In this study we show, using a conscious guinea-pig model of cough, that centrally administered BK sensitizes the cough reflex via B2receptors. We also show that the central BK-induced sensitization occurs via activation of both TRPV1 and TRPA1 channels through metabolites of COX and 12-LOX enzymes. Furthermore, our data show that combined blockade of both TRPV1 and TRPA1 channels results in greater degree of inhibition of both cough and airway obstruction.
BK is an important inflammatory mediator that has been shown to be involved in cough mechanisms. Administration of BK to the airways of animals and humans results in both induction and sensitization of cough [28, 36, 39, 50, 93]. Whether BK can result in the sensitization of the cough reflex, in conscious animal models, via a central mechanism has not been addressed. Our findings show that acute exposure of BK, i.c.v., significantly increased citric acid-induced cough in a dose dependent manner, within a short period of time following BK infusion. This adds further support to the findings showing that treatment with lisinopril up-regulates the cough reflex via a BK-dependent mechanism in an anesthetized cough model [24]. Our observations with BK are also similar to the cough sensitizing actions of centrally administered NGF previously reported [31]. Similarly, substance P (SP) microinjected into the nTS has been shown to up-regulate the cough reflex. These findings suggest that cough can be centrally enhanced and that central regions, particularly the nTS, represents key sites for sensitization of cough [24, 81].
Our data is also in keeping with the central effects of BK in hyperalgesia. For example, it has been shown that central administration of BK, at low doses, results in hyperalgesia 15 min later [14]. Mice lacking BK receptors also have a 70% reduction in acute acetic acid-induced nociception [20].
In addition to effects on cough, our findings also show that BK administration results in an enhancement of the citric acid-induced airway obstruction. The exact mechanism by which this may occur is not clear. However, it is possible that BK stimulates a specific set of second order neurons in the nTS which in turn activate the airway-related vagal preganglionic neuron (AVPNs). The AVPNs are the final common pathway from brain stem/CNS to the airways and transmit signals to the tracheobronchial ganglia which lie in close proximity to effector systems such as blood vessels, submucosal glands and airway smooth muscle. AVPNs are indeed central integrators of several excitatory (glutamatergic, tachykinergic) and inhibitory (GABAergic, serotonergic and noradrenergic pathways) inputs which regulate cholinergic outflow to the airways [42,43,44,45,46,47,48,49]. One possible mechanism by which BK can affect airway tone is via modulation of central SP release. Indeed, SP injected into the fourth ventricle has been shown to remarkably increase tracheal cholinergic tone [70]. Of relevance also is that i.c.v. administration of a non-selective neurokinin antagonist prevents the BK-induced potentiation of histamine-mediated increased airway cholinergic tone [70].
Our data also show that the BK effects on cough and airway obstruction are mediated mainly via B2 receptors since pretreatment with the selective B2 receptor blocker, HOE-140, significantly inhibited the BK enhanced tussive effects by approximately 80%. This is generally in agreement with studies, in both cough and pain, showing that the B2 receptor, a constitutively expressed receptor, mediates BK-induced tussive and nociceptive effects in normal animals, respectively [23, 24, 36, 82]. Interestingly, a study has reported a role for B1receptor in enalapril-induced cough. However, in this study, the drug treatment was done over 20–30 days which may likely explain the difference between their findings and those of ours and other groups [51]. Similar to the cough effects, pretreatment with HOE-140 also significantly blocked BK-enhancement of airway obstruction indicating that this response was also mediated via the B2 receptor.
Our next question was whether TRP channels, specifically TRPV1 and TRPA1, were involved in the BK sensitization of cough and airway obstruction. The reason for investigating the link between BK and the TRP channels is twofold. Firstly, several studies have shown that both TRPV1 and TRPA1 channels are involved in the transducing mechanisms of cough inducing stimuli [3, 7, 11, 13, 69, 74, 75, 87]. Secondly, TRPV1 and TRPA1 channels have been shown to be critical in mediating BK-induced cough and hyperalgesia [5, 11, 18, 39]. Our data show that pretreatment with the selective and potent TRPV1 antagonist, JNJ-17203212 [10], dose dependently inhibited the BK-induced sensitization of the cough reflex. Similarly, pretreatment with HC-030031, a selective and potent TRPA1 antagonist [27], also inhibited the BK-induced sensitization of cough response in a dose dependent manner. These findings are in line with data showing a role for both TRPV1 and TRPA1 in inhaled BK-induced cough [39].
