No abstract

Alteration in the intracellular signal transduction pathway in primary afferent neurons may contribute to pain hypersensitivity. We demonstrated that very rapid phosphorylation of extracellular signal-regulated protein kinases (pERK) occurred in DRG neurons that were taking part in the transmission of various noxious signals. The electrical stimulation of Adelta fibers induced pERK primarily in neurons with myelinated fibers. c-Fiber activation by capsaicin injection induced pERK in small neurons with unmyelinated fibers containing vanilloid receptor-1 (VR-1), suggesting that pERK labeling in DRG neurons is modality specific. Electrical stimulation at the c-fiber level with different intensities and frequencies revealed that phosphorylation of ERK is dependent on the frequency. We examined the pERK in the DRG after application of natural noxious stimuli and found a stimulus intensity-dependent increase in labeled cell size and in the number of activated neurons in the c- and Adelta-fiber population. Immunohistochemical double labeling with phosphorylated ERK/VR-1 and pharmacological study demonstrated that noxious heat stimulation induced pERK in primary afferents in a VR-1-dependent manner. Capsaicin injection into the skin also increased pERK labeling significantly in peripheral fibers and terminals in the skin, which was prevented by a mitogen-activated protein kinase/ERK kinase inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminopheylthio)butadiene (U0126). Behavioral experiments showed that U0126 dose-dependently attenuated thermal hyperalgesia after capsaicin injection and suggested that the activation of ERK pathways in primary afferent neurons is involved in the sensitization of primary afferent neurons. Thus, pERK in primary afferents by noxious stimulation in vivo showed distinct characteristics of expression and may be correlated with the functional activity of primary afferent neurons.

Proinflammatory agents trypsin and mast cell tryptase cleave and activate PAR2, which is expressed on sensory nerves to cause neurogenic inflammation. Transient receptor potential A1 (TRPA1) is an excitatory ion channel on primary sensory nerves of pain pathway. Here, we show that a functional interaction of PAR2 and TRPA1 in dorsal root ganglion (DRG) neurons could contribute to the sensation of inflammatory pain. Frequent colocalization of TRPA1 with PAR2 was found in rat DRG neurons. PAR2 activation increased the TRPA1 currents evoked by its agonists in HEK293 cells transfected with TRPA1, as well as DRG neurons. Application of phospholipase C (PLC) inhibitors or phosphatidylinositol-4,5-bisphosphate (PIP 2 ) suppressed this potentiation. Decrease of plasma membrane PIP 2 levels through antibody sequestration or PLC-mediated hydrolysis mimicked the potentiating effects of PAR2 activation at the cellular level. Thus, the increased TRPA1 sensitivity may have been due to activation of PLC, which releases the inhibition of TRPA1 from plasma membrane PIP 2 . These results identify for the first time to our knowledge a sensitization mechanism of TRPA1 and a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A metabotropic ATP receptor, P2Y 12 , has been shown to be expressed in spinal microglia constitutively and be involved in chemotaxis. Activation of p38 mitogen-activated protein kinase (MAPK) occurs in spinal microglia after nerve injury and may be related to the production of cytokines and other mediators, resulting in neuropathic pain. However, it remains unknown whether any type of P2Y receptor in microglia is involved in the activation of p38 MAPK and the pain behaviors after nerve injury.Using the partial sciatic nerve ligation (PSNL) model in the rat, we found that P2Y 12 mRNA and protein increased in the spinal cord and peaked at 3 d after PSNL. Double labeling studies revealed that cells expressing increased P2Y 12 mRNA and protein after nerve injury were exclusively microglia. Both pharmacological blockades by intrathecal administration of P2Y 12 antagonist and antisense knockdown of P2Y 12 expression suppressed the development of pain behaviors and the phosphorylation of p38 MAPK in spinal microglia after PSNL. The intrathecal infusion of the P2Y 12 agonist 2-(methythio) adenosine 5Ј-diphosphate trisodium salt into naive rats mimicked the nerve injury-induced activation of p38 in microglia and elevated pain behaviors.These data suggest a new mechanism of neuropathic pain, in which the increased P2Y 12 works as a gateway of the following events in microglia after nerve injury. Activation of this receptor by released ATP or the hydrolyzed products activate p38 MAPK pathway and may play a crucial role in the generation of neuropathic pain.

To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small-to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.

Bradykinin is an inflammatory mediator that plays a pivotal role in pain and hyperalgesia in inflamed tissues by exciting and/or sensitizing nociceptors. TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain. Here, using electrophysiological, immunocytochemical and behavioural analyses, we showed a functional interaction of these two inflammation-related molecules in both heterologous expressing systems and primary sensory neurons. We found that bradykinin increased the TRPA1 currents evoked by allyl isothiocyanate (AITC) or cinnamaldehyde in HEK293 cells expressing TRPA1 and bradykinin receptor 2 (B2R). This potentiation was inhibited by phospholipase C (PLC) inhibitor or protein kinase A (PKA) inhibitor, and mimicked by PLC or PKA activator. The functional interaction between B2R and TRPA1, as well as the modulation mechanism, was also observed in rat dorsal root ganglia neurons. In an occlusion experiment, the PLC activator could enhance AITC-induced TRPA1 current further even in saturated PKA-mediated potentiation, indicating the additive potentiating effects of the PLC and PKA pathways. These data for the first time indicate that a cAMP-PKA signalling is involved in the downstream from B2R in dorsal root ganglia neurons in addition to PLC. Finally, subcutaneous pre-injection of a sub-inflammatory dose of bradykinin into rat hind paw enhanced AITC-induced pain behaviours, which was consistent with the observations in vitro. Collectively, these results represent a novel mechanism through which bradykinin released in response to tissue inflammation might trigger the sensation of pain by TRPA1 activation.

During the preparation of the manuscript, the doses of ET-18-OCH3, U73122, and GF were incorrectly reported in the legend for Figure 3. The correct sentence appears below.In some experiments, the bath solution was perfused with either a PLC inhibitor -ET-18-OCH3 (ET; 2 mM) or U73122 (2 mM) -or a PKC inhibitor, GF (0.5 mM or 10 mM) 120 seconds before SL-NH2 reapplication.The authors regret the error.

To investigate the intracellular signal transduction pathways involved in regulating the gene expression of brain-derived neurotrophic factor (BDNF) in primary afferent neurons, we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons after peripheral inflammation and sciatic nerve transection. Peripheral inflammation induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons. The treatment of the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126 reversed the pain hypersensitivity and the increase in phosphorylated-ERK (p-ERK) and BDNF in DRG neurons induced by complete Freund's adjuvant. On the other hand, axotomy induced the activation of ERK mainly in medium-and large-sized DRG neurons and in satellite glial cells. U0126 suppressed the axotomy-induced autotomy behavior and reversed the increase in p-ERK and BDNF. The intrathecal application of nerve growth factor (NGF) induced an increase in the number of p-ERK-and BDNF-labeled cells, mainly small neurons, and the application of anti-NGF induced an increase in p-ERK and BDNF in some medium-to-large-diameter DRG neurons. The activation of MAPK in the primary afferents may occur in different populations of DRG neurons after peripheral inflammation and axotomy, respectively, through alterations in the target-derived NGF. These changes, including the changes in BDNF expression, might be involved in the pathophysiological changes in primary afferent neurons.