Different role of TTX-sensitive voltage-gated sodium channel (NaV 1) subtypes in action potential initiation and conduction in vagal airway nociceptors.

KEY POINTS: The action potential initiation in the nerve terminals and its subsequent conduction along the axons of afferent nerves are not necessarily dependent on the same voltage-gated sodium channel (NaV 1) subunits. The action potential initiation in jugular C-fibres within airway tissues is not blocked by TTX; nonetheless, conduction of action potentials along the vagal axons of these nerves is often dependent on TTX-sensitive channels. This is not the case for nodose airway Aδ-fibres and C-fibres, where both action potential initiation and conduction is abolished by TTX or selective NaV 1.7 blockers. The difference between the initiation of action potentials within the airways vs. conduction along the axons should be considered when developing NaV 1 blocking drugs for topical application to the respiratory tract. ABSTRACT: The action potential (AP) initiation in the nerve terminals and its subsequent AP conduction along the axons do not necessarily depend on the same subtypes of voltage-gated sodium channels (NaV 1s). We evaluated the role of TTX-sensitive and TTX-resistant NaV 1s in vagal afferent nociceptor nerves derived from jugular and nodose ganglia innervating the respiratory system. Single cell RT-PCR was performed on vagal afferent neurons retrogradely labelled from the guinea pig trachea. Almost all of the jugular neurons expressed the TTX-sensitive channel NaV 1.7 along with TTX-resistant NaV 1.8 and NaV 1.9. Tracheal nodose neurons also expressed NaV 1.7 but, less frequently, NaV 1.8 and NaV 1.9. NaV 1.6 were expressed in ∼40% of the jugular and 25% of nodose tracheal neurons. Other NaV 1 α subunits were only rarely expressed. Single fibre recordings were made from the vagal nodose and jugular nerve fibres innervating the trachea or lung in the isolated perfused vagally-innervated preparations that allowed for selective drug delivery to the nerve terminal compartment (AP initiation) or to the desheathed vagus nerve (AP conduction). AP initiation in jugular C-fibres was unaffected by TTX, although it was inhibited by NaV 1.8 blocker (PF-01247324) and abolished by combination of TTX and PF-01247324. However, AP conduction in the majority of jugular C-fibres was abolished by TTX. By contrast, both AP initiation and conduction in nodose nociceptors was abolished by TTX or selective NaV 1.7 blockers. Distinction between the effect of a drug with respect to inhibiting AP in the nerve terminals within the airways vs. at conduction sites along the vagus nerve is relevant to therapeutic strategies involving inhaled NaV 1 blocking drugs.

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

KEY POINTS: 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. ABSTRACT: 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 Cß3 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.