Qihai Gu

Sensitivity of vagal afferent endings to chemical irritants in the rat lung

This study was carried out to investigate the relationship between the conduction velocity of the vagal afferents arising from the rat lungs and their sensitivities to capsaicin, other chemical irritants, and lung inflation. We recorded single-unit activities of vagal pulmonary afferents (n = 205) in anesthetized, open-chest rats, and distinguished C fibers (conduction velocity < 2 m/sec) from myelinated afferents; the latter group was further classified into rapidly adapting pulmonary receptors (RARs) and slowly adapting pulmonary stretch receptors (SARs) on the basis of their adaptation indexes to lung inflation. Right-atrial injection of capsaicin (1 microg/kg) evoked an abrupt and intense stimulatory effect in 88.9% (64/72) of the pulmonary C fibers tested, but only a mild stimulation in 6.3% (3/48) of the RARs and none of the SARs. Other inhaled and injected chemical stimulants (e.g., cigarette smoke, lactic acid) activated 68.9% (42/61) of the pulmonary C fibers. The same chemical irritants exerted a mild stimulatory effect in only 14.5% (8/55) of the RARs; this subgroup of RARs exhibited a low or no baseline activity, and half of them were located near the hilum. Chemical stimulants had little or no effect on SARs. The response of pulmonary C fibers to lung inflation (tracheal pressure = 30 cm H2O) was not only extremely weak, but also showed a longer onset latency and an irregular pattern. In a sharp contrast, lung inflation evoked rapid and vigorous discharges in both RARs and SARs. In conclusion, C fibers are the primary type of chemosensitive vagal pulmonary afferents in rat lungs.

Role of TRPV1 in inflammation-induced airway hypersensitivity

Airway hypersensitivity is a common pathophysiological feature in various airway inflammatory diseases. Increasing evidence suggests that activation of the transient receptor potential vanilloid type 1 receptor (TRPV1) plays an important part in the manifestation of various symptoms of airway hypersensitivity. This mini-review focuses on recent studies that have revealed several potential contributing factors to the increase in TRPV1 sensitivity in pulmonary sensory neurons during airway inflammatory reaction. In addition, chronic allergic airway inflammation induces a pronounced overexpression of TRPV1 in neurofilament-positive pulmonary sensory neurons in nodose ganglia. A better understanding of the mechanisms underlying the increase in sensitivity and/or expression of TRPV1 during acute and chronic airway inflammation should generate the necessary information for developing effective therapeutic interventions to alleviate airway hypersensitivity.

Heavy metals zinc, cadmium, and copper stimulate pulmonary sensory neurons via direct activation of TRPA1

Airway exposure to zinc dust and zinc-containing ambient particulates can cause symptoms of airway irritation and inflammation, but the underlying molecular and cellular mechanisms are largely unknown. Transient receptor potential A1 (TRPA1) is selectively expressed in a subpopulation of pulmonary C-fiber afferents and has been considered as a major irritant sensor in the lung and airways. Using whole cell patch-clamp recording and Ca(2+) imaging, we have demonstrated that application of ZnCl(2) concentration dependently evoked inward current and Ca(2+) transient in isolated vagal pulmonary sensory neurons; both responses were almost completely inhibited after pretreatment with AP18, a specific TRPA1 antagonist. In anesthetized spontaneously breathing animals, intratracheal instillation of ZnCl(2) (2 mM, 25 microl) induced pronounced respiratory depression in wild-type mice, and the effect was essentially absent in TRPA1-deficient mice. In addition, our study showed that two other heavy metals, cadmium and copper, also stimulated pulmonary sensory neurons via a direct activation of TRPA1. In summary, our results suggest that activation of TRPA1 in pulmonary C-fiber sensory nerves may contribute to the respiratory toxicity induced by airway exposure to these three heavy metals.

Calcium transient evoked by TRPV1 activators is enhanced by tumor necrosis factor-α in rat pulmonary sensory neurons

TNFα, a proinflammatory cytokine known to be involved in the pathogenesis of allergic asthma, has been shown to induce hyperalgesia in somatic tissue via a sensitizing effect on dorsal root ganglion neurons expressing transient receptor potential vanilloid type 1 receptor (TRPV1). Because TRPV1-expressing pulmonary sensory neurons play an important role in regulating airway function, this study was carried out to determine whether TNFα alters the sensitivity of these neurons to chemical activators. Responses of isolated nodose and jugular ganglion neurons innervating the rat lungs were determined by measuring the transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). Our results showed the following. 1) A pretreatment with TNFα (50 ng/ml) for ∼24 h increased significantly the peak Δ[Ca(2+)](i) evoked by capsaicin (Cap) in these neurons. A pretreatment with the same concentration of TNFα for a longer duration (∼48 h) did not further increase the response, but pretreatment for a shorter duration (1 h) or with a lower concentration (25 ng/ml, 24 h) failed to enhance the Cap sensitivity. 2) The same TNFα pretreatment also induced similar but less pronounced and less uniform increases in the responses to acid (pH 6.5-5.5), 2-aminoethoxydiphenyl borate (2-APB), a common activator of TRPV1, V2, and V3 channels, and allyl isothiocyanate (AITC), a selective activator of TRPA1 channel. 3) In sharp contrast, the responses to ATP, ACh, and KCl were not affected by TNFα. 4) The TNFα-induced hypersensitivity to Cap was not prevented by pretreatment with indomethacin (30 μM). 5) The immunoreactivity to both TNF receptor types 1 and 2 were detected in rat vagal pulmonary sensory neurons. In conclusion, prolonged treatment with TNFα induces a pronounced potentiating effect on the responses of isolated pulmonary sensory neurons to TRPV1 activators. This action of TNFα may contribute in part to the airway hyperresponsiveness induced by this cytokine.

