Allergen-induced histaminergic and non-histaminergic activation of itch C-fiber nerve terminals in mouse skin.

Acute cutaneous exposure to allergen often leads to itch, but seldom pain. The effect of mast cell activation on cutaneous C-fibers was studied using innervated isolated mouse skin preparation that allows for intra-arterial delivery of chemicals to the nerve terminals in the skin. Allergen (ovalbumin) injection into the isolated skin of actively sensitized mice strongly stimulated chloroquine (CQ)-sensitive C-fibers (also referred to as "itch" nerves); on the other hand, CQ-insensitive C-fibers were activated only modestly, if at all. The histamine H1 receptor antagonist pyrilamine abolished itch C-fibers response to histamine, but failed to significantly reduce the response to ovalbumin. Ovalbumin also strongly activated itch C-fibers in skin isolated from Mrgpr-cluster Δ-/- mice. When pyrilamine was studied in the Mrgpr-cluster Δ-/- mice thereby eliminating the influence of both histamine H1 and Mrgpr receptors (MrgprA3 and C11 are selectively expressed by itch nerves), the ovalbumin response was very nearly eliminated. The data indicate that the acute activation of itch C-fibers in mouse skin is largely secondary to the combined effect of activation of histamine H1 and Mrpgr receptors.

Jurcakova D ，Ru F ，Undem BJ 《-》

Mechanisms of pruritogen-induced activation of itch nerves in isolated mouse skin.

Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G-protein coupled receptor (GPCR) MrgprA3; it is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that transient receptor potential A1 (TRPA1) gene deletion blocks CQ-induced scratching. In the present study we used a novel dorsal skin-nerve preparation to evaluate mechanisms underlying CQ- and histamine-induced action potential discharge in itch nerve terminals. We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, but TRPA1 or other TRP channel are not required. Evidence is provided for a role for calcium-activated chloride channels such as TMEM16a in GPCR-activation of itch nerve terminals. The mechanism by which TRP channels participate in pruritogen-induced scratching may involve sites of action other than the primary afferent terminals. Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin-nerve preparation was used to evaluate chloroquine (CQ)- and histamine-induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C-fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr-cluster Δ-/- mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ-induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild-type and TRPA1-deficient mice. A selective inhibitor of the calcium-activated chloride channel TMEM16A, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), inhibited CQ-induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role.

Ru F ，Sun H ，Jurcakova D ，Herbstsomer RA ，Meixong J ，Dong X ，Undem BJ ... -  《-》

Voltage-Gated Sodium Channels Regulating Action Potential Generation in Itch-, Nociceptive-, and Low-Threshold Mechanosensitive Cutaneous C-Fibers.

We evaluated the effect of voltage-gated sodium channel 1 (NaV1) blockers in three nonoverlapping C-fiber subtypes in the mouse skin: chloroquine (CQ)-sensitive C-fibers with high mechanical thresholds-itch C-fibers; second, CQ-insensitive, capsaicin-sensitive C-fibers with high mechanical thresholds-nociceptors; and CQ and capsaicin-insensitive C-fibers with a very low mechanical threshold-C-LTMs. NaV1-blocking drugs were applied to the nerve terminal receptive fields using an innervated isolated dorsal mouse skin-nerve preparation where the drugs are delivered into the skin intra-arterially. We combined these studies with an analysis of the mRNA expression of the α-subunits of NaV1 in individual dorsal root ganglia neurons labeled from the same region of the skin. Our results show that virtually all nociceptors and itch C-fibers expressed the tetrodotoxin (TTX)-resistant channels NaV1.8 and NaV1.9. However, TTX applied selectively into the skin abolished the action potential firing in response to mechanical stimulation in 75% of the itch C-fibers, 100% of the nociceptors, and 100% of C-LTMs. NaV1.7 was the most commonly expressed TTX-sensitive NaV1 in all three C-fiber subtypes innervating the dorsal skin. Selectively blocking NaV1.7 abolished responses in about 40% of itch C-fibers, 65% of nociceptors, but only 20% of C-LTMs. Blocking NaV1.8 alone had no affect on the firing sensitivity of the C-fibers. However, in itch and nociceptive C-fibers where the activation was not inhibited with a NaV1.7 blocker, adding the NaV1.8 blocker silenced action potential discharge.

Jurcakova D ，Ru F ，Kollarik M ，Sun H ，Krajewski J ，Undem BJ ... -  《-》

Phospholipase Cbeta 3 mediates the scratching response activated by the histamine H1 receptor on C-fiber nociceptive neurons.

Han SK ，Mancino V ，Simon MI 《NEURON》

Activation of Mast-Cell-Expressed Mas-Related G-Protein-Coupled Receptors Drives Non-histaminergic Itch.

Classical itch studies have focused on immunoglobulin E (IgE)-mediated mast cell activation and histamine release. Recently, members of the Mas-related G-protein-coupled receptor (Mrgpr) family have been identified as mast cell receptors, but their role in itch is unclear. Here, we report that mast cell activation via Mrgprb2 evoked non-histaminergic itch in mice independently of the IgE-Fc epsilon RI (FcεRI)-histamine axis. Compared with IgE-FcεRI stimulation, Mrgprb2 activation of mast cells was distinct in both released substances (histamine, serotonin, and tryptase) and the pattern of activated itch-sensory neurons. Mrgprb2 deficiency decreased itch in multiple preclinical models of allergic contact dermatitis (ACD), a pruritic inflammatory skin disorder, and both mast cell number and PAMP1-20 concentrations (agonist of the human Mrgprb2 homolog, MRGPRX2) were increased in human ACD skin. These findings suggest that this pathway may represent a therapeutic target for treating ACD and mast-cell-associated itch disorders in which antihistamines are ineffective.

Meixiong J ，Anderson M ，Limjunyawong N ，Sabbagh MF ，Hu E ，Mack MR ，Oetjen LK ，Wang F ，Kim BS ，Dong X ... -  《-》