OC triggers an unusual irritation in the pharynx when ingested. Unlike most known chemical irritants, OC does not significantly irritate the oral cavity; instead, the sting is restricted to the upper airways and is often accompanied by throat clearing and coughing. Our goal in this work was to understand the molecular and physiological basis for this unique pungency. We show here that OC, as well as ibuprofen, another compound whose irritancy is primarily restricted to the throat, activate the ion channel hTRPA1 ex vivo, and their ability to excite the trigeminal nervous system depends upon functional TRPA1 in sensory neurons. Our perceptual studies in humans show that OC triggers irritation in the throat and nasal cavities with high potency compared to the anterior tongue. Consistent with the hypothesis that the OC sensory properties in vivo are the result of TRPA1 activation, immunohistochemical imaging of human tissues with hTRPA1-specific antibodies revealed poor reactivity in neural fibers of the anterior tongue relative to the reactivity in neural fibers of the sensitive pharyngeal and nasal epithelia.
Previous perceptual studies have indicated that the oral mucosae along the rostrocaudal axis are not uniformly sensitive to chemical irritants (Rentmeister-Bryant and Green, 1997
). The problem is that in vitro
neural studies of primary sensory neurons derive largely from analysis of complete neuronal populations prepared from whole sensory ganglia such as the trigeminal ganglion. Thus, different functional properties of primary trigeminal afferents from the different sub-regions of the face remain largely uninvestigated. Using a viral tracing technique to identify nasal and cutaneous cultured mouse trigeminal neurons, Damann et al. observed a larger fraction of nasal trigeminal neurons exhibiting sensitivity for menthol and capsaicin. This indicates that neurons expressing TRPM8 and TRPV1 are not equally distributed among trigeminal fibers innervating different regions of the mouse head (Damann et al., 2006
). In a comparable fashion, we showed heterogeneity of TRPA1 channel expression in human trigeminal afferents with poor expression of TRPA1 in the mandibular branch. Previous immunohistochemical studies have shown the presence of TRPA1 channel in nerve bundles of mouse tongues ((Nagatomo and Kubo, 2008
), in the human lingual nerve ((Morgan et al., 2009
). TRPA1 mRNAs have also been identified in the mandibular branch of the trigeminal nerve in mouse ((Kobayashi et al., 2005
). To our knowledge, however, the expression of TRPA1 channels on neuronal fibers innervating the human anterior tongue have not been reported.
As demonstrated, the concentration differences among compounds are unlikely to account for the pattern of OC irritation. Lipophilicity, and hence access to nerve fibers, also does not account for the differences in anterior tongue sensations, as AITC and OC have comparable partition coefficients. Moreover, OC is similarly effective in water and in oil. That irritation from other TRPA1 agonists is not restricted to the throat is consistent with a higher specificity of OC to the hTRPA1 channel we assessed. We demonstrated that OC does not bind to the ‘traditional’ cysteine residues of TRPA1 that are required for cinnamaldehyde and AITC activation of TRPA1. In addition, we showed that OC differs from these other agonists in that OC requires both aldehyde groups to activate TRPA1. The selectivity for the TRPA1 channel highly expressed in nasal and pharyngeal tissues is thus likely explained by differences in how OC interacts with the channel. Thus, the high specificity of OC for this receptor and the restricted expression pattern of the receptor underlie the unusual pungency of extra-virgin olive oil.
Our explanation for these observations is consistent with other data on receptor specificity. Many compounds presumed to be specific to a given TRP channel have subsequently been shown to activate other TRP channels involved in irritation transduction (Macpherson et al., 2006
). For example, allicin can activate TRPV1 in addition to TRPA1 (Macpherson et al., 2005
; Salazar et al., 2008
), and cinnamaldehyde, carvacrol and thymol are also TRPV3 agonists (Macpherson et al., 2006
; Xu et al., 2006
; Lee et al., 2008
). In several studies, AITC-activated sensory neurons have been shown to be more numerous than those stimulated by other TRPA1 agonists such as zinc (Hu et al., 2009
) or cinnamaldehyde (Bandell et al., 2004
) and AITC specificity toward TRPA1 has been previously questioned (Bandell et al., 2004
; Kwan et al., 2006
). AITC has also been reported to activate porcine pTRPV1 at concentrations (mM) found in food products (Ohta et al., 2007
). Therefore, it is possible that sensory receptor(s) other than the hTRPA1 protein we assessed, possibly a splice variant of TRPA1, contribute to the anterior oral pungency evoked by AITC and other canonical TRPA1 ligands in humans.
What is the functional or ecological significance of the pungency to the human upper airways? One explanation is that the posterior location of toxin and irritant detectors can protect against their intake either by inhalation or ingestion. Many bitter tasting toxins are perceived more strongly in the posterior oral cavity than the anterior (Danilova and Hellekant, 2003
). For instance, the bitter iso-α-acids from hop cone flowers found in beers stimulate taste receptors almost exclusively in the pharynx. Detection in the posterior mouth guards against intake of toxins and irritants at the last possible checkpoint. Many air pollutants such as acrolein are TRPA1 agonists (Bautista et al., 2006
) and will elicit cough. Like TRPV1, TRPA1 has been shown to be expressed both in the upper and the lower airways (Fajardo et al., 2008
; Nassenstein et al., 2008
). Thus, TRPA1 is well positioned to protect the lungs by triggering defensive cough responses to reactive agents (Bessac and Jordt, 2008
; Taylor-Clark et al., 2008
But if the role of these ion channels is to protect tissue from harmful compounds, then it is a mystery how the TRPA1-mediated throat irritation of extra virgin olive oils came to be valued as a positive sensory attribute by those who consume them. Indeed, this pungency is an important quality that distinguishes particularly good olive oils in the European Union standards. Similarly, other common food irritants (e.g. capsaicin, menthol, AITC and so forth) are also important positive components in many cuisines, so this is a very general question. Pungency is believed to signal potentially harmful compounds in our food but consumption of many compounds eliciting this sensation is also linked to decreased risks of cancer and degenerative and cardiovascular diseases (Boyd et al., 2006
; Peng and Li, 2010
). Oleocanthal has been shown to be a potent anti-inflammatory agent (Beauchamp et al., 2005
), implicating potential medicinal value to this compound. It is, therefore, perhaps no coincidence that the only other known restricted throat irritants are NSAID molecules such as ibuprofen. At least in the case of extra-virgin olive oils, we suggest that by a process not yet well understood people have come, perhaps unconciously, to transform an inherently unpleasant sensation into a positive one because it has beneficial health effects (Peyrot des Gachons et al., 2009
). This broad speculation requires considerably more investigation before it can be demonstrated.