Capsaicin and the respiratory system.
The pungent ingredient in red pepper fruits of the genus Capsicum, which includes paprika, jalapen˜ o, and cayenne, is called capsaicin. Capsaicin is known to stimulate sensory nerves leading to the activation of nociceptive and protective reflex responses (e.g., cough, bronchospasm) and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion.
The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; TRPV1). It has previously been suggested that there is an upregulation of TRPV1 expression in inflammatory diseases and that inappropriate activation of this receptor may lead to sensory nerve hyperresponsiveness. Thus, it would appear that airway inflammatory diseases (e.g., asthma and chronic obstructive pulmonary disease) may respond to treatment with an effective and selective inhibitor of TRPV1 and to this end much work is being carried out to develop novel inhibitors.
Sensory nerves in the airways regulate central and local reflex events such as bronchoconstriction, airway plasma leakage, and cough. Sensory nerve activity may be enhanced during inflammation such that these protective reflexes become exacerbated and deleterious. Sensory nerve reflexes are under the control of at least two different classes of sensory fiber, the myelinated rapidly adapting stretch receptors (RARs) and nonmyelinated capsaicin-sensitive C fibers. In the airways, activation of RARs and C fibers elicits cough, bronchoconstriction, and mucus secretion via an afferent central reflex pathway.
Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as bronchoconstriction, mucus secretion, plasma exudation, and vasodilatation via the peripheral release of neuropeptides, a phenomenon known as ‘neurogenic inflammation’. A characteristic feature of many nociceptive sensory fibers is their sensitivity to capsaicin. However, until recently the molecular mechanisms involved in activation of sensory nociceptive fibers were unknown.
Pharmacological evidence for the presence of a ‘capsaicin receptor’ in sensory nerves was provided by the discovery of two capsaicin analogs, resiniferatoxin (potent agonist) and capsazepine (an antagonist). First, specific binding sites for resiniferatoxin were demonstrated on dorsal root ganglion membranes and second, capsazepine has been found to inhibit numerous capsaicin-evoked neuronal responses including those in the airways. The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; transient receptor potential vanilloid 1 (TRPV1)).
Capsaicin and Functional Responses in the Airways
Inhalation challenge with capsaicin and low pH (e.g., citric acid) has been shown to evoke cough and has been used as a model for the last 50 years to investigate the action of potential antitussive therapies in clinical trials. In fact, agonists at TRPV1 such as capsaicin and resiniferatoxin are the most potent stimulants of the cough reflex so far described in man. Therefore, it has been suggested that TRPV1 activation may be one of the primary sensory mechanisms in cough. However, if activation of TRPV1 is an important initiating factor for the cough reflex then an endogenous capsaicin-like ligand must be present.
A number of putative endogenous ligands that are known to activate TRPV1 have been demonstrated to cause cough. TRPV1 has been shown to be sensitive to a fall in the extracellular pH, and Hþ ions not only stimulate the receptor directly but also increase the sensitivity of the receptor to capsaicin and low pH solutions, which are known to elicit cough in animals and humans. Interestingly, the TRPV1 antagonists capsazepine and iodo-resiniferatoxin have been shown to inhibit capsaicin, citric acid, and anandamide-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, that is, activation of TRPV1 (Figure 1). However, it is unlikely that TRPV1 activation is the only stimulus for cough since some tussigenic agents such as hypertonic saline are not inhibited by TRPV1 antagonism.
Capsaicin activates sensory nerves leading to the activation of nociceptive and protective reflex responses and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion.
Early experiments using the relatively weak TRPV1 antagonist capsazepine provided pharmacological validation that capsaicin-induced bronchospasm in guinea pig bronchi did indeed involve the activation of TRPV1. Contractile responses to resiniferatoxin and capsaicin were unaffected by the neurokinin (NK)-1 antagonist CP 96345, partially inhibited by the NK-2 antagonist SR 48968, but nearly abolished by a combination of the antagonists. These data suggest that resiniferatoxin and capsaicin both release tachykinins that act on both NK-1 and NK-2 receptor subtypes in a TRPV1-dependent manner. More recently, a more potent and selective agent has been used to confirm these observations.
Interestingly, contractile and relaxant responses to capsaicin and resiniferatoxin have also been examined in human isolated bronchus (5–12mm outside diameter). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The capsaicin-induced contractions were mimicked by resiniferatoxin and inhibited by capsazepine. The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968, whereas responses to concentrations of NKA, NKB, substance P, neuropeptide g, and neuropeptide K, which produced contractions of a similar size, were almost abolished by SR 48968. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone in a TRPV1-dependent manner, but this response does not appear to involve substance P or related neurokinins.
