-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats. RESULTS: Genioglossus (GG) muscle activity was evoked by pulpal application of 100 mM α,ß-meATP and was significantly larger than GG activity following vehicle (phosphate-buffered saline PBS) application (p < 0.01). The enhanced GG muscle activity following 100 mM α,ß-meATP was significantly reduced (p < 0.05) by co-application of 1 mM TNP-ATP (P2X1, P2X3 and, P2X2/3 antagonist). A large number of pERK-LI cells were expressed in the Vc, Vi/Vc, C1/C2 and Pa5 at 5 min following pulpal application of 100 mM α,ß-meATP compared to PBS application to the pulp (p < 0.05). The pERK-LI cell expression and GG muscle activity induced by 100 mM α,ß-meATP pulpal application were significantly reduced after intrathecal injection of the MAPK/ERK kinase (MEK) inhibitor PD 98059 and by pulpal co-application of 1 mM TNP-ATP (p < 0.05). CONCLUSIONS: The present findings suggest that activation of P2X3 and P2X2/3 receptors in the tooth pulp is sufficient to elicit nociceptive behavioral responses and trigeminal brainstem neuronal activity.

Peripheral formalin injury induces 2 stages of microglial activation in the spinal cord.

UNLABELLED: The formalin test produces 2 well-known acute phases of nociceptive behavior. Recently, we have shown that this same formalin test produces a third phase of nociceptive behavior consisting of prolonged thermal and mechanical hyperalgesia beginning days after formalin injection and lasting for at least 3 weeks. Here we investigated the activity of 3 MAPKs (p38, ERK and JNK) in the spinal dorsal horn following 5% formalin injection into rat hind paw. The p38 MAPK was rapidly activated in the spinal microglia minutes after injection and the activation persisted for 1 hour. In addition, this same injury induced a secondary increase of phospho-p38 expression in spinal microglia that was maximal 3 to 7 days postinjection. Intrathecal administration of p38 inhibitor SB203580 not only inhibited the early acute spontaneous nociceptive behaviors, but also inhibited the long-term formalin injury-induced mechanical hyperalgesia. Our results suggest that peripheral formalin injection induces 2 stages of microglial activation, and p38 activation in spinal microglia plays key roles in central pain modulation in formalin test respectively for the early acute phases and the late secondary long-term pain state as well. PERSPECTIVE: This article presents unique properties of spinal microglial activation in a pain animal model. This finding could potentially help clinicians to further understand the contributions of spinal microglia to acute and chronic pain state.