This study was carried out to determine the effect of 2-aminoethoxydiphenyl borate (2-APB), a common activator of transient receptor potential vanilloid (TRPV) type 1, 2, and 3 channels, on cardiorespiratory reflexes, pulmonary C fiber afferents, and isolated pulmonary capsaicin-sensitive neurons. In anesthetized, spontaneously breathing rats, intravenous bolus injection of 2-APB elicited the pulmonary chemoreflex responses, characterized by apnea, bradycardia, and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 2-APB no longer evoked any of these reflex responses. In open-chest and artificially ventilated rats, 2-APB evoked an abrupt and intense discharge in vagal pulmonary C fibers in a dose-dependent manner. The stimulation of C fibers by 2-APB was attenuated but not abolished by capsazepine, a selective antagonist of the TRPV1, which completely blocked the response to capsaicin in these C fiber afferents. In isolated pulmonary capsaicinsensitive neurons, 2-APB concentration dependently evoked an inward current that was partially inhibited by capsazepine but almost completely abolished by ruthenium red, an effective blocker of all TRPV channels. In conclusion, 2-APB evokes a consistent and distinct stimulatory effect on pulmonary C fibers in vivo and on isolated pulmonary capsaicin-sensitive neurons in vitro. These results establish the functional evidence demonstrating that TRPV1, V2, and V3 channels are expressed on these sensory neurons and their terminals. pulmonary chemoreflex; vagal C fiber; pulmonary sensory neuron; transient receptor potential channels THE TRANSIENT RECEPTOR POTENTIAL (TRP) superfamily is a novel and rapidly expanding group of ion channel proteins. Mammalian homologs of the TRP channel gene encode a family of Ͼ20 related ion channels that are further divided into TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) subfamilies (12,32,33). These channels are widely distributed in a variety of mammalian organisms, tissues, and cell types and sense local changes in stimuli ranging from light, olfaction, temperature, pH, and osmolarity to mechanical, chemical, and metabolic stress (31, 43). Currently, the TRPV subfamily has six members. Like members in other TRP subfamilies, they are all cation channels; however, they vary substantially in their selectivity and mode of activation (32,33). Except temperature as the common stimulus of thermosensitive TRPV channels, namely TRPV1-4, most TRPV channels can be activated by nonthermal stimuli (11,16,20,40). For example, TRPV1 can be activated by capsaicin, protons, or endocannabinoids (8,10,21,22,27). TRPV4 can be activated by 4␣-phorbol 12,13-didecanoate and several arachidonic acid metabolites (11,35,44,45). 2-Aminoethoxydiphenyl borate (2-APB), which has been used extensively to investigate the effects of inositol 1,4,5-trisphosphate (IP 3 )-induced Ca 2ϩ release and Ca 2ϩ influx in a variety of cell types (15, 30, 46), was recently found to be a common and potent activato...

, an arachidonic acid derivative produced during inflammatory conditions, is an endogenous agonist of both transient receptor potential vanilloid 1 (TRPV1) receptors and cannabinoid CB1 receptors. Sensitization of capsaicin-sensitive lung vagal afferent (CSLVA) fibers by chemical mediators is important in the pathogenesis of hyperreactive airway diseases. We investigated the effect of the intravenous infusion of AEA (2 mg ⅐ kg Ϫ1 ⅐ ml Ϫ1 , 0.5 ml/min for 2 min) on the sensitivity of CSLVA fibers to chemical and mechanical stimulation in anesthetized rats. In artificially ventilated rats, AEA infusion only mildly elevated the baseline activity of CSLVA fibers. However, CSLVA fiber responses to right atrial injection of capsaicin, AEA, or adenosine and to lung inflation (tracheal pressure ϭ 30 cmH2O) were all markedly potentiated during AEA infusion, which reverted 20 min after termination of the infusion. The potentiating effect on the sensitivity of CSLVA fibers to adenosine injection or lung inflation was completely blocked by pretreatment with capsazepine (a TRPV1 receptor antagonist) but was unaffected by pretreatment with AM281 (a CB1 receptor antagonist). In spontaneously breathing rats, right atrial injection of adenosine evoked an apneic response that is presumably mediated through CSLVA fibers. Similarly, the adenosine-evoked apneic response was potentiated during AEA infusion, and this potentiating effect was also completely prevented by pretreatment with capsazepine. These results suggest that AEA infusion at the dose tested produces a mild activation of TRPV1 receptors and this nonspecifically increases CSLVA fiber sensitivity to chemical and mechanical stimulation.capsaicin-sensitive lung vagal afferent fibers; afferent sensitization CAPSAICIN-SENSITIVE LUNG VAGAL afferents (CSLVAs), mainly C fibers and some A-␦ fibers, are nociceptive-like free nerve endings that play an important role in the regulation of respiratory functions under both normal and pathophysiological conditions (6,30,33,45). These lung afferent fibers can be stimulated by a variety of chemical mediators (30,34,40,45) or inhaled irritants (6,26,27,30), leading to elicitation of various airway reflexes including cough and bronchoconstriction (6, 30). Thus the sensitization of CSLVA fibers by inflammatory mediators has been implicated in the pathogenesis of hyperreactive airway diseases such as chronic cough and asthma (31,50).Studies of the afferent responses of CSLVA fibers to certain agonists have revealed that various types of pharmacological receptors may be located on the membrane of their nerve terminals (30, 45). Activation of several types of these pharmacological receptors by chemical mediators may result in sensitization of the CSLVA fibers. For examples, previous investigations have shown that bradykinin (14), histamine (29), prostaglandin E 2 (19, 25), adenosine (18), and epinephrine (17), possibly via activation of their specific receptors, increase the excitability of CSLVA fibers or vagal airway capsaicinsensitive affe...

