Bradley J. Undem

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus.

The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side effect of this widely used antimalarial drug. Here, we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and coexpress gastrin-releasing peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with the MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

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.

Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways

1. The present study addressed the hypothesis that jugular and nodose vagal ganglia contain the somata of functionally and anatomically distinct airway afferent fibres. 2. Anatomical investigations were performed by injecting guinea‐pig airways with the neuronal tracer Fast Blue. The animals were killed 7 days later, and the ganglia were removed and immunostained with antisera against substance P (SP) and neurofilament protein (NF). In the nodose ganglion, NF‐immunoreactive neurones accounted for about 98% of the Fast Blue‐labelled cells while in the jugular ganglion they accounted for approximately 48%. SP and NF immunoreactivity was never (n = 100) observed in the same cell suggesting that the antisera labelled distinct populations. 3. Electrophysiological investigations were performed using an in vitro guinea‐pig tracheal and bronchial preparation with intact afferent vagal pathways, including nodose and jugular ganglia. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in either ganglion. 4. The nodose ganglion contained the somata of mainly fast‐conducting tracheal A delta fibres whereas the jugular ganglion contained equal numbers of C fibre and A delta fibre tracheal afferent somata. The nodose A delta neurones adapted rapidly to mechanical stimulation, had relatively low mechanical thresholds, were not activated by capsaicin and adapted rapidly to a hyperosmotic stimulus. By contrast, jugular A delta and C fibres adapted slowly to mechanical stimulation, were often activated by capsaicin, had higher mechanical thresholds and displayed a slow adaptation to a hyperosmotic stimulus. 5. The anatomical, physiological and pharmacological data provide evidence to support the contention that the vagal ganglionic source of the fibre supplying the airways ultimately dictates its neurochemical and physiological phenotype.

Prostaglandin-Induced Activation of Nociceptive Neurons via Direct Interaction with Transient Receptor Potential A1 (TRPA1)

Inflammation contributes to pain hypersensitivity through multiple mechanisms. Among the most well characterized of these is the sensitization of primary nociceptive neurons by arachidonic acid metabolites such as prostaglandins through G protein-coupled receptors. However, in light of the recent discovery that the nociceptor-specific ion channel transient receptor potential A1 (TRPA1) can be activated by exogenous electrophilic irritants through direct covalent modification, we reasoned that electrophilic carbon-containing A- and J-series prostaglandins, metabolites of prostaglandins (PG) E2 and D2, respectively, would excite nociceptive neurons through direct activation of TRPA1. Consistent with this prediction, the PGD2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) activated heterologously expressed human TRPA1 (hTRPA1-HEK), as well as a subset of chemosensitive mouse trigeminal neurons. The effects of 15dPGJ2 on neurons were blocked by both the nonselective TRP channel blocker ruthenium red and the TRPA1 inhibitor (HC-030031), but unaffected by the TRPV1 blocker iodo-resiniferatoxin. In whole-cell patch-clamp studies on hTRPA1-HEK cells, 15dPGJ2 evoked currents similar to equimolar allyl isothiocyanate (AITC) in the nominal absence of calcium, suggesting a direct mechanism of activation. Consistent with the hypothesis that TRPA1 activation required reactive electrophilic moieties, A- and J-series prostaglandins, and the isoprostane 8-iso-prostaglandin A2-evoked calcium influx in hTRPA1-HEK cells with similar potency and efficacy. It is noteworthy that this effect was not mimicked by their nonelectrophilic precursors, PGE2 and PGD2, or PGB2, which differs from PGA2 only in that its electrophilic carbon is rendered unreactive through steric hindrance. Taken together, these data suggest a novel mechanism through which reactive prostanoids may activate nociceptive neurons independent of prostaglandin receptors.

Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs

Transient receptor potential (TRP) A1 and TRPM8 are ion channels that have been localized to afferent nociceptive nerves. These TRP channels may be of particular relevance to respiratory nociceptors in that they can be activated by various inhaled irritants and/or cold air. We addressed the hypothesis that mouse vagal sensory nerves projecting to the airways express TRPA1 and TRPM8 and that they can be activated via these receptors. Single cell RT‐PCR analysis revealed that TRPA1 mRNA, but not TRPM8, is uniformly expressed in lung‐labelled TRPV1‐expressing vagal sensory neurons. Neither TRPA1 nor TRPM8 mRNA was expressed in TRPV1‐negative neurons. Capsaicin‐sensitive, but not capsaicin‐insensitive, lung‐specific neurons responded to cinnamaldehyde, a TRPA1 agonist, with increases in intracellular calcium. Menthol, a TRPM8 agonist, was ineffective at increasing cellular calcium in lung‐specific vagal sensory neurons. Cinnamaldehyde also induced TRPA1‐like inward currents (as measured by means of whole cell patch clamp recordings) in capsaicin‐sensitive neurons. In an ex vivo vagal innervated mouse lung preparation, cinnamaldehyde evoked action potential discharge in mouse vagal C‐fibres with a peak frequency similar to that observed with capsaicin. Cinnamaldehyde inhalation in vivo mimicked capsaicin in eliciting strong central‐reflex changes in breathing pattern. Taken together, our results support the hypothesis that TRPA1, but not TRPM8, is expressed in vagal sensory nerves innervating the airways. TRPA1 activation provides a mechanism by which certain environmental stimuli may elicit action potential discharge in airway afferent C‐fibres and the consequent nocifensor reflexes.

