The present studies from in vivo to in vitro revealed several new findings. (1) PGE2-evoked cough was suppressed by inhalation of aerosolized TRPV1 or EP3 receptor antagonist, while CA-evoked cough was only inhibited by TRPV1 antagonist. (2) Approximately 1/4 of vagal pulmonary C-neurons marked by TRPV1 co-expressed EP3 with a cell size often smaller than 20 µm. (3) PGE2 at 20 µM could trigger immediate inward currents in vagal pulmonary neurons that are sensitive to CAP. (4) PGE2-induced currents were inhibited by both EP3 receptor and TRPV1 antagonists, but CAP-induced currents were uniquely suppressed by TRPV1 antagonists. (5) CAP-induced currents were facilitated by pre-ejection of PGE2 acting on EP3 receptor, but PGE2-induced currents were inhibited by pre-ejection of CAP (desensitization). Taken together, our results suggest that full expression of PGE2-induced vagal pulmonary C-neuronal excitation in vitro and cough in vivo requires interaction of EP3 and TRPV1 receptors, while EP3 receptors were not involved in CAP-induced neuronal currents and cough in guinea pigs.
Studies have shown that PCE2-induced coughs are largely attenuated by IP injection of JNJ 17203212 (100 mg/kg) or L-826266 (300 mg/kg) in unanesthetized guinea pigs [14, 28]. CAP-induced coughs are also suppressed and eliminated by IP injection of JNJ 17203212 at lower (10–30 mg/kg) and higher doses (> 100 mg/kg) respectively , but no study has been carried out to test the effect of EP3 receptor antagonist on CAP-induced cough. To relatively focus on the airways, we tested if TRPV1 or EP3 receptor antagonist administrated via aerosol inhalation was sufficient to suppress CA- and/or PGE2-induced coughs. We found that PGE2-induced coughs were significantly decreased by 75% and 50% after inhaling JNJ 17203212 and L-798106 respectively, and CA-evoked coughs were reduced by 58% after JNJ 17203212 and unchanged after L-798106 application. This finding suggests that activation of airway TRPV1 and EP3 receptor is essential for fully expressing PGE2-induced cough. The fact that inhaling L-798106 fails to affect CA-induced cough reveals, for the first time, that EP3 receptors under pathogen free condition has a limited contribution to this type of cough. There are several advances in inhalation approach used in this study. First, compared to IP injection of JNJ 17203212 and L-826266 (30 and 300 mg/kg) [14, 28], inhalation of the same antagonists at much lower doses (3.75 mg/2.5 ml for both JNJ 17203212 and L-798106) produces similar antitussive effects. Second, the post-administration duration required to produce the antitussive effect is shorter through inhalation than that via IP injection (5 min vs. 40 min). Third, owing to the presence of TRPV1 and EP3 receptor in a variety of organs, such as the brains (for a review, see [51, 52]), inhalation of these antagonists should have less possible side effects as compared to systemic administration. In summary, our results not only reveal the higher antitussive efficacy of these antagonists via inhalation than systemic administration, but also support the interaction of TRPV1 and EP3 receptor in cough mainly occurring in airway sensory fibers.
PGE2-induced depolarization of the isolated vagal nerve in human, guinea pig, and mouse is reportedly reduced by local application of JNJ 17203212 or L-826266 in vitro [14, 28]. Because the vagal nerve contains sensory fibers innervate airways/lungs and other visceral organs, and because PCFs play a critical role in both CA/CAP- and PGE2-induced coughs, a fundamental question raised is whether the interaction of TRPV1 and EP3 receptor occurs in PCFs. The expression of TRPV1 has been identified in vagal pulmonary C-neurons [32, 33] and that of EP3 receptors in the nodose ganglion and dorsal root ganglion neurons [34, 35, 53]. In this study, we confirmed the co-expression of TRPV1 and EP3 in ~ 1/4 of vagal pulmonary C-neurons (cell size < 20 µm with TRPV1 labeling) (Fig. 3). In agreement, mRNA expression of EP3 has been identified in rat nodose ganglionic neurons with small cell size [34, 54]. Our morphological data showing the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons provide a strong rationale for our following electrophysiological studies.
CA/CAP and PGE2 are capable of stimulating PCFs respectively [9, 55, 56, 57, 58]. However, it is unclear whether the same vagal pulmonary C-neurons are responsive to both CAP and PGE2, and if so, whether there is a functional interaction of TRPV1 and EP3 receptor in the neural excitation. In this study, both CAP- and PGE2-induced currents were observed in some vagal pulmonary C-neurons (34%). Different from our data, PGE2 (up to 10 µM) itself failed to evoke any response in afferent sensory neurons innervating rat intestinal wall, but enhanced the serotonin (5- HT)-evoked currents . This discrepancy may be due to the different sensory neurons tested (gastrointestinal vs. pulmonary) and lower concentration of PGE2 used (10 vs. 20 µM) compared to our study. We also found that CAP-induced currents were suppressed by 74% after JNJ 17203212 and unchanged by L-798106, while PGE2-induced currents were inhibited by 67% and 47% after JNJ 17203212 and L-798106 respectively (Fig. 5C and D). These cellular data are highly similar to our cough data showing suppression of CAP-induced cough by 58% after inhaling JNJ 17203212 and PGE2-induced cough by 75% and 50% after inhalation JNJ 17203212 and L-798106 respectively (Fig. 2). The similarity of the effects on cough (via inhaling the antagonists mainly acting on the airways) and vagal pulmonary C-neurons (via focal ejecting the same antagonists) forms a new conception (i.e., the interaction of TRPV1 and EP3 receptor of vagal pulmonary C-neurons contributes, at least in part, to their interaction in cough). It is well known that inhalation of CA/CAP provokes individual loud coughs (Type I), while inhalation of PGE2 induces bout(s) of smaller and quieter coughs (Type II) in humans and unanesthetized guinea pigs [14, 15, 37, 43, 44, 45, 46, 48]. However, the mechanisms underlying the genesis of the two distinct cough patterns remain unexplored. Our morphological and electrophysiological findings mentioned above raise the possibility that the two groups of vagal pulmonary C-neurons expressing TRPV1 alone and TRPV1 + EP3 receptor may be accountable for the different cough patterns generated. Further studies are needed to define whether different loops of second-order neurons in the NTS and/or synaptic neurotransmissions are also involved in the genesis of the distinct cough patterns in response to CA/CAP and PGE2.
