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.

Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. Capsazepine (10 μm) reversibly reduced or abolished the current response to capsaicin (500 nm) of voltage‐clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 μm γ‐aminobutyric acid (GABA) and 5 μm adenosine 5′‐triphosphate (ATP) were unaffected. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nm)‐induced ⁴⁵Ca²⁺ uptake in cultures of rat DRG neurones with an IC₅₀ of 420 ± 46 nm (mean ± s.e.mean, n = 6). The ⁴⁵Ca²⁺ uptake evoked by resiniferatoxin (RTX), a potent capsaicin‐like agonist was also inhibited. (Log concentration)‐effect curves for RTX (0.3 nm‐1 μm) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 ± 0.15 (s.e.) and gave an apparent K_d estimate for capsazepine of 220 nm (95% confidence limits, 57–400 nm). Capsazepine also inhibited the capsaicin‐ and RTX‐evoked efflux of ⁸⁶Rb⁺ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent K_d estimates of 148 nm (95% confidence limits, 30–332 nm) with capsaicin as the agonist and 107 nm (95% confidence limits, 49–162 nm) with RTX as agonist. A similar competitive inhibition by capsazepine was seen for capsaicin‐induced [¹⁴C]‐guanidinium efflux from segments of adult rat vagus nerves (apparent K_d = 690 nm; 95% confidence limits, 63 nm‐1.45 μm). No significant difference was noted in the apparent K_d estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits.6 Capsazepine, at concentrations up to 10 μm, had no significant effects on the efflux of ⁸⁶Rb⁺ from cultured DRG neurones evoked either by depolarization with high (50 mm) K⁺ solutions or by acidification of the external medium to pH 5.0–5.6. Similarly capsazepine had no significant effect on the depolarization (50 mm KCl)‐induced efflux of [¹⁴C]‐guanidinium from vagus nerve preparations.7 Ruthenium Red was also tested for antagonism against capsaicin evoked [¹⁴C]‐guanidinium release from vague nerves and capsaicin induced ⁴⁵Ca²⁺ uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10–500 nm in DRG and 0.5–10 μm in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex, non‐competitive inhibition.

A recent major conceptual advance has been the recognition of the importance of immune system–neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS-induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.