Subtypes of vagal afferent C‐fibres in guinea‐pig lungs

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C‐fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C‐fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C‐fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C‐fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C‐fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C‐fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C‐fibre population responded with action potential discharge to the P2X selective receptor agonist α,β‐methylene‐ATP. Whole cell patch clamp recording of capsaicin‐sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C‐fibre neurones express functional P2X receptors, whereas lung specific jugular C‐fibres do not. The data support the hypothesis that both neural crest‐derived neurones (jugular ganglia) and placode‐derived neurones (nodose ganglia) project C‐fibres in the vagus, and that these two C‐fibre populations represent distinct phenotypes.

Cysteinyl leukotrienes in asthma: old mediators up to new tricks

The cysteinyl leukotrienes have long been suspected to play a role in the pathogenesis of asthma. This speculation was based largely on their release in human lung following antigen challenge as well as their potent bronchoconstrictor activity. However, there is increasing evidence that the cysteinyl leukotrienes also produce several pro-inflammatory effects and alter the activity of neuronal pathways in the airways. Douglas Hay, Theodore Torphy and Bradley Undem review these recent data and discuss the therapeutic possibilities of cysteinyl leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary C‐fibres by the endogenous autacoid 4‐oxononenal

Transient receptor potential (TRP) A1 channels are cation channels found preferentially on nociceptive sensory neurones, including capsaicin‐sensitive TRPV1‐expressing vagal bronchopulmonary C‐fibres, and are activated by electrophilic compounds such as mustard oil and cinnamaldehyde. Oxidative stress, a pathological feature of many respiratory diseases, causes the endogenous formation of a number of reactive electrophilic alkenals via lipid peroxidation. One such alkenal, 4‐hydroxynonenal (4HNE), activates TRPA1 in cultured sensory neurones. However, our data demonstrate that 100 μm 4HNE was unable to evoke significant action potential discharge or tachykinin release from bronchopulmonary C‐fibre terminals. Instead, another endogenously produced alkenal, 4‐oxononenal (4ONE, 10 μm), which is far more electrophilic than 4HNE, caused substantial action potential discharge and tachykinin release from bronchopulmonary C‐fibre terminals. The activation of mouse bronchopulmonary C‐fibre terminals by 4ONE (10–100 μm) was mediated entirely by TRPA1 channels, based on the absence of responses in C‐fibre terminals from TRPA1 knockout mice. Interestingly, although the robust increases in calcium caused by 4ONE (0.1–10 μm) in dissociated vagal neurones were essentially abolished in TRPA1 knockout mice, at 100 μm 4ONE caused a large TRPV1‐dependent response. Furthermore, 4ONE (100 μm) was shown to activate TRPV1 channel‐expressing HEK cells. In conclusion, the data support the hypothesis that 4‐ONE is a relevant endogenous activator of vagal C‐fibres via an interaction with TRPA1, and at less relevant concentrations, it may activate nerves via TRPV1.

Mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig

The mechanisms underlying the response of airway afferent nerves to low pH were investigated in an isolated guinea‐pig airway nerve preparation. Extracellular recordings were made from single jugular or nodose vagal ganglion neurons that projected their sensory fibers into the airways. The airway tissue containing the mechanically sensitive receptive fields was exposed into acidic solutions. Rapid and transient (∼3 s) administration of 1 mm citric acid to the receptive field consistently induced action potential discharge in nociceptive C‐fibers (41/44) and nodose Aδ fibres (29/30) that are rapidly adapting low threshold mechanosensors (RAR‐like fibres). In contrast, citric acid activated only 8/17 high threshold mechanosensitive jugular Aδ fibres. The RAR‐like fibres were slightly more sensitive than C‐fibres to acidic solutions (pH threshold > 6.7). The RAR‐like fibres response to the ∼3 s acid treatment was not affected by a vanilloid receptor 1 (VR1) antagonist, capsazepine (10 μM), and was rapidly inactivating (action potential discharge terminated before the acid administration was completed). Gradual reduction of pH did not activate the RAR‐like fibres even when the pH was reduced to ∼5.0. The C‐fibres responded to the gradual reduction of pH with persistent action potential discharge that was nearly abolished by capsazepine (10 μM) and inhibited by over 70% with another VR1 antagonist iodo‐resiniferatoxin (1 μM). In contrast the C‐fibre response to the transient ∼3 s exposure to pH ∼5.0 was not affected by the VR1 antagonists. We conclude that activation of guinea‐pig airway afferents by low pH is mediated by both slowly and rapidly inactivating mechanisms. We hypothesize that the slowly inactivating mechanism, present in C‐fibres but not in RAR‐like fibres, is mediated by VR1. The rapidly inactivating mechanism acts independently of VR1, has characteristics similar to acid sensing ion channels (ASICs) and is found in the airway terminals of both C‐fibres and RAR‐like fibres.