Sensitization of isolated rat vagal pulmonary sensory neurons by eosinophil-derived cationic proteins

It has been shown that airway exposure to eosinophil-derived cationic proteins stimulated vagal pulmonary C fibers and markedly potentiated their responses to lung inflation in anesthetized rats (Lee LY, Gu Q, Gleich GJ, J Appl Physiol 91: 1318-1326, 2001). However, whether the effects resulted from a direct action of these proteins on the sensory nerves was not known. The present study was therefore carried out to determine the effects of these proteins on isolated rat vagal pulmonary sensory neurons. Our results obtained from perforated whole cell patch-clamp recordings showed that pretreatment with eosinophil major basic protein (MBP; 2 microM, 60 s) significantly increased the capsaicin-evoked inward current in these neurons; this effect peaked approximately 10 min after MBP and lasted for >60 min; in current-clamp mode, MBP substantially increased the number of action potentials evoked by both capsaicin and electrical stimulation. Pretreatment with MBP did not significantly alter the input resistance of these sensory neurons. In addition, the sensitizing effect of MBP was completely abolished when its cationic charge was neutralized by mixing with a polyanion, such as low-molecular-weight heparin or poly-L-glutamic or poly-L-aspartic acid, before its delivery to the neurons. Moreover, a similar sensitizing effect was also generated by other eosinophil granule-derived proteins (e.g., eosinophil peroxidase). These results demonstrate a direct, charge-dependent, and long-lasting sensitizing effect of cationic proteins on pulmonary sensory neurons, which may contribute to the airway hyperresponsiveness associated with airway infiltration of eosinophils under pathophysiological conditions.

Hypersensitivity of pulmonary chemosensitive neurons induced by activation of protease-activated receptor-2 in rats.

This study was carried out to determine the effect of protease‐activated receptor‐2 (PAR2) activation on the pulmonary chemoreflex responses and on the sensitivity of isolated rat vagal pulmonary chemosensitive neurons. In anaesthetized, spontaneously breathing rats, intratracheal instillation of trypsin (0.8 mg ml−1, 0.1 ml), an endogenous agonist of PAR2, significantly amplified the capsaicin‐induced pulmonary chemoreflex responses. The enhanced responses were completely abolished by perineural capsaicin treatment of both cervical vagi, suggesting the involvement of pulmonary C‐fibre afferents. In patch‐clamp recording experiments, pretreatment with trypsin (0.1 μm, 2 min) potentiated the capsaicin‐induced whole‐cell inward current in isolated pulmonary sensory neurons. The potentiating effect of trypsin was mimicked by PAR2‐activating peptide (PAR2‐AP) in a concentration‐dependent manner. PAR2‐AP pretreatment (100 μm, 2 min) also markedly enhanced the acid‐evoked inward currents in these sensory neurons. Furthermore, the sensitizing effect of PAR2 was completely abolished by pretreatment with either U73122 (1 μm, 4 min), a phospholipase C inhibitor, or chelerythrine (10 μm, 4 min), a protein kinase C (PKC) inhibitor. In summary, our results have demonstrated that activation of PAR2 upregulates the pulmonary chemoreflex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro, and this effect of PAR2 activation was mediated through the PKC‐dependent transduction pathway. These results further suggest that the hypersensitivity of these neurons may play a part in the development of airway hyper‐responsiveness resulting from PAR2 activation.