We studied acute effects of tumor necrosis factor-α (TNFα) on the sensitivity of isolated rat vagal pulmonary sensory neurons. Our results showed the following. First, a brief pretreatment with a low dose of TNFα (1.44 nM, 9 min) enhanced the sensitivity of transient receptor potential vanilloid type 1 (TRPV1) receptors in these neurons in two distinct phases: the inward current evoked by capsaicin was amplified (Δ = 247%) immediately following the TNFα pretreatment, which gradually declined toward control and then increased again reaching another peak (Δ = 384%) after 60-90 min. Second, the immediate phase of this potentiating effect of TNFα was completely abolished by a pretreatment with a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, whereas the delayed potentiation was only partially attenuated. Third, in sharp contrast, TNFα did not generate any potentiating effect on the responses to non-TRPV1 chemical activators of these neurons. Fourth, the selectivity of the TNFα action on TRPV1 was further illustrated by the responses to acid (pH 6.0); TNFα did not affect the rapid transient current mediated by acid-sensing ion channels but significantly augmented the slow sustained current mediated by TRPV1 in the same neurons. Fifth, in anesthetized rats, a similar pattern of acute sensitizing effects of TNFα on pulmonary C-fiber afferents and the involvement of COX-2 were also clearly shown. In conclusion, a brief pretreatment with TNFα induced both immediate and delayed potentiating effects on the TRPV1 sensitivity in pulmonary sensory neurons, and the production of COX-2 arachidonic acid metabolites plays a major role in the immediate sensitizing effect of TNFα.

Stimulatory effect of CO 2 on vagal bronchopulmonary C-fiber afferents during airway inflammation. J Appl Physiol 99: 1704 -1711, 2005. First published June 30, 2005; doi:10.1152/japplphysiol.00532.2005.-This study investigated 1) whether pulmonary C fibers are activated by a transient increase in the CO 2 concentration of alveolar gas; and 2) if the CO 2 sensitivity of these afferents is altered during airway inflammation. Single-unit pulmonary C-fiber activity was recorded in anesthetized, open-chest rats. Transient alveolar hypercapnia (HPC) was induced by administering a CO 2-enriched gas mixture (25-30% CO2, 21% O2, balance N 2) for five to eight breaths, which increased alveolar CO2 concentration progressively to near or above 13% for 3-5 s and lowered the arterial pH transiently to 7.10 Ϯ 0.05. Our results showed the following. 1) HPC evoked only a mild stimulation in a small fraction (4/47) of pulmonary C fibers, and there was no significant change in fiber activity (change in fiber activity ϭ 0.22 Ϯ 0.16 imp/s; P Ͼ 0.1, n ϭ 47).2) In sharp contrast, after airway exposure to poly-L-lysine, a cationic protein known to induce mucosal injury, the same challenge of transient HPC activated 87.5% of the pulmonary C fibers tested and evoked a distinct stimulatory effect on these afferents (change in fiber activity ϭ 6.59 Ϯ 1.78 imp/s; P Ͻ 0. 01, n ϭ 8). 3) Similar potentiation of the C-fiber response to HPC was also observed after acute exposure to ozone (n ϭ 6) and during a constant infusion of inflammatory mediators such as adenosine (n ϭ 15) or prostaglandin E 2 (n ϭ 12). 4) The enhanced C-fiber sensitivity to CO 2 after poly-L-lysine was completely abrogated by infusion of NaHCO 3 (1.82 mmol⅐kg Ϫ1 ⅐min Ϫ1) that prevented the reduction in pH during HPC (n ϭ 6). In conclusion, only a small percentage (Ͻ10%) of the bronchopulmonary C fibers exhibit CO 2 sensitivity under control conditions, but alveolar HPC exerts a consistent and pronounced stimulatory effect on the C-fiber endings during airway inflammation. This effect of CO 2 is probably mediated through the action of hydrogen ions. hydrogen ion; airway mucosal injury; airway hyperreactivity; hypercapnia PREVIOUS INVESTIGATORS HAVE suggested the existence of sensory receptors that can detect an increase in CO 2 in the lung (32). However, no direct and definitive evidence has been established in identifying the presence of "CO 2 sensors" in the lung structures. Experiments employing various techniques to block the conduction of myelinated fibers in the vagus have yielded compelling evidence suggesting the involvement of bronchopulmonary C fibers in the hyperpneic response to CO 2 (26, 28). One possibility is that the increase in alveolar CO 2 concentration in those experiments led to a decrease in the pulmonary interstitial pH, which then activated the pulmonary C fibers. This hypothesis is supported by a recent study demonstrating that pulmonary C fibers are consistently activated when pH in the arterial blood (pH a ) (pulmonary venous blood) is lowered to...

We studied the respiratory responses to an increase in airway temperature in patients with allergic rhinitis (AR). Responses to isocapnic hyperventilation (40% of maximal voluntary ventilation) for 4 minutes of humidified hot air (HA; 49 °C) and room air (RA: 21 °C) were compared between AR patients (n=7) and healthy subjects (n=6). In AR patients, cough frequency increased pronouncedly from 0.10±0.07 before to 2.37±0.73 during, and 1.80±0.79 coughs/min for the first 8 minutes after the HA challenge, but not during the RA challenge. In contrast, neither HA nor RA had any significant tussive effect in healthy subjects. The HA challenge also caused respiratory discomfort (mainly throat irritation) measured by the handgrip dynamometry in AR patients, but not in healthy subjects. Bronchoconstriction was not detected after the HA challenge in either group of subjects. In conclusion, hyperventilation of HA triggered vigorous cough response and throat irritation in AR patients, indicating the involvement of sensory nerves innervating upper airways.