Sensory Detection and Responses to Toxic Gases

Olfactory and Gustatory Chemoreceptors

The major sensations induced by exogenous chemicals are smell, detected by olfactory neurons; taste, detected by the taste receptor cells in the taste buds; and pungency/irritation/pain that are mediated by peripheral sensory neurons (50). Most TIHs are associated with an odor and many elicit a taste sensation, such as the sour taste of acids and the bitter taste of cyanide. In addition to being an important aspect of consciously identifying TIHs, odors and tastes may have a role in initiating peripheral physiological responses. This is probably a more important mechanism in animals with more sophisticated olfactory systems than humans, such as rodents and certain insects (51). The odors of the volatile organic sulfur compounds and some other TIHs are innately repulsive and nauseating (52). However, this repulsive sensation can desensitize with continued exposure, and toxic exposures have occurred without victims becoming aware of the presence of the sulfide compound (53). Certain TIHs have an agreeable smell and elicit no acute noxious sensations, so their avoidance behavior has to be learned. For example, the faint and agreeable odor of certain nerve gases is the only physiological mechanism to recognize them until the hypercholinergic effects occur (54). Carbon monoxide, some simple asphyxiates, and the chemical weapon quinuclidinyl benzilate (BZ) are odorless and can go undetected until victims show effects from their toxicity (2).

The chemoreceptors for odors are specific ligand-gated G protein–coupled receptors (GPCRs) on olfactory neurons that stimulate adenylate cyclase to synthesize cAMP via a G protein called G_olf. Insects also express ion channel odorant receptors (55). The receptor for bitter-tasting chemicals is the GPCR TAS2Rn, which is coupled with the G protein gustducin. Interestingly, these receptors are also expressed in the motile cilia of the airway epithelium. The α-subunit of gustducin facilitates the activity of cAMP phosphodiesterase by binding inhibitory subunits and thereby decreasing intracellular cAMP levels, while the β-subunit activates PLC β2 and subsequently increases intracellular Ca²⁺. The rise in Ca²⁺ gates the monovalent TRP ion channel TRPM5. This produces a bitter taste sensation mediated by the taste buds and increases the activity of cilia to expel mucous-bound-toxin up and out of the lung (56–58).

The acidity-invoked sour sensation is not completely understood at the receptor level. It is likely due to H⁺ and H₃O⁺ cations activating specific ion channels. Different acid receptor proteins have been proposed, including members of the ASICs (a sub-family of the amiloride-sensitive Na⁺ channels), hyperpolarization-activated cyclic nucleotide–gated K⁺ channels (HCNs), 2-pore domain K⁺ leak conductance channels, and the TRP ion channel TRPP3 and its partner PKD1L3 (59). It is possible that ASIC channels are directly activated by low pH, while TRPP3 and PKD1L3 are primed by acidity, and subsequently activated by return to physiological pH (60, 61).

Sensory Neuron Chemoreceptors

Most TIHs (80–90% of theTIHs in Haz-Map) are reactive chemicals and produce similar sensations of a pungent “burning” irritation and pain by activating chemoreceptor sensory neurons. The airway mucosal epithelium is densely innervated with projections from the peripheral nerves of the somatic sensory system, including two nerve fiber types involved in chemical nociception (62). These fibers are anatomically distinguishable from each other by their diameter and degree of myelination. A-δ fibers have a slightly larger diameter and are encased in thin myelin sheaths, while the C-fiber neurons are unmyelinated with smaller diameters. Activation of A-δ fibers nerves produces a fast, sharp pain sensation, while the slow velocity C-fiber activation induces a slow “burning” pain. In the respiratory system, these fibers correspond through the trigeminal or vagus nerve with brainstem nuclei to coordinate sensations. This results in reflexes and responses to deter further inhalation, expel toxicants by coughing and sneezing, dilute and neutralize reactive toxins by parasympathetic glandular secretions, and alter breathing patterns and other brain-mediated airway responses to reactive TIHs (10, 16–21). In rodents, TIHs activation of sensory neurons induces a decrease in respiratory rate by increasing the pause between individual breaths. This is used as a common model to measure and determine respiratory irritation by TIHs (16, 18, 63).

