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Voltage-gated Na + channels (Nav) are the targets of a variety of scorpion toxins. Here, we investigated the effects of Amm VIII, a toxin isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus , on pain-related behaviours in mice. The effects of Amm VIII were compared with the classic scorpion α-toxin AaH II from Androctonus australis . Contrary to AaH II, intraplantar injection of Amm VIII at relatively high concentrations caused little nocifensive behaviours. However, Amm VIII induced rapid mechanical and thermal pain hypersensitivities. We evaluated the toxins’ effects on Nav currents in nociceptive dorsal root ganglion (DRG) neurons and immortalized DRG neuron-derived F11 cells. Amm VIII and AaH II enhanced tetrodotoxin-sensitive (TTX-S) Nav currents in DRG and F11 cells. Both toxins impaired fast inactivation and negatively shifted activation. AaH II was more potent than Amm VIII at modulating TTX-S Nav currents with EC 50 of 5 nM and 1 μM, respectively. AaH II and Amm VIII also impaired fast inactivation of Nav1.7, with EC 50 of 6.8 nM and 1.76 μM, respectively. Neither Nav1.8 nor Nav1.9 was affected by the toxins. AaH II and Amm VIII reduced first spike latency and lowered action potential threshold. Amm VIII was less efficient than AaH II in increasing the gain of the firing frequency-stimulation relationship. In conclusion, our data show that Amm VIII, although less potent than AaH II, acts as a gating-modifier peptide reminiscent of classic α-toxins, and suggest that its hyperalgesic effects can be ascribed to gain-of-function of TTX-S Na + channels in nociceptors.

The recently identified IL-6 family member cardiotrophin-like cytokine (also named novel neurotrophin-1 or B cell stimulating factor-3) forms a secreted complex with cytokine-like factor-1 which binds and activates the tripartite ciliary neurotrophic factor receptor. The striking differences between the phenotype of mice in which either the ciliary neurotrophic factor or its receptor are inactivated suggest that the cardiotrophin-like cytokine/cytokine-like factor-1 complex could be the developmentally important ciliary neurotrophic factor receptor ligand. Cardiotrophin-like cytokine is also produced in the immune system and has been reported to activate B cells in vivo and in vitro. B cells do not express the ciliary neurotrophic factor receptor suggesting the existence of an alternative receptor. We produced the cardiotrophin-like cytokine/cytokine-like factor-1 complex tagged with a Bir A biotin ligase AviTag peptide substrate. This cytokine could be efficiently biotinylated in vitro with Bir A. It was subsequently validated as a sensitive tool for ciliary neurotrophic factor receptor detection by flow cytometry and for magnetic-activated cell sorting. It was also shown to allow the detection of a specific receptor by activated B cells. Whereas binding to cells expressing the ciliary neurotrophic factor receptor could be prevented by competition with ciliary neurotrophic factor, binding to B cells was not. The biotinylated cardiotrophin-like cytokine/cytokine-like factor-1 complex therefore represents a new reagent to study ciliary neurotrophic factor and cardiotrophin-like cytokine receptor expression and for the identification of the putative cardiotrophin-like cytokine B cell receptor. It further validates the use of biotin ligase catalysed biotinylation for the detection of cytokine receptors.

