Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

Transient receptor potential A1 (TRPA1) is expressed in a subset of nociceptive sensory neurons where it acts as a sensor for environmental irritants, including acrolein, and some pungent plant ingredients such as allyl isothiocyanate and cinnamaldehyde. These exogenous compounds activate TRPA1 by covalent modification of cysteine residues. We have used electrophysiological methods and measurements of intracellular calcium concentration ([Ca²⁺]_i) to show that TRPA1 is activated by several classes of endogenous thiol-reactive molecules. TRPA1 was activated by hydrogen peroxide (H₂O₂; EC₅₀, 230 μm), by endogenously occurring alkenyl aldehydes (EC₅₀: 4-hydroxynonenal 19.9 μm, 4-oxo-nonenal 1.9 μm, 4-hydroxyhexenal 38.9 μm) and by the cyclopentenone prostaglandin, 15-deoxy-Δ^12,14-prostaglandin J₂(15d-PGJ₂, EC₅₀: 5.6 μm). The effect of H₂O₂was reversed by treatment with dithiothreitol indicating that H₂O₂acts by promoting the formation of disulfide bonds whereas the actions of the alkenyl aldehydes and 15d-PGJ₂were not reversed, suggesting that these agents form Michael adducts. H₂O₂and the naturally occurring alkenyl aldehydes and 15d-PGJ₂acted on a subset of isolated rat and mouse sensory neurons [∼25% of rat dorsal root ganglion (DRG) and ∼50% of nodose ganglion neurons] to evoke a depolarizing inward current and an increase in [Ca²⁺]_iin TRPA1 expressing neurons. The abilities of H₂O₂, alkenyl aldehydes and 15d-PGJ₂to raise [Ca²⁺]_iin mouse DRG neurons were greatly reduced in neurons fromtrpa1^−/−mice. Furthermore, intraplantar injection of either H₂O₂or 15d-PGJ2 evoked a nocifensive/pain response in wild-type mice, but not intrpa1^−/−mice. These data demonstrate that multiple agents produced during episodes of oxidative stress can activate TRPA1 expressed in sensory neurons.

Acetaminophen (paracetamol) is a popular domestic analgesic and antipyretic agent with a weak anti-inflammatory action and a low incidence of adverse effects as compared with aspirin and other non-steroidal anti-inflammatory drugs. Here we show that acetaminophen, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and the spinal cord to form the potent TRPV₁ agonist N-arachidonoylphenolamine (AM404). This conjugation is absent in mice lacking the enzyme fatty acid amide hydrolase. AM404 also inhibits purified cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages. This novel metabolite of acetaminophen also acts on the endogenous cannabinoid system, which, together with TRPV₁ and COX, is present in the pain and thermoregulatory pathways. These findings identify fatty acid conjugation as a novel pathway for drug metabolism and provide a molecular mechanism for the occurrence of the analgesic N-acylphenolamine AM404 in the nervous system following treatment with acetaminophen.