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.

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 neuron's 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 patchclamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to ␣,␤-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 ␣,␤-methylene ATP (30 M) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of ␣,␤-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.

Mediators of inflammation, such as PGE(2), are known to sensitize the airways to inhaled irritants and circulating autacoids. Evidence from in vivo studies has shown the involvement of vagal pulmonary C-fiber afferents in the PGE(2)-elicited airway hypersensitivity. However, whether PGE(2) acts directly on these sensory nerves is unclear. The present study aimed to investigate whether PGE(2) has direct potentiating effects on nodose and jugular pulmonary C neurons cultured from adult Sprague-Dawley rats and, if so, determine whether the EP(2) prostanoid receptor is involved. Pulmonary neurons were identified by retrograde labeling with a fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Using perforated patch-clamp technique, our results showed that 1) PGE(2) pretreatment (1 microM) increased the whole cell current density elicited by capsaicin and phenylbiguanide, chemical agents known to stimulate pulmonary C fibers; 2) selective activation of the EP(2) prostanoid receptor by butaprost (3-10 microM) increased the whole cell current density elicited by capsaicin; and 3) PGE(2), as well as butaprost, increased the number of action potentials evoked by current injection. Therefore, we conclude that PGE(2) directly sensitizes vagal pulmonary C neurons to chemical and electrical stimulation. Furthermore, butaprost modulates the neurons in a manner similar to that of PGE(2), suggesting that the effects of PGE(2) are mediated, at least in part, through the EP(2) prostanoid receptor.

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 Na V 1.8 and Na V 1.9 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.

No abstract

Intravenous administration of adenosine (Ado) to patients can cause dyspnoea, chest discomfort and bronchoconstriction. To assess the role of vagal pulmonary C fibres in evoking these adverse reactions, the effect of Ado on single pulmonary C fibres was studied in anaesthetized and artificially ventilated rats. Right‐atrial injection of Ado (320 μg kg−1) activated 68 % (73/107) of pulmonary C fibres; the total number of action potentials during a period of 15 s increased from a baseline of 0.2 ± 0.1 impulses to a peak of 16.4 ± 2.6 impulses (P < 0.01, n= 107) after Ado. Inosine, the metabolite of Ado, did not activate any of eleven C fibres tested in six rats. Furthermore, C fibres were activated only by right‐atrial and not by left‐ventricular injection of the same dose of Ado. Unlike the immediate and transient stimulation of C fibres by capsaicin, the C fibre stimulation by Ado had a latency of 6.5 ± 0.3 s (range, 3‐18 s) and lasted longer. The stimulation of C fibres by Ado was significantly attenuated by pretreatment with aminophylline, a non‐selective Ado receptor antagonist, was completely prevented by 1,3‐dipropyl‐8‐cyclopentylxanthine, an Ado A1 receptor antagonist, but was unaffected by 3,7‐dimethy‐1‐propargylxanthine, an A2 receptor antagonist. None of these Ado receptor antagonists prevented capsaicin‐induced C fibre stimulation. In conclusion, Ado stimulates pulmonary C fibre terminals through an activation of A1 receptors. The stimulation of pulmonary C fibres may play an important role in Ado‐induced adverse respiratory effects.

No abstract

The effect of prostaglandin E(2) (PGE(2)) on chemical stimulation-evoked calcium (Ca(2+)) transient was investigated in isolated vagal sensory neurons of the rat using fura-2-based ratiometric Ca(2+) imaging. Application of capsaicin (3 x 10(-8) to 10(-7) M; 15 s) caused a rapid surge of intracellular Ca(2+) concentration in small- and medium-size neurons; the response was reproducible when >10 min elapsed between two challenges and was absent in nominally Ca(2+)-free solution. After pretreatment with PGE(2) (3 x 10(-7) M; 5 min), the peak of this capsaicin-evoked Ca(2+) transient was increased by almost fourfold, and its duration was also prolonged. This augmented response to capsaicin induced by PGE(2) gradually declined but remained higher than control after 15-min washout. Similarly, PGE(2) pretreatment also markedly enhanced the Ca(2+) transients induced by other chemical stimulants to C neurons, such as phenylbiguanide (PBG), adenosine 5'-triphosphate (ATP), and KCl. The Ca(2+) transients evoked by PBG, ATP, and KCl were potentiated after the pretreatment with PGE(2) to 242, 204, and 163% of their control, respectively. This potentiating effect of PGE(2) could be mimicked by forskolin (10(-6) M; 5 min), an activator of adenylyl cyclase, and 8-(4-chlorophenylthio)adenosine-3'-5'-cyclic monophosphate (CPT-cAMP; 3 x 10(-6) M, 10 min), a membrane-permeable cAMP analogue. Furthermore, the potentiating effects of PGE(2), forskolin, and CPT-cAMP were abolished by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 10(-5) M; 15-20 min), a protein kinase A (PKA) inhibitor. In summary, these results show that PGE(2) reversibly potentiates the chemical stimuli-evoked Ca(2+) transients in cultured rat vagal sensory neurons, and this potentiating effect is mediated through the cyclic AMP/PKA transduction cascade.