Sonya Meeker

Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea‐pigs

We have identified the tracheal and laryngeal afferent nerves regulating cough in anaesthetized guinea‐pigs. Cough was evoked by electrical or mechanical stimulation of the tracheal or laryngeal mucosa, or by citric acid applied topically to the trachea or larynx. By contrast, neither capsaicin nor bradykinin challenges to the trachea or larynx evoked cough. Bradykinin and histamine administered intravenously also failed to evoke cough. Electrophysiological studies revealed that the majority of capsaicin‐sensitive afferent neurones (both Aδ‐ and C‐fibres) innervating the rostral trachea and larynx have their cell bodies in the jugular ganglia and project to the airways via the superior laryngeal nerves. Capsaicin‐insensitive afferent neurones with cell bodies in the nodose ganglia projected to the rostral trachea and larynx via the recurrent laryngeal nerves. Severing the recurrent nerves abolished coughing evoked from the trachea and larynx whereas severing the superior laryngeal nerves was without effect on coughing. The data indicate that the tracheal and laryngeal afferent neurones regulating cough are polymodal Aδ‐fibres that arise from the nodose ganglia. These afferent neurones are activated by punctate mechanical stimulation and acid but are unresponsive to capsaicin, bradykinin, smooth muscle contraction, longitudinal or transverse stretching of the airways, or distension. Comparing these physiological properties with those of intrapulmonary mechanoreceptors indicates that the afferent neurones mediating cough are quite distinct from the well‐defined rapidly and slowly adapting stretch receptors innervating the airways and lungs. We propose that these airway afferent neurones represent a distinct subtype and that their primary function is regulation of the cough reflex.

Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions

Mast cells are primary effectors in allergic reactions, and may have important roles in disease by secreting histamine and various inflammatory and immunomodulatory substances. Although they are classically activated by immunoglobulin (Ig)E antibodies, a unique property of mast cells is their antibody-independent responsiveness to a range of cationic substances, collectively called basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. The pathogenic roles of these substances have prompted a decades-long search for their receptor(s). Here we report that basic secretagogues activate mouse mast cells in vitro and in vivo through a single receptor, Mrgprb2, the orthologue of the human G-protein-coupled receptor MRGPRX2. Secretagogue-induced histamine release, inflammation and airway contraction are abolished in Mrgprb2-null mutant mice. Furthermore, we show that most classes of US Food and Drug Administration (FDA)-approved peptidergic drugs associated with allergic-type injection-site reactions also activate Mrgprb2 and MRGPRX2, and that injection-site inflammation is absent in mutant mice. Finally, we determine that Mrgprb2 and MRGPRX2 are targets of many small-molecule drugs associated with systemic pseudo-allergic, or anaphylactoid, reactions; we show that drug-induced symptoms of anaphylactoid responses are significantly reduced in knockout mice; and we identify a common chemical motif in several of these molecules that may help predict side effects of other compounds. These discoveries introduce a mouse model to study mast cell activation by basic secretagogues and identify MRGPRX2 as a potential therapeutic target to reduce a subset of drug-induced adverse effects.

TRPV1 induction in airway vagal low-threshold mechanosensory neurons by allergen challenge and neurotrophic factors.

