Marian Kollarik

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.

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.

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.

Activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and TRPV1-/- mice

The vanilloid receptor TRPV1 (formerly VR1) has been implicated in the activation of nociceptive sensory nerves by capsaicin, noxious heat, protons, bradykinin, cannabinoids such as anandamide, and certain metabolites of arachidonic acid. Using TRPV1 knockout mouse (TRPV1–/–) we address the question of whether TRPV1 is obligatory for action potential discharge in vagal C‐fibre terminals evoked by capsaicin, anandamide, acid and bradykinin. The response of a defined subtype of the vagal afferent bronchopulmonary C‐fibres (conduction velocity < 0.7 ms−1) to the putative TRPV1 activators was studied in vitro in the mouse isolated/perfused lung–nerve preparation. Capsaicin (1 μm) evoked action potential discharge of ∼90% (28/31) of C‐fibres in the TRPV1+/+ mice, but failed to activate bronchopulmonary C‐fibres in TRPV1–/– animals (n= 10). Anandamide (3–100 μm) induced concentration‐dependent activation of capsaicin‐sensitive TRPV1+/+ C‐fibres with a threshold of 3–10 μm, but failed to evoke substantive discharge in TRPV1–/– C‐fibres. In the TRPV1+/+ mice, the B2 receptor‐mediated activation by bradykinin (1 μm) was restricted to the capsaicin‐sensitive C‐fibres. Bradykinin was effective in evoking B2 receptor‐mediated action potential discharge in TRPV1–/– C‐fibres, but the response was significantly (P < 0.05) less persistent than in TRPV1+/+ C‐fibres. Exposing the tissue to acid (pH = 5) excited both TRPV1+/+ and TRPV1–/– C‐fibres. We conclude that TRPV1 is obligatory for vagal C‐fibre activation by capsaicin and anandamide. By contrast, whereas TRPV1 may have a modulatory role in bradykinin and acid‐induced activation of bronchopulmonary C‐fibres, it is not required for action potential discharge evoked by these stimuli.

Vagal afferent nerves with nociceptive properties in guinea-pig oesophagus

Some vagal afferent nerves are thought to mediate autonomic responses evoked by noxious oesophageal stimuli and participate in the perception of pain originating in the oesophagus. However, the vagal nociceptive nerve phenotypes implicated in this function have yet to be identified. In this study, nociceptive fibres were defined by the capacity to discriminate noxious mechanical stimuli (wide range of oesophageal distension with pressure up to 100 mmHg) and detect noxious chemical stimuli (the activators of capsaicin receptor TRPV1). Using immunohistochemical techniques with retrogradely labelled oesophagus‐specific neurones and performing extracellular recordings from the isolated vagally innervated oesophagus, we show that in the guinea‐pig, the vagus nerves supply the oesophagus with a large population of nociceptive‐like afferent nerve fibres. Vagal nociceptive‐like fibres in the guinea‐pig oesophagus are derived from two embryonically distinct sources: neurones situated in the nodose vagal ganglia and neurones situated in the jugular vagal ganglia. Nodose (placode‐derived) nociceptive‐like fibres are exclusively C‐fibres sensitive to a P2X receptors agonist and rarely express the neuropeptide substance P. In contrast, jugular (neural crest‐derived) nociceptive‐like fibres include both A‐fibres and C‐fibres, are insensitive to P2X receptors agonist and mostly express substance P. The non‐nociceptive vagal tension mechanoreceptors are distinguished from nociceptors by their saturable response to oesophageal distension and by the lack of TRPV1. These tension mechanoreceptors are exclusively A‐fibres arising from the nodose ganglion. We conclude that the vagus nerves supply the guinea‐pig oesophagus with nociceptors in addition to tension mechanoreceptors. The vagal nociceptive‐like fibres in the oesophagus comprise two distinct subtypes dictated by the ganglionic location of their cell bodies.

