Marcello Trevisani

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of “hot” chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous “capsaicin-like” substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 ≈ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 = 1.5 ± 0.3 μg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous α,β-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Protease-Activated Receptor 2 Sensitizes the Capsaicin Receptor Transient Receptor Potential Vanilloid Receptor 1 to Induce Hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Protease‐activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease‐activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2‐mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene‐related peptide (CGRP), mediators of pain transmission. In PAR2‐expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4α‐phorbol 12,13‐didecanoate (4αPDD) and hypotonic solutions. PAR2‐agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cβ and protein kinases A, C and D inhibited PAR2‐induced sensitization of TRPV4 Ca2+ signals and currents. 4αPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4‐dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4αPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist‐induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4‐dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca2+‐ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1‐dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1‐mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1‐mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5‐triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.

Activation and sensitisation of the vanilloid receptor: role in gastrointestinal inflammation and function

The exquisite specific excitatory and desensitising actions of capsaicin on a subpopulation of primary sensory neurons have been instrumental in identifying the roles of these neurons in nociception, reflex responses and neurogenic inflammation. Structure activity studies with capsaicin‐like molecules have suggested that a ‘receptor’ should mediate the effects of capsaicin on sensory neurons. The cloning of the vanilloid receptor‐1 (VR1) has confirmed this hypothesis. VR1 (TRPV1) belongs to the transient receptor potential (TRP) family of channels, and its activation by various xenobiotics, noxious temperature, extracellular low pH and high concentration of certain lipid derivatives results in cation influx and sensory nerve terminal excitation. TRPV1 may dimerise or form tetramers or heteromers with PLC‐γ and TrkA or even with other TRPs. TRPV1 is markedly upregulated and/or ‘sensitised’ under inflammatory conditions via protein kinase C‐ɛ‐, cAMP‐dependent PK‐ and PLC‐γ‐dependent pathways or by exposure to dietary agents as ethanol. TRPV1 is expressed on sensory neurons distributed in all the regions of the gastrointestinal tract in myenteric ganglia, muscle layer and mucosa. There is evidence of TRPV1 expression also in epithelial cells of the gastrointestinal tract. High expression of TRPV1 has been detected in several inflammatory diseases of the colon and ileum, whereas neuropeptides released upon sensory nerve stimulation triggered by TRPV1 activation seem to play a role in intestinal motility disorders. TRPV1 antagonists, which will soon be available for clinical testing, may undergo scrutiny for the treatment of inflammatory diseases of the gut.

Hydrogen sulfide causes vanilloid receptor 1-mediated neurogenic inflammation in the airways

1 Hydrogen sulfide (H2S) is described as a mediator of diverse biological effects, and is known to produce irritation and injury in the lung following inhalation. Recently, H2S has been found to cause contraction in the rat urinary bladder via a neurogenic mechanism. Here, we studied whether sodium hydrogen sulfide (NaHS), used as donor of H2S, produces responses mediated by sensory nerve activation in the guinea‐pig airways. 2 NaHS evoked an increase in neuropeptide release in the airways that was significantly attenuated by capsaicin desensitization and by the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. In addition, NaHS caused an atropine‐resistant contraction of isolated airways, which was completely prevented by capsaicin desensitization. Furthermore, NaHS‐induced contraction was reduced by TRPV1 antagonism (ruthenium red, capsazepine and SB366791), and was abolished by pretreatment with the combination of tachykinin NK1 (SR140333) and NK2 (SR48968) receptor antagonists. 3 In anesthetized guinea‐pigs, intratracheal instillation of NaHS increased the total lung resistance and airway plasma protein extravasation. These two effects were reduced by TRPV1 antagonism (capsazepine) and tachykinin receptors (SR140333 and SR48968) blockade. 4 Our results provide the first pharmacological evidence that H2S provokes tachykinin‐mediated neurogenic inflammatory responses in guinea‐pig airways, and that this effect is mediated by stimulation of TRPV1 receptors on sensory nerves endings. This novel mechanism may contribute to the irritative action of H2S in the respiratory system.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ2, PGA2, and PGA1, formed by dehydration of their respective parent PGs, PGD2, PGE2, and PGE1, possess a highly reactive α,β-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1+/+) or deficient (TRPA1−/−) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1+/+, but not of TRPA1−/− mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1+/+ but not in TRPA1−/− mice. The classical proalgesic PG, PGE2, caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1+/+ and TRPA1−/− mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.