Reactive TIHs also induce peripheral endings of C-fiber neurons to secrete peptides and other molecules at and near the site of contact, such as Substance P (SP) and calcitonin gene-related peptide (CGRP). SP preferentially binds to the tachykinin receptor NK1 receptors, while CGRP binds to calcitonin receptor–like receptor (CRLR) coupled with the receptor activity modifying protein (RAMP1 or 3). NK1 receptors are GPCRs coupled to the Gq subunit, which actives PLC and increases intracellular Ca²⁺. CGRP receptors (CRLR+RAMP) are coupled to the Gs subunit, which activates adenylate cyclase, increasing intracellular cAMP. These peptide receptors show partially overlapping expression patterns. In certain instances their actions are synergistic, while in other instances they counteract each other (34, 64, 65). Extensive studies on SP and CGRP have shown that they are predominantly proinflammatory factors that increase bronchoconstriction, immune cell infiltration, vasodilation, mast cell degranulation, plasma extravasation, and other local inflammatory responses (34). The acute pneumonitis from certain reactive TIHs most likely has a neurogenic inflammatory component. Possibly, the pulmonary edema resulting from reactive TIH exposure also has a sensory neuronal component. Substance P and CGRP have been proposed to be involved in the pathology of allergic asthma (66, 67). Occupational asthma and reactive airway dysfunction syndrome may also have a neurogenic inflammatory component (68).

Acid Sensing: ASICs

Acidic TIHs stimulate both A-δ and C-fiber sensory neurons. This results in acidic TIH induction of a sharp, immediate pungent pain and reflex-like breath withdrawal as well as burning pain, pneumonitis, coughing, mucus secretion, and other responses (69–71). C-fibers appear to have at least two distinct electrophysiological currents sensitive to acidic pH, suggesting at least two distinct ion channel populations responding to acidic stimuli. An influx of Na⁺ occurs at pH levels slightly below the homeostatic level that rapidly inactivates and a mixed Ca²⁺/Na⁺ influx occurs at more acidic conditions. Various combinations of splice-variants of the three members of the ASIC (ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3) family of ion channels appear to be responsible for the first currents. These channels are expressed in both A-δ and C-fiber sensory neurons and show similar electrophysiological properties in heterologous expression systems, with conducting Na⁺ currents induced between pH 6 and 7 that rapidly inactivate. Different combinations of subunits and splice variants of the three ASIC genes may explain the observed differences in kinetics and pH sensitivity in native neurons (59).

Acid-Sensing Ion Channels: TRPV1

At lower pH (∼5) the nonspecific cation channel TRPV1 is activated in sensory neurons (72). While the various heteromeric combinations of ASIC-mediated acid-sensitive channels are ubiquitous in both chemosensory fiber types, the splice-variant and subtype distribution of ASICs have some specificity. TRPV1-like acid-sensitive currents are primarily observed in unmyelinated C-fiber neurons (59). These currents correlate with TRPV1 expression and the currents are not observed in the sensory neurons of mice devoid of TRPV1. While TRPV1 is activated at relatively low pH, inflammatory mediators and injury signals can augment the sensitivity of TRPV1 to acidity, and acidity can in turn hypersensitize TRPV1 (73). While it is unlikely that the pH in the lung would drop low enough to activate TRPV1 during inhalation of a TIH, repeated inhalation or underlying inflammation may facilitate TRPV1 to become hypersensitive and therefore triggered by an exposure to a gaseous acid (73–80). For instance, TRPV1 is sensitized by the potent proinflammatory signal, bradykinin (81).

TRPV1 is well established to have a crucial role in the nociception of heat. It is the site of action for the active ingredient in chili peppers, capsaicin. There are symptom similarities between high-level exposures to capsaicin as pepper spray (OC tear gas) and exposures to reactive chemical TIHs. However, studies of TRPV1 gene–deleted mice still show normal irritant responses to TIHs mediated by sensory neurons (82).

Most C fibers express either TRPV1 (80–90%) or the cold activated ion channel, TRPM8 (8%). TRPM8 is sensitive to cool temperatures and is at least partially responsible for the sensation elicited by cool temperatures and by menthol and related compounds. Similar to TRPP3 and TRPM5, these proteins belong to the Transient Receptor Potential (TRP) superfamily of cation channels. TRP proteins are believed to form tetrameric cation channels in a manner similar to the voltage-gated K⁺ channels, with six transmembrane spans and an ion selectivity filter-pore between the fifth and the sixth span. Both the amino and carboxyl terminal spans are cytosolic. Most TRP channels conduct and are regulated by calcium ions. Predictably, their physiological functions often correlate with the dynamic roles that calcium performs in cells, organisms, and disease, especially in sensing and signaling (83).