Although the mechanisms underlying PGE2-induced currents via acting EP3 receptor are not clear, we reason that PGE2 evokes currents via activating the ion channels including TRPV1. PGE2 receptors comprise of four subtypes (EP1- EP4), among which EP3 receptors uniquely couple to Gi protein [59, 60]. Activation of EP3 receptors is reported to decrease the intracellular cAMP concentration and increase the intracellular calcium concentration , which are capable of potentiating the activity of TRPV1 . Actually, the importance of the opening of TRPV1 channel in generation of PGE2-currents is evident in the present study. Our data demonstrated that PGE2-induced currents were largely blunted by blockade or desensitization of TRPV1, strongly suggesting that TRPV1 is a critical component responsible for generating PGE2-currents. Because blockade of TRPV1 by JNJ 17203212 largely reduces, but does not eliminate, PGE2-induced currents, other ion channels may also be involved in generation of the currents. The lack of effects of L-826266 on CAP-induced currents in our study in vitro suggests that the activation of EP3 receptors is not required for CAP-induced currents, consistent with absence of the effect of L-826266 on CAP-induced cough in vivo.
Because both TRPV1 and EP3 receptor exist in the same vagal pulmonary C-neuron, we further tested whether CAP- or PGE2-induced currents would be facilitated by pre-ejecting PGE2 or CAP 30 s ahead. Our results showed that the amplitudes of CAP-induced currents in vagal pulmonary C-neurons were doubled by PGE2 pre-ejection and this augmentation was abolished after blockade of EP3 receptor (Fig. 6A), consistent with PGE2 enhancing the sensitivity of CAP-induced cough . PGE2 is reported to be able to enhance CAP-induced apneic response in rats via acting on EP2 receptor of PCFs [62, 63, 64]. Thus, it is possible that the apneic response to PGE2 is mediated by EP2 receptor, while the cough response is mediated by EP3 receptor. Our results also showed that PGE2-induced currents were inhibited by ~ 33% after CAP pretreatment. A previous report has shown that incubation of both DRG neurons and TRPV1-expressing HEK293 cells with 0.1 µM capsaicin for 20 min induces a significant TRPV1 desensitization . In agreement, we found a remarkable desensitization of vagal pulmonary C-neurons in response to the second ejection of CAP 30 s after the first one (Fig. 7). Because of the dependency of PGE2-induced currents on activation of TRPV1, the desensitized TRPV1 30 s after CAP pretreatment is accountable for the reduction of the subsequent PGE2-induced currents. In the present study, CAP pre-ejection alone blunted PGE2-induced currents by 33%, while that coupled with blockade of TRPV1 attenuated these currents by 69%. The combination of TRPV1 desensitization (pre-ejection of CAP) and blockade (pretreatment with JNJ 17203212) in the latter, different from TRPV1 desensitization alone in the former, may explain the greater attenuation of PGE2-induced currents caused by the latter. No attempt was made in this study to determine whether CAP pretreatment would suppress PGE2-induced cough because CAP/CA at the threshold concentration evokes significant bronchoconstriction and mucosal secretion [66, 67] that could confound the subsequent cough response to inhalation of PGE2.
Significance of our results is evident. Elevation of PGE2 levels (up to 10-fold) in the airways has been observed in a variety of diseases, for instance chronic obstructive pulmonary disease, cough variant asthma, idiopathic cough and cough associated with post-nasal drip, gastrooesophageal reflux disease, and eosinophilic bronchitis [68, 69, 70, 71, 72]. Interestingly, these patients often have hypoxemia and pulmonary inflammation that could lead to certain lipoxygenase products and lactic acid, etc. in the airways to stimulate TRPV1 of PCFs [20, 73, 74, 75, 76]. In fact, the co-presence of elevated pulmonary PGE2 and TRPV1stimulants has been believed to responsible for coughs in patients with asthma, idiopathic pulmonary fibrosis, idiopathic chronic cough and COPD ( and for review, see ). Our results show that inhalation of aerosolized TRPV1 or EP3 receptor antagonist sufficiently suppresses PGE2-evoked cough and that ¼ vagal pulmonary C-neurons co-express TRPV1 and EP3 receptor with the interaction of the two receptors in the neural excitability similarly to the cough. These results suggest that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the interaction of the two receptors in the cough response to PGE2. Therefore, inhalation of aerosolized TRPV1 and EP3 receptor antagonists capable of targeting vagal pulmonary sensory fibers may be an effective antitussive therapy in these patients. Further studies are required to determine which other ion channels, in addition to TRPV1, are also involved in forming PGE2-induced currents and what the cellular/molecular mechanisms are for the interaction of TRPV1 and EP3 receptor.