Capsaicin-sensitive and -insensitive vagal bronchopulmonary C-fibres in the mouse

We developed an isolated tracheally perfused (35‐37 °C) nerve‐lung preparation for the study of bronchopulmonary afferent nerve activity in the mouse. Extracellular recordings were made from the vagal sensory neurons located in the jugular‐nodose ganglia complex (JNC) with identified receptive fields in the lungs. Analysis of the vagal compound action potential revealed that the mouse vagal C‐fibre conduction velocities range from 0.3 to 1.5 m s−1. A total of 83 bronchopulmonary C‐fibres were studied. The sensitivity of the bronchopulmonary C‐fibres to the vanilloid receptor 1 (VR1) agonist capsaicin was dependent on conduction velocity. Thus C‐fibres with conduction velocities between 0.3 and 0.7 m s−1 responded to capsaicin (1 μM) while C‐fibres with conduction velocities between 0.7 and 1.5 m s−1 were capsaicin insensitive. Similarly, bradykinin (1 μM) excited only those C‐fibres with conduction velocities < 0.7 m s−1. The response to bradykinin was not mimicked by the B1 receptor agonist [des‐Arg9]bradykinin (1 μM) and was abolished by the bradykinin B2 receptor antagonist HOE 140 (1 μM). Adenosine 5′‐triphosphate (ATP, 30 μM) activated the C‐fibres irrespective of the conduction velocities. This response was mimicked by the selective P2X agonist α,β‐methylene‐adenosine 5′‐triphosphate (30 μM). Consistent with the electrophysiology, morphological analysis revealed that only ˜40 % of the lung‐specific small diameter (< 20 μm) JNC neurons consistent with the C‐fibre cell bodies display VR1 immunoreactivity. This study describes a convenient in vitro method for the study of mouse bronchopulmonary C‐fibres. The results indicate that C‐fibres in the mouse lungs are not homogeneous, but can be subclassified into capsaicin‐sensitive and capsaicin‐insensitive phenotypes.

P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neurons embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.

Acidification of the oesophagus acutely increases the cough sensitivity in patients with gastro-oesophageal reflux and chronic cough.

Abstract  Gastro‐oesophageal reflux disease (GORD) is one of the most common causes of chronic cough; however, the mechanisms by which GOR initiates coughing are incompletely understood. We address the hypothesis that acidification of oesophagus acutely increases the cough reflex sensitivity in patients with GORD and chronic cough. Nine patients with GORD with chronic cough and 16 patients with GORD without cough were recruited. In a randomized double blind study, saline and acid (HCl, 0.1 mol L−1) were separately infused into oesophagus via naso‐oesophageal catheter. Cough reflex sensitivity to inhaled capsaicin was determined immediately after completion of each infusion. Infusion of acid into oesophagus increased capsaicin cough reflex sensitivity in patients with GORD and chronic cough. In contrast, acid had no effect on the cough sensitivity in patients with GORD without cough. In a separate study, acid infusion into oesophagus did not affect the cough sensitivity in 18 healthy subjects. We conclude that acid in the oesophagus acutely increases the cough reflex sensitivity to capsaicin in patients with GORD and chronic cough. This phenomenon may contribute to the pathogenesis of cough due to GORD.

Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

We have identified a distinct subtype of airway vagal afferent nerve that plays an essential role in regulating the cough reflex. These afferents are exquisitely sensitive to punctate mechanical stimuli, acid, and decreases in extracellular chloride concentrations, but are insensitive to capsaicin, bradykinin, histamine, adenosine, serotonin, or changes in airway intraluminal pressures. In this study we used intravital imaging, retrograde neuronal tracing, and electrophysiological analyses to characterize the structural basis for their peculiar mechanical sensitivity and to further characterize the regulation of their excitability. In completing these experiments, we uncovered evidence for an essential role of an isozyme of Na+-K+ ATPase in regulating cough. These vagal sensory neurons arise bilaterally from the nodose ganglia and are selectively and brilliantly stained intravitally with the styryl dye FM2-10. Cough receptor terminations are confined and adherent to the extracellular matrix separating the airway epithelium and smooth muscle layers, a site of extensive remodeling in asthma and chronic obstructive pulmonary disease. The cough receptor terminals uniquely express the α3 subunit of Na+-K+ ATPase. Intravital staining of cough receptors by FM2-10, cough receptor excitability in vitro, and coughing in vivo are potently and selectively inhibited by the sodium pump inhibitor ouabain. These data provide the first detailed morphological description of the peripheral terminals of the sensory nerves regulating cough and identify a selective molecular target for their modulation.