Hyperthermia increases sensitivity of pulmonary C-fibre afferents in rats

This study was carried out to investigate whether an increase in tissue temperature alters the excitability of vagal pulmonary C‐fibres. Single‐unit afferent activities of 88 C‐fibres were recorded in anaesthetized and artificially ventilated rats when the intrathoracic temperature (Tit) was maintained at three different levels by isolated perfusion of the thoracic chamber with saline: control (C: ∼36°C), medium (M: ∼38.5°C) and high (H: ∼41°C), each for 3 min with 30 min recovery. Our results showed: (1) The baseline fibre activity (FA) of pulmonary C‐fibres did not change significantly at M, but increased drastically (>5‐fold) at H. (2) The C‐fibre response to right‐atrial injection of capsaicin (0.5 μg kg−1) was markedly elevated at H (ΔFA = 5.94 ± 1.65 impulses s−1 at C and 13.13 ± 2.98 impulses s−1 at H; P < 0.05), but not at M. Similar increases in the C‐fibre responses to other chemical stimulants (e.g. adenosine, etc.) were found at H; all the enhanced responses returned to control in 30 min. (3) The C‐fibre response to lung inflation was also significantly potentiated at H. In sharp contrast, there was no detectable change in either the baseline activity or the responses to lung inflation and deflation in 10 rapidly adapting pulmonary receptors and 10 slowly adapting pulmonary receptors at either M or H. (4) The enhanced C‐fibre sensitivity was not altered by pretreatment with indomethacin or capsazepine, a selective antagonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor, but was significantly attenuated by ruthenium red that is known to be an effective blocker of all TRPV channels. (5) The response of pulmonary C‐fibres to a progressive increase in Tit in a ramp pattern further showed that baseline FA started to increase when Tit exceeded 39.2°C. In conclusion, a pronounced increase in the baseline activity and excitability of pulmonary C‐fibres is induced by intrathoracic hyperthermia, and this enhanced sensitivity probably involves activation of temperature‐sensitive ion channel(s), presumably one or more of the TRPV receptors, expressed on the C‐fibre endings.

Expression of neuronal nicotinic acetylcholine receptors in rat vagal pulmonary sensory neurons.

It is known that cigarette smoke inhalation causes airway irritation and cough, and the effect is caused by both direct and indirect stimulatory effects of nicotine on bronchopulmonary sensory nerves. However, little is known about the expression of nicotinic acetylcholine receptors (nAChRs) in these afferents. In the present study, whole-cell patch-clamp recording and RT-PCR were carried out to examine the expression and function of nAChRs in isolated rat vagal pulmonary sensory neurons that were identified by retrograde labeling with a fluorescent tracer. Patch-clamp recordings demonstrated that application of acetylcholine concentration-dependently evoked an inward current in a subset of pulmonary sensory neurons, which was inhibited by hexamethonium. Application of nicotine or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also activated these neurons, evoking an inward current in voltage-clamp configuration and causing depolarization and action potential in current-clamp recordings. RT-PCR analysis further demonstrated the expression of mRNA encoding for the nAChR subunits alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4, but not alpha2 and alpha3 in these neurons.

Mechanisms of eosinophil major basic protein-induced hyperexcitability of vagal pulmonary chemosensitive neurons.

We have reported recently that eosinophil-derived basic proteins directly enhance the capsaicin- and electrical stimulation-evoked whole cell responses in rat pulmonary sensory neurons (19). Our present study further elucidates the mechanisms underlying the sensitization of pulmonary afferent nerves induced by these cationic proteins. Our results show that pretreatment with eosinophil major basic protein (MBP; 2 microM, 60 s) significantly enhanced the excitability of isolated rat vagal pulmonary chemosensitive neurons to acid and ATP in the current-clamp mode, but this potentiating effect was absent in the voltage-clamp recordings. The hyperexcitability induced by MBP was not prevented by the blockade of either transient receptor potential vanilloid type-1 receptor (TRPV1) selectively (inhibitor: AMG 9810; 1 microM, 2 min) or all TRPV1-4 channels (inhibitor: ruthenium red; 5 microM, 2 min). In addition, MBP also markedly potentiated the excitability of mouse pulmonary chemosensitive neurons, and no detectable difference was found between those isolated from wild-type and TRPV1 knockout mice. Furthermore, MBP pretreatment affected the decay time and recovery phase of the action potentials evoked by current injections and significantly inhibited both the sustained delayed-rectifier voltage-gated K(+) current (IK(dr)) and the A-type, fast-inactivating K(+) current (IK(a)) in these sensory neurons. In conclusion, our results indicate that the inhibition of IK(dr) and IK(a) should, at least in part, account for the hyperexcitability of pulmonary chemosensitive neurons induced by eosinophil-derived cationic proteins, whereas an interaction with TRPV1 channels does not seem to be required for the sensitizing effect of these proteins.

Cough Sensors. IV. Nicotinic Membrane Receptors on Cough Sensors

Cigarette smoke is undoubtedly one of the most common inhaled irritants in the human respiratory tract, and invariably evokes coughing in both smokers and nonsmokers. Results obtained from the studies in human volunteers and from single-fiber recording of vagal bronchopulmonary afferents in animals clearly indicate that nicotine is primarily responsible for the airway irritation and coughing caused by inhalation of cigarette smoke. Furthermore, both nicotine and acetylcholine can evoke inward current, membrane depolarization, and action potentials in isolated pulmonary sensory neurons, and these responses are blocked by hexamethonium. Taken together, these findings suggest that the tussive effect of nicotine is probably mediated through an activation of nicotinic acetylcholine receptors (nAChRs) expressed on the sensory terminals of cough receptors located in the airway mucosa. Indeed, the expressions of alpha4-alpha7 and beta2-beta4 subunits of nAChR transcripts in pulmonary sensory neurons have lent further support to this conclusion. The specific subtypes of the neuronal nAChRs and their subunit compositions expressed on the cough sensors remain to be determined.