An important role for TRPV1-dependent sensitization of the cough reflex has also been previously demonstrated for NGF, in this same model, in both peripheral and central sensitization [31]. Together, these data suggest that TRPV1, and also TRPA1, channels may act as a signaling hub for stimuli that sensitize the cough reflex. This assertion is in line with the important role that both these channels play in centrally mediated hyperalgesia [2, 18, 94].
In addition to the effects on cough, our data show that blockade of both the TRPV1 and TRPA1 channels (by JNJ-17203212 and HC-030031, respectively) resulted in a significant inhibition of the BK-induced enhancement of airway obstruction by 77 and 80%, respectively. The degree of inhibition, through blockade of both these channels, was similar thus indicating that both channels are equally important in this response. The link between TRPV1 and TRPA1 activation and increased airway obstruction is not clear, However, multimodal activation of TRPV1 channels trigger increased spontaneous glutamate release within the nTS [26, 56, 78]. Furthermore, TRPA1 channels agonists have also been reported to increase CNS neuronal excitability, For example, in the spinal dorsal horn, TRPA1 agonist not only increases the frequency but also amplifies the excitatory postsynaptic currents (EPSCs) [60]. Therefore, such action, in the nTS, may result in increased activation of AVPNs and increased airway obstruction.
Based on the fact that BK-enhanced cough and airway obstruction were dependent on both TRPV1 and TRPA1 channels activation, in our next experiments we assessed whether combined treatment with sub-maximal doses of the TPRV1 and TRPA1 antagonists would achieve greater inhibitory effects [35]. Our findings show that, in contrast to the sub-maximal dose of each drug administered alone, where the degree of inhibition of cough was 26 and 24% for JNJ-17203212 and HC-030031, respectively, combined pretreatment with both antagonists significantly reduced the BK sensitization of cough by 83%. This finding is in-line with data from a previous study showing that the combined blockade of TRPV1 and TRPA1 produced a significant degree of cough inhibition compared to each individual drug [39]. This confirms that both TRPV1 and TRPA1 channels are involved in the BK-induced sensitization of the cough reflex. Similarly, our finding shows that the combination of JNJ-17203212 and HC-030031 significantly blocked BK-enhanced airway obstruction compared to each drug administered alone. Of interest, several studies have reported that both TRPV1 and TRPA1 channels are co-expressed on sensory and dorsal root ganglia (DRG) neurons [38, 59, 75, 76]. It has also been reported that TRPA1 channels can be activated via calcium inflow through TRPV1 channels suggesting that calcium influx from one channel can result in activation of the other channel [19, 54, 61, 68, 95], thus implying that these two channel are closely linked, both physically and functionally. In view of the fact that current clinical studies with TRPV1 antagonists have failed to show any significant effects [6, 58], it is tempting to speculate, based on our findings and that of others, that both TPRA1 and TPRV1 channels may need to be blocked in order to see significant inhibitory effects on cough.
Our next question was whether the coupling of B2 receptors to TRPV1 and TRPA1 activation was mediated by the metabolites of COX, 12-LOX and/or 15-LOX-1 enzyme. Several studies have shown that BK activation of B2 receptors results in prostanoid production, such as PGE2, by the COX enzyme [37, 92]. Furthermore, PGE2, which is one of the major prostanoids, seems to play an important role in BK-induced cough and pain [39, 77, 83]. Our data show that pretreatment with indomethacin blocked the BK sensitization of cough by 70% which suggests that COX metabolites are involved in the BK sensitization of cough. This finding is in agreement with studies showing that several metabolites of the COX enzymes such as PGD2 and PGE2 can induce cough in conscious animals via mainly DP1 and EP3 receptors, respectively [64,65,66]. Furthermore, in support of our findings, a double-blind, randomized, cross-over study showed that treatment with indomethacin, of patients who developed cough as a side effect of chronic captopril therapy, significantly inhibited their cough [34]. This suggests that metabolic products of the COX enzyme may couple the activation of B2 receptors to the sensitization of TRPV1 and TRPA1 channels. In support of this assertion, zaltoprofen, an NSAID, has been shown to inhibit the enhancement effects of BK on capsaicin-induced 45Ca2+ uptake in DRG neurons [86]. Also, PGE2 activation of isolated guinea pig sensory ganglia was partially inhibited by blockade of either TRPA1 or TRPV1, and completely inhibited in the presence of both blockers [39].