International audience; Inflammation is known to be responsible for the sensitization of peripheral sensory neurons, leading to spontaneous pain and invalidating pain hypersensitivity. Given its role in regulating neuronal excitability, the voltage-gated Nav1.9 channel is a potential target for the treatment of pathological pain, but its implication in inflammatory pain is yet not fully described. In the present study, we examined the role of the Nav1.9 channel in acute, subacute and chronic inflammatory pain using Nav1.9-null mice and Nav1.9 knock-down rats. In mice we found that, although the Nav1.9 channel does not contribute to basal pain thresholds, it plays an important role in heat pain hypersensitivity induced by subacute paw inflammation (intraplantar carrageenan) and chronic ankle inflammation (complete Freund's adjuvant-induced monoarthritis). We showed for the first time that Nav1.9 also contributes to mechanical hypersensitivity in both models, as assessed using von Frey and dynamic weight bearing tests. Consistently, antisense-based Nav1.9 gene silencing in rats reduced carrageenan-induced heat and mechanical pain hypersensitivity. While no changes in Nav1.9 mRNA levels were detected in dorsal root ganglia (DRGs) during subacute and chronic inflammation, a significant increase in Nav1.9 immunoreactivity was observed in ipsilateral DRGs 24 hours following carrageenan injection. This was correlated with an increase in Nav1.9 immunolabeling in nerve fibers surrounding the inflamed area. No change in Nav1.9 current density could be detected in the soma of retrolabeled DRG neurons innervating inflamed tissues, suggesting that newly produced channels may be non-functional at this level and rather contribute to the observed increase in axonal transport. Our results provide evidence that Nav1.9 plays a crucial role in the generation of heat and mechanical pain hypersensitivity, both in subacute and chronic inflammatory pain models, and bring new elements for the understanding of its regulation in those models.

International audience; Menthol is a natural compound of plant origin known to produce cool sensation via the activation of the TRPM8 channel. It is also frequently part of topical analgesic drugs available in a pharmacy, although its mechanism of action is still unknown. Compelling evidence indicates that voltage-gated Na(+) channels are critical for experiencing pain sensation. We tested the hypothesis that menthol may block voltage-gated Na(+) channels in dorsal root ganglion (DRG) neurons. By use of a patch clamp, we evaluated the effects of menthol application on tetrodotoxin (TTX)-resistant Nav1.8 and Nav1.9 channel subtypes in DRG neurons, and on TTX-sensitive Na(+) channels in immortalized DRG neuron-derived F11 cells. The results indicate that menthol inhibits Na(+) channels in a concentration-, voltage-, and frequency-dependent manner. Menthol promoted fast and slow inactivation states, causing use-dependent depression of Na(+) channel activity. In current clamp recordings, menthol inhibited firing at high-frequency stimulation with minimal effects on normal neuronal activity. We found that low concentrations of menthol cause analgesia in mice, relieving pain produced by a Na(+) channel-targeting toxin. We conclude that menthol is a state-selective blocker of Nav1.8, Nav1.9, and TTX-sensitive Na(+) channels, indicating a role for Na(+) channel blockade in the efficacy of menthol as topical analgesic compound.

Chondroitin sulfate is referred as a symptomatic slow-acting drug for osteoarthritis because it improves articular function, and reduces joint swelling and effusion. In addition, chondroitin sulfate prevents joint space narrowing of the knee. We hypothesized that the anti-inflammatory effect of chondroitin sulfate is associated to a decrease in the activation of mitogen-activated protein kinases (MAPK) and of the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Cultured rabbit chondrocytes were stimulated with interleukin-1beta (IL-1beta) in presence of chondroitin sulfate. Nuclear translocation of NF-kappaB and AP-1, and nitrite concentrations (as an index for nitric oxide) was assessed 48 hr later. The effect of chondroitin sulfate on IL-1beta activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38MAPK was documented by immunoblot. The effect of chondroitin sulfate on sodium nitroprusside-induced apoptosis was evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay. Chondroitin sulfate reduced IL-1beta-induced NF-kappaB nuclear translocation, but not AP-1 translocation, it decreased IL-1beta-induced phosphorylation of Erk1/2 and abrogated p38MAPK phosphorylation, but did not prevent IL-1beta-induced increase in nitrite. Finally, chondroitin sulfate decreased nitroprusside-induced apoptosis of the chondrocytes. These results suggest that some of the biological activities of chondroitin sulfate may be associated to the reduction in Erk1/2 and p38MAPK phosphorylation and nuclear transactivation of NF-kappaB.