Airway hyperresponsiveness (AHR) to bronchoconstrictor agents is recognized as a critical feature of bronchial asthma and sensory nerves in the airway are strongly implicated in the hyperresponsiveness. Several studies have demonstrated that pretreatment with capsaicin (which depletes sensory neuropeptides) significantly inhibited the late bronchial response that was observed after ovalbumin inhalation, AHR, and eosinophil accumulation in an allergic guinea pig model, and AHR to histamine in a rabbit model. It remains to be seen whether TRPV1 antagonists are effective in this regard.
Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as plasma exudation and vasodilatation via the peripheral release of neuropeptides. Plasma extravasation has been shown to be induced in rats or guinea pigs by intravenous injections of substance P (SP) and capsaicin. The effect of intravenous capsaicin was absent in capsaicin-desensitized animals and in those pretreated with capsazepine. Capsaicin-induced plasma extravasation was also markedly inhibited by CP 96345, a nonpeptide antagonist of tachykinin NK-1 receptors. These data suggest that capsaicin-induced plasma leakage is mediated by the release of neuropeptides and the activation of NK-1 receptors via a TRPV1- dependent mechanism.
When stimulated capsaicin-sensitive C fibers (afferents) containing the neuropeptides SP, NKA, and calcitonin gene-related peptide have also been shown to evoke neurogenic secretion from airway mucussecreting cells. However, although capsaicin has been shown to elicit mucus secretion, there is no data available describing the effect of TRPV1 antagonists on this response.
Molecular Mechanism of Action of Capsaicin
The Type 1 Vanilloid Receptor
TRPV1 is a membrane-associated vanilloid receptor. It is a nociceptor-specific ligand-gated ion channel expressed on the neuronal plasma membrane of nociceptive C fibers and is required for the activation of sensory nerves by vanilloids such as capsaicin, the pungent extract from plants in the genus Capsicum (e.g., hot chilli peppers). TRPV1 also mediates the response to painful heat, extracellular acidosis, protons, and tissue injury. TRPV1 is an outwardly rectifying cation-selective ion channel with a preference for calcium (PCa/PNaB10) and magnesium (PMg/PNaB5).
TRPV1 is activated by heat (4431C), but is effectively dormant at normal body temperature and low pH (o5.9) and may act as an integrator of chemical and physical pain-eliciting stimuli. When activated, TRPV1 produces depolarization through the influx of Naþ, but the high Ca2þ permeability of the channel is also important for mediating the response to pain. Gating by heat is direct but the receptor can be opened by ligands or stimuli such as mild acidosis, which also reduces the threshold for temperature activation and potentiates the response to capsaicin. To summarize, TRPV1 mediates nociception and contributes to the detection and integration of diverse chemical and thermal stimuli.
The expected role for TRPV1 is in pain pathways and recent data from a study with TRPV1 knockouts showed impaired inflammatory thermal hyperalgesia. This has led to a growing interest in developing small molecule antagonists for this target. Recent preclinical data demonstrating efficacy in rodent models of both thermal and mechanical neuropathic or inflammatory pain has fuelled this enthusiasm. The established role of sensory nerve activation in the cough reflex and the role of TRPV1 in inflammatory pain has also alerted the respiratory community to the therapeutic potential of TRPV1 antagonists as antitussives and as therapy for airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD).
Ligands for TRPV1
Exogenous agonists of TRPV1 include capsaicin and resiniferatoxin. Endogenous agonists include the cannabinoid receptor agonist anandamide, N-arachidonoyl- dopamine, inflammatory mediators (bradykinin, 5-HT, and prostaglandin E2 (PGE2)), hydrogen ions, heat, arachidonic acid, lipoxin A4, prostacyclin, ethanol, and several eicosanoid products of lipoxygenases including 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-(S)-hydroxyeicosatetraenoic acid and leukotriene B4. Interestingly, the effect of bradykinin may be indirect given that data exists suggesting that it appears to activate bradykinin B2 receptors on afferent neurons leading to the generation of lipoxygenase metabolites that have agonist activity at TRPV1.