We addressed the hypothesis that allergic inflammation in guinea pig airways leads to a phenotypic switch in vagal tracheal cough-causing, low-threshold mechanosensitive Aδ neurons, such that they begin expressing functional transient receptor potential vanilloid (TRPV1) channels. Guinea pigs were actively sensitized to ovalbumin (OVA) and beginning 21 days later exposed via aerosol to OVA daily for 3 days. Tracheal-specific neurons were identified in the nodose ganglion using retrograde tracing techniques. Tracheal specific neurons were isolated, and mRNA expression was evaluated at the single-neuron level using RT-PCR analysis. Electrophysiological studies have revealed that the vast majority of vagal nodose afferent nerves innervating the trachea are capsaicin-insensitive Aδ-fibers. Consistent with this, we found <20% of these neurons express TRPV1 mRNA or respond to capsaicin in a calcium assay. Allergen exposure induced de novo TRPV1 mRNA in a majority of the tracheal-specific nodose neurons (P < 0.05). The allergen-induced TRPV1 induction was mimicked by applying either brain-derived neurotrophic factor (BDNF) or glial-derived neurotrophic factor (GDNF) to the tracheal lumen. The BDNF-induced phenotypic change observed at the level of mRNA expression was mimicked using a calcium assay to assess functional TRPV1 ion channels. Finally, OVA exposure induced BDNF and GDNF production in the tracheal epithelium, the immediate vicinity of the nodose Aδ -fibers terminations. The induction of TRPV1 in nodose tracheal Aδ -fibers would substantively expand the nature of stimuli capable of activating these cough-causing nerves.

Voltage-gated sodium channels in nociceptive versus non-nociceptive nodose vagal sensory neurons innervating guinea pig lungs

Lung vagal sensory fibres are broadly categorized as C fibres (nociceptors) and A fibres (non‐nociceptive; rapidly and slowly adapting low‐threshold stretch receptors). These afferent fibre types differ in degree of myelination, conduction velocity, neuropeptide content, sensitivity to chemical and mechanical stimuli, as well as evoked reflex responses. Recent studies in nociceptive fibres of the somatosensory system indicated that the tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channels (VGSC) are preferentially expressed in the nociceptive fibres of the somatosensory system (dorsal root ganglia). Whereas TTX‐R sodium currents have been documented in lung vagal sensory nerves fibres, a rigorous comparison of their expression in nociceptive versus non‐nociceptive vagal sensory neurons has not been carried out. Using multiple approaches including patch clamp electrophysiology, immunohistochemistry, and single‐cell gene expression analysis in the guinea pig, we obtained data supporting the hypothesis that the TTX‐R sodium currents are similarly distributed between nodose ganglion A‐fibres and C‐fibres innervating the lung. Moreover, mRNA and immunoreactivity for the TTX‐R VGSC molecules NaV1.8 and NaV1.9 were present in nearly all neurons. We conclude that contrary to findings in the somatosensory neurons, TTX‐R VGSCs are not preferentially expressed in the nociceptive C‐fibre population innervating the lungs.

Pirt, a TRPV1 modulator, is required for histamine-dependent and -independent itch.

Itch, or pruritus, is an important clinical problem whose molecular basis has yet to be understood. Recent work has begun to identify genes that contribute to detecting itch at the molecular level. Here we show that Pirt, known to play a vital part in sensing pain through modulation of the transient receptor potential vanilloid 1 (TRPV1) channel, is also necessary for proper itch sensation. Pirt−/− mice exhibit deficits in cellular and behavioral responses to various itch-inducing compounds, or pruritogens. Pirt contributes to both histaminergic and nonhistaminergic itch and, crucially, is involved in forms of itch that are both TRPV1-dependent and -independent. Our findings demonstrate that the function of Pirt extends beyond nociception via TRPV1 regulation to its role as a critical component in several itch signaling pathways.

Mast cell‐cholinergic nerve interaction in mouse airways

We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 μg ml−1) caused histamine release (∼70% total tissue content), and smooth muscle contraction that was rapid in onset and short‐lived (t1/2 < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5‐HT, and responses to both OVA and 5‐HT were sensitive to 10 μm‐ketanserin (5‐HT2 receptor antagonist) and strongly inhibited by atropine (1 μm). Epithelial denudation had no effect on the OVA‐induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5‐HT. There were virtually no mast cells in the mast cell‐deficient (sash−/−) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell‐deficient (sash−/−) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin‐sensitive 5‐HT‐induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen‐induced contraction of mouse trachea is epithelium‐independent, and requires mast cell‐derived 5‐HT to activate 5‐HT2 receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction.

Activation of mouse bronchopulmonary C-fibres by serotonin and allergen-ovalbumin challenge.

