Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.

A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems.

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 mea- Ϫ/Ϫ 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 TRPV 1 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 TRPV 1 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.Acetaminophen was introduced into clinical medicine more than a century ago, but its mechanism of action is still a matter of debate. The analgesic, antipyretic, and anti-inflammatory effects of non-steroidal anti-inflammatory drugs are believed to depend on their ability to inhibit COX 1 (1, 2). However, acetaminophen differs from most non-steroidal anti-inflammatory drugs in that it is a weak anti-inflammatory agent with a low incidence of COX-related adverse effects (2-4).Although this may seem incompatible with an action on COX, studies in vitro clearly show that acetaminophen is able to inhibit both COX-1 and COX-2, provided that the ambient concentration of peroxides is kept low (5-7). Such a peroxidedependent inhibition of COX could explain why acetaminophen does not suppress inflammation and platelet activity (5-7). However, final proof that the analgesic and antipyretic effects of acetaminophen are dependent on COX is still lacking. There are also indications that the analgesic effect of acetaminophen is mediated by molecular targets distinct from COX (8 -10).In this study we have explored the possibility that acetaminophen undergoes a two-step metabolic transformation to form the bioactive N-acylphenolamine AM404. AM404 is a potent activator of TRPV 1 , a ligand at cannabinoid CB 1 receptors and an inhibitor of cellular anandamide uptake, the inhibition of which leads to increased levels of endogenous cannabinoids (11-15). TRPV 1 and cannabinoid CB 1 receptors are both present in the pain and thermoregulatory pathways, and much interest has been focused on these receptors as potential drug targets for the treatment of pain and inflammation (11, 14, 16 -19).AM404 belongs to a group of bioactive N-acylamines that also includes the endogenous lipids anandamide (20), N-arachidonoyldopamine (21), and N-arachidonoylglycine (22) and the synthetic compounds olvanil (23) and arvanil (24). These drugs all display analgesic activity in a ...

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1 − / − mice. The electrophilic metabolites N-acetylp-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-pbenzoquinoneimine metabolite l-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1 − / − mice. Intrathecal injection of a non-electrophilic cannabinoid, ∆ 9 -tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

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