The evidence to support the suggestion that TRPV1 can be gated not only by vanilloids such as capsaicin, but also by protons, heat, agonists at certain G-protein-coupled receptors, ethanol, cannabinoids, and lipoxygenase products of arachidonic acid has come from electrophysiological patch-clamp recording studies on the cell bodies of TRPV1-expressing cells. Furthermore, pharmacological antagonism of TRPV1 has been shown to inhibit action potential discharge evoked by each of these stimuli. However, due to the nonselective effects of several of these tool compounds, this pharmacological approach has its limitations.
Furthermore, if the antagonist inhibits but does not completely block a given response, it is not clear whether activation of the TRPV1 is obligatory for the response or merely contributes to the end response. In studies using TRPV1–/– mice it was concluded that whereas TRPV1 is required for action potential discharge of C fiber terminals evoked by capsaicin and anandamide, it plays a contributory role in responses evoked by low pH of bradykinin. These data suggest that TRPV1 is one of multiple ion channels responsible for the bradykinin-evoked generator potentials (i.e., membrane depolarization) in C fiber terminals.
TRPV1 Receptor Antagonists
Antagonists of TRPV1 include ruthenium red, a dye that exhibits properties of noncompetitive antagonism for the TRPV1, and has a poorly defined mechanism of action and limited selectivity. Capsazepine is another agent characterized as a weak but relatively selective, competitive TRPV1 receptor antagonist (over other TRPVs). However, at the concentrations required to antagonize the TRPV1 (approximately 10 mM), capsazepine has demonstrated non-specific effects such as inhibition of voltage-gated calcium channels and nicotinic receptors. Furthermore, this ligand has poor metabolic and pharmacokinetic properties in rodents, where it undergoes extensive firstpass metabolism when given orally.
Recently, a number of antagonists with improved potency and/or selectivity have been described and these include antagonists that are structurally related to agonists such as iodo-resiniferatoxin, which is 100-fold more potent than capsazepine. Previous evidence has indicated that iodo-resiniferatoxin is a potent antagonist at the TRPV1 both in vitro and in vivo. Since then several studies have used this antagonist as a pharmacological tool in order to explore the role for TRPV1 in various physiological settings. Recently, however, although the in vitro antagonistic activity of iodo-resiniferatoxin has been confirmed, in vivo studies have demonstrated some agonist activity of this agent at high doses. Therefore, there is a possibility that this agent retains some agonistic activity and that an agonist-dependent desensitization effect on sensory nerves may well contribute to some of the antagonistic activity observed.
The GlaxoSmithKline lead antagonist SB-366791, which has a good selectivity profile in a series of assays, and potent vanilloid agonist AM-404 should provide useful tools for probing the physiology and pharmacology of TRPV1.
Several TRPV1 receptor antagonists that share no obvious structural resemblance to any class of vanilloid agonist have also been developed. These compounds have been discovered by high-throughput random screening projects. At present this group of antagonists includes: (1) N-(haloanilino)carbonyl-N-alkyl-N-arylethylendiamines discovered at SKB; (2) N-diphenyl-Nnapthylureas discovered at Bayer; and (3) pyridol [2,3-d]-pyrimidin-4-ones discovered at Novartis.
Localization of TRPV1
The capsaicin-sensitive vanilloid receptor is expressed mainly in sensory nerves including those emanating from the dorsal root ganglia and afferent fibers that innervate the airway, which originate from the vagal ganglia. In the dorsal root and trigeminal ganglion, TRPV1 is localized to small and mediumsized neurons. Somatic sensory neurons of the vagus nerve are located in the jugular ganglion, whereas visceral sensory neurons of the nerve are located in the nodose ganglion. Previous studies have demonstrated that TRPV1-containing neurons are abundant in these ganglia.
The traditional view that TRPV1 is simply a marker of primary sensory nerves is now being challenged. The TRPV1 receptor-has been detected in guinea pig and human airways by receptor-binding assays and has been identified in nonneuronal cell types such as mast cells, fibroblasts, and smooth muscle.
Role of TRPV1 in Airway Inflammatory Disease
A role for TRPV1 in airway inflammatory disease would depend on the presence of endogenous activators of this channel under physiological and pathophysiological conditions. In fact, it is quite possible that this situation does, in fact, exist in the ‘disease’ scenario given that TRPV1 activation can be initiated and responses to other activators potentiated in an acidic environment, which has been shown to be present in diseases such as asthma and COPD. Interestingly, it has previously been established that the airway response to TRPV1 activation is enhanced in certain disease conditions. In particular, it has been reported that the cough response elicited in response to capsaicin is exaggerated in diseases such as asthma and COPD.