It is worthy to note that not all studies have been able to document a role for PGE2 in BK induced-cough. For example, meclofenamic acid pretreatment failed to attenuate the inhaled BK-evoked cough [50]. Whilst the reasons for this discrepancy are not known, a possible explanation is that some NSAIDs can actually activate TRP channels, such as TRPA1 [55]. For example, in HEK293 cells that express TRPA1 channels, extracellular application of several NSAIDs rapidly activate TRPA1 channels. Indeed, fenamates were the most potent NSAIDs in activating TRPA1 channels, an effect blocked by pretreatment with HC-030031 [55]. Interestingly, naproxen, the only NSAIDs not reported to activate TRPA1 channels, has been shown to improve viral-induced cough [1, 55, 84].
Similar to the effects on cough, pretreatment with indomethacin inhibited the BK-enhanced airway obstruction indicating that COX metabolites are involved not only in the BK-enhanced cough but airway obstruction as well. The mechanisms by which central COX metabolites result in increased airway obstruction is not known. However, PGE2 induced glutamate release was noted in several regions in the CNS including the nTS [9, 91]. Indeed, i.c.v. administration of PGE2 results in an increased c-Fos expression in several brain regions including the brainstem [72, 80]. This implies that increased PGE2 release can increase neuronal activity in the brain stem which may in turn affect the airway tone.
Our data show that central pretreatment with baicalein, an inhibitor of 12-LOX significantly reduced the BK-enhanced cough response by 74%. This implies that metabolites of 12-LOX activate TRP channels such as TRPV1 and TRPA1 which then result in sensitization of the cough reflex. In support of this, BK has been previously shown to activate TRPV1 receptors via a 12-HPETE-dependent mechanism. Indeed, it has also been reported that, in DRG neurons expressing B2 receptors, 12-lipoxygenase and TRPV1, activation of B2 receptors induced synthesis of 12-HPETE which was followed by TRPV1 channel opening [82]. In addition, 12-HPETE has been also shown to activate TRPA1 via hepoxilins A3 and B3 formation [40].
Our data also shows that pretreatment with baicalein significantly inhibited the BK-enhancement of airway obstruction. The mechanism by which products of central 12-LOX enhance airway obstruction is not known. However, there is evidence that, in the spinal cord, hydroxynonenal, a metabolic product of 12-LOX, activates TRPA1 via the release of SP. Therefore, it is possible that BK-induced SP release, centrally, can activate TRPA1 channels via a 12-LOX dependent pathway which may consequently enhance the airway tone [89].
Based on the fact that products of both COX and 12-LOX appear to be involved in BK sensitization of the cough reflex and airway obstruction, we asked whether combined treatment with sub-maximal doses of the COX and 12-LOX inhibitor, would achieve greater degree of inhibition than when each drug is given alone. Our data show that the combination treatment with indomethacin and baicalein reduced the BK enhanced cough response by 76% compared to 35 and 28%, respectively, with each drug administered alone. The findings confirm that metabolites of both COX and 12-LOX are involved in the coupling of B2 receptors to TRPV1 and TRPA1 activation. Similarly, the combined sub-maximal doses significantly blocked the BK-enhanced airway obstruction, again confirming that both COX and 12-LOX metabolites are involved in the BK sensitization of airway obstruction.
The role of 15-LOX-1 metabolites in sensitization of cough was also investigated. In our study, pretreatment with ML-351, a selective and highly potent 15-LOX-1(12/15-LOX) inhibitor [79], failed to inhibit either BK sensitization of cough or airway obstruction. This suggests that metabolites of 15-LOX-1 are not major contributors to the BK-induced central sensitization of either cough or airway obstruction. This lack of effect of ML-351 is unlikely to be dose related as the doses used in our experiment were based on doses previously shown to have clear effects [79]. Moreover, we were unable to use higher doses of ML-351 due to solubility limitation.
In summary, our data show that centrally administered BK activates B2 receptors which results in enhanced citric acid-induced cough and airway obstruction via the sensitization of TRPV1 and/or TRPA1 channels through metabolites of COX and/or 12-LOX (Fig. 10). Collectively, our findings point to an important role for BK, via B2 receptors, in the central sensitization of airway responses namely cough and airway obstruction and further identify TRPV1, TRPA1 and metabolites of COX and 12-LOX as key molecules in the sensitization process. An important finding in this study is that simultaneous blockade of TRPV1 and TRPA1 results in a synergistic inhibitory effect on cough and airway obstruction.