•  Mast cell‐derived serotonin is a principal mediator of allergic reactions in rodents. Serotonin can also be released from platelets during various pathological conditions. •  Vagal C‐fibres innervating the respiratory tract can be subdivided into nodose (placodal) C‐fibres or jugular (neural crest) C‐fibres). Activation of vagal placodal and/or neural crest vagal C‐fibres probably contributes to the dyspnoea, cough and reflex parasympathetic drive commonly associated with serotonin. •  Serotonin used different receptor subtypes to evoke strong action potential discharge in placodal (5‐HT3 receptors) versus neural crest (non‐5‐HT3 receptors)‐derived vagal C‐fibres in the mouse lung. •  Mast cell derived serotonin may activate neural crest C‐fibres, but did not stimulate placodal C‐fibres. •  The effect of extracellular serotonin on vagal afferent activation therefore depends on its cellular source and the C‐fibre phenotype.

Thrombin and trypsin directly activate vagal C‐fibres in mouse lung via protease‐activated receptor‐1

The nature of protease‐activated receptors (PARs) capable of activating respiratory vagal C‐fibres in the mouse was investigated. Infusing thrombin or trypsin via the trachea strongly activated vagal lung C‐fibres with action potential discharge, recorded with the extracellular electrode positioned in the vagal sensory ganglion. The intensity of activation was similar to that observed with the TRPV1 agonist, capsaicin. This was mimicked by the PAR1‐activating peptide TFLLR‐NH2, whereas the PAR2‐activating peptide SLIGRL‐NH2 was without effect. Patch clamp recording on cell bodies of capsaicin‐sensitive neurons retrogradely labelled from the lungs revealed that TFLLR‐NH2 consistently evokes a large inward current. RT‐PCR revealed all four PARs were expressed in the vagal ganglia. However, when RT‐PCR was carried out on individual neurons retrogradely labelled from the lungs it was noted that TRPV1‐positive neurons (presumed C‐fibre neurons) expressed PAR1 and PAR3, whereas PAR2 and PAR4 were rarely expressed. The C‐fibres in mouse lungs isolated from PAR1−/− animals responded normally to capsaicin, but failed to respond to trypsin, thrombin, or TFLLR‐NH2. These data show that the PAR most relevant for evoking action potential discharge in vagal C‐fibres in mouse lungs is PAR1, and that this is a direct neuronal effect.

Control of Neurotransmission by NaV1.7 in Human, Guinea pig, and Mouse airway Parasympathetic Nerves

Little is known about the neuronal voltage-gated sodium channels (NaVs) that control neurotransmission in the parasympathetic nervous system. We evaluated the expression of the α subunits of each of the nine NaVs in human, guinea pig, and mouse airway parasympathetic ganglia. We combined this information with a pharmacological analysis of selective NaV blockers on parasympathetic contractions of isolated airway smooth muscle. As would be expected from previous studies, tetrodotoxin potently blocked the parasympathetic responses in the airways of each species. Gene expression analysis showed that that NaV 1.7 was virtually the only tetrodotoxin-sensitive NaV1 gene expressed in guinea pig and human airway parasympathetic ganglia, where mouse ganglia expressed NaV1.1, 1.3, and 1.7. Using selective pharmacological blockers supported the gene expression results, showing that blocking NaV1.7 alone can abolish the responses in guinea pig and human bronchi, but not in mouse airways. To block the responses in mouse airways requires that NaV1.7 along with NaV1.1 and/or NaV1.3 is blocked. These results may suggest novel indications for NaV1.7-blocking drugs, in which there is an overactive parasympathetic drive, such as in asthma. The data also raise the potential concern of antiparasympathetic side effects for systemic NaV1.7 blockers.

Different role of TTX‐sensitive voltage‐gated sodium channel (NaV1) subtypes in action potential initiation and conduction in vagal airway nociceptors

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 (NaV1) 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 NaV1.7 blockers. The difference between the initiation of action potentials within the airways vs. conduction along the axons should be considered when developing NaV1 blocking drugs for topical application to the respiratory tract.