Oxidant and Electrophile Sensing: TRPA1

TRPA1, a nonspecific cation channel expressed in 30–40% of the TRPV1-expressing C-fiber neurons, is directly activated by volatile reactive organic electrophiles or oxidative agents (∼40% TIHs on Haz-Map site shown in Table 1) (84). This has been demonstrated in vitro and in vivo in numerous publications using TRPA1-gene deleted mice or treatments with TRPA1-antagonists. Table 2 provides a relatively inclusive list of all the TIHs and related chemicals found thus far to activate TRPA1. However, TRPA1's role in the airway responses to many of these reactive chemical TIHs has yet to be characterized. The most potent and specific TRPA1 agonists characterized so far are the CS-, CR-, and CN-tear gases (riot control agents) (85, 86). These agents appear to target TRPA1 to induce their intended effects of incapacitating pain, glandular secretions, coughing, acute neurogenic inflammation, and other symptoms associated with exposures to reactive chemical TIHs.

Just before the first putative transmembrane span of TRPA1 a short protein moiety is located containing three cysteine residues and a lysine residue. These amino acid residues form a “binding site” for the protein modifying TIHs. It is believed that covalent modification of these residues leads to activation of the channel, although additional residues have been shown to also be involved (87–89). Figure 1 shows the likely modification of cysteine residues by certain types of TIH molecules that are electrophiles or oxidants. As mentioned, some of these reactive organic molecules have yet to be examined for activity on TRPA1 (90–92). Most likely, if the molecule can form covalent bonds with the cysteine residues of glutathione, then it will also react with the cysteines in the “covalent modification binding site” and thereby activate TRPA1 and the C-type sensory neuron. Interestingly, ammonia also activates TRPA1, probably through alkalinization of the cytosolic spans (93).

Similar to many TRP channels, TRPA1 conducts both divalent and monovalent cations. TRPA1-carried cations depolarize the membrane, thereby activating voltage-gated Na⁺ and Ca²⁺ channels to propagate action potentials and consequentially signal the spinal and brainstem nerves to initiate reflexes and autonomic and other protective responses of the airways and the lungs (94). In TRPA1-deficient (Trpa1^−/−) mice or mice treated with a TRPA1-antagonist, pain, neurogenic inflammation, bronchial constriction, and respiratory depression in response to reactive chemicals are absent or abated (see Table 2, second to last row). Activation of TRPA1 also results in the secretion of proinflammatory factors such as Substance P and CGRP (95). Neuronal peptide release is thought to be triggered by the influx of Ca²⁺ into the airway nerve ending, either directly through TRPA1 or by TRPA1-triggered voltage-gated Ca²⁺ channels. However, other mechanisms may be involved. About half of TRPA1's amino acid sequence is dedicated to a span of about 17 ankyrin repeat domains on the amino terminal span that may interact with signaling complexes and the vesicle release machinery.

Some of the necrotic cell–derived factors and mediators released as a consequence of chemical injury and inflammation modulate the sensitivity and activity of TRPA1 (see Table 2). Hyperactive or hypersensitive TRPA1 channels may contribute to chronic bronchitis, occupational asthma, and other inflammatory lung diseases associated with exposures to reactive TIHs (96). TRPA1 is crucially involved in allergenic respiratory sensitivity to ovalbumin, an inducer of allergic asthma in animal models (97). These findings show that airway sensory neurons actively modulate the local immune response to allergens.

Phosgene, Ozone, and Nitrogen Dioxide

The relatively hydrophobic reactive chemicals, phosgene, ozone, and nitrogen dioxide, do not readily diffuse through the mucus and epithelium to reach underlying sensory nerve endings (2). Therefore, they are usually not detected by sensory neurons and do not trigger defensive responses, thereby permitting the toxicants to penetrate more deeply into the lungs to the delicate alveolar sacs. The inability of the chemosensory system to detect these hydrophobic TIHs may compound the damage these chemicals inflict in the respiratory system. An additional factor to their toxicity is that hydrophobic TIHs do not readily interact with the aqueous compounds in the airway fluids that sequester and neutralize reactive chemicals.