Anandamide is also an endogenous ligand known to be produced in central neurons. However, more recent studies have suggested that anandamide is synthesized in lung tissue as well as a result of calcium stimulation suggesting that this mediator could also be involved in the activation of TRPV1 under normal and disease conditions. Inflammatory agents (e.g., bradykinin, ATP, PGE2, and nerve growth factor (NGF)) can indirectly sensitize TRPV1 to cause hyperalgesia. Thus, bradykinin and NGF, which activate phospholipase C (PLC), release TRPV1 from phosphatidylinositol 4,5-biphosphate (PIP2)-mediated inhibition. However, PLC also regulates TRPV1 by diacylglycerol (DAG) formation and subsequent activation of protein kinase C (PKC), which in turn phosphorylates and sensitizes TRPV1. Inflammatory stimuli, including prostaglandins and NGF among others, have also been shown to upregulate the expression and function of TRPV1 via the activation of p38 mitogen-activated protein-kinase (MAPK)- and protein kinase A (PKA)-dependent pathways.
Previous data has suggested that there is an upregulation of TRPV1 in inflammatory diseases. For example, TRPV1 immunoreactivity is greatly increased in colonic nerve fibers of patients with active inflammatory bowel disease. Furthermore, a recent study has found an increase in TRPV1 expression in sensory nerves found in the airway epithelial layer in biopsy specimens from patients with chronic cough compared to noncoughing, healthy volunteers. There was a significant correlation between the tussive response to capsaicin and the number of TRPV1- positive nerves in the patients with cough. Therefore, it has been postulated that TRPV1 expression may be one of the determinants of the enhanced cough reflex found in patients with chronic cough and that the recently described TRPV1 antagonists could be effective in the treatment of chronic persistent cough due to diverse causes.
Thus, it would appear that airway inflammatory diseases (e.g., asthma and COPD) may respond to treatment with an effective and selective inhibitor of the ‘capsaicin receptor’ TRPV1 and to this end much work is being carried out to develop novel inhibitors. Interestingly, capsaicin-sensitive nerve stimulation in subjects with active allergic rhinitis produces reproducible and dose-dependent leukocyte influx, albumin leakage, and glandular secretion. These results provide in vivo evidence for the occurrence of neurogenic inflammation in the human upper airway with active allergic disease and may therefore suggest the therapeutic utility of TRPV1 antagonists in the management of this disease. In addition, the treatment of persistent cough is a facet of airway diseases that is sorely in need of effective treatment and a TRPV1 inhibitor may prove extremely effective against cough induced by gastroesophageal acid reflux, for example, as well as that associated with asthma and other diseases of the airways described above.
Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154.
Belvisi MG (2003) Airway sensory innervation as a target for novel therapies: an outdated concept? Current Opinion in Pharmacology 3: 239–243.
Belvisi MG (2003) Sensory nerves and airway inflammation: role of A delta and C-fibres. Pulmonary Pharmacology and Therapeutics 16: 1–7.
Belvisi MG and Geppetti P (2004) Cough * 7: current and future drugs for the treatment of chronic cough. Thorax 59: 438–440.
Caterina MJ and Julius D (2001) The vanilloid receptor: a molecular gateway to the pain pathway. Annual Review of Neuroscience 24: 487–517.
Coleridge HM and Coleridge JC (1994) Pulmonary reflexes: neural mechanisms of pulmonary defence. Annual Review of Physiology 56: 69–91.
Holzer P (1991) Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacological Reviews 43: 143–201.
Hwang SW and Oh U (2002) Hot channels in airways: pharmacology of the vanilloid receptor. Current Opinion in Pharmacology 2: 235–242.
Karlsson JA (1993) A role for capsaicin sensitive, tachykinin containing nerves in chronic coughing and sneezing but not in asthma: a hypothesis. Thorax 48: 396–400.
Maggi CA and Meli A (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. General Pharmacology 19: 1–43.
Morice AH and Geppetti P (2004) Cough 5: the type 1 vanilloid receptor: a sensory receptor for cough. Thorax 59: 257–258.
Rogers DF (2002) Pharmacological regulation of the neuronal control of airway mucus secretion. Current Opinion in Pharmacology 2: 249–255.
Szallasi A and Blumberg PM (1999) Vanilloid (Capsaicin) receptors and mechanisms. Pharmacological Reviews 51: 159–212.
Szolcsanyi J (2004) Forty years in capsaicin research for sensory pharmacology and physiology. Neuropeptides 38(6): 377–384.