Ruolo dei canali Transient Receptor Potential (TRP) espressi in cellule neuronali e non neuronali, nelle patologie dolorose di origine infiammatoria e neuropatica.

The TRPA1 represents a key target in pain modulation. It is manly expressed in primary sensory neurons, where It can be activated by a wide series of exogenous chemicals, and also detects and can be sensitized by a series of endogenous molecules, including oxidative/nitrative/carbonylic stress (ROS/RNS/RCS) byproducts deriving from inflammatory processes. TRPA1 and ROS are emerging as a major pathway in different pain conditions, including migraine headache, which affects almost 15% of the adult worldwide population, with an enormous social and economic cost. In the first study presented here, we examined the effect of the butterbur component [Petasites hybridus (L.)] isopetasin on TRPA1. Butterbur is currently indicated as level A recommendation for migraine prophylaxis by the American Headache Society guidelines, and its major constituents, petasin and isopetasin, are considered responsible for its antimigraine effects. Our results showed that isopetasin is able to gate mouse, rat and human (native and recombinant) TRPA1 channel. In addition, isopetasin acts as a partial agonist on the channel. A chronic treatment in mice with isopetasin showed that this molecule can target TRPA1 and induce desensitization of the channel and defunctionalization of trigeminal nerve terminals, attenuating their ability to release the calcitonin gene-related peptide (CGRP), the main mediator of migraine pain. Successful treatment and prevention with isopetasin, may provide a solid basis for future investigations of TRPA1-tropic approaches for migraine. In the second part, we investigated the possibility that some constituents of saffron [Crocus sativus (L.)] could as well target TRPA1. Saffron has been used for centuries not only for food flavoring and coloring, but also to treat headache in Indian traditional medicine. Furthermore, saffron constituents safranal, crocin and picrocrocin, have been reported to possess analgesic properties in various animal models of pain. We showed that safranal can activate human and rodent TRPA1, acting as a partial agonist. Desensitization experimental protocols in vitro showed that safranal is able to desensitize TRPA1, in a homologous manner. Chronic treatment with safranal in mice confirmed this hypothesis. This mechanism could account for the antinociceptive properties of safranal. Another part of this three years study aimed at investigating glyceryl trinitrate (nitroglycerin, GTN)-induced migraine-like attacks. GTN has long been used as a clinical provocation test for migraine. In most migraineurs, GTN administration evokes headaches that fulfill the criteria of a typical migraine attack. Moreover, GTN administration in rodents and humans induces a delayed and prolonged hyperalgesia, and is able to release the active vasodilator nitric oxide (NO), which is a known TRPA1 agonist. Our study provided evidence that the delayed and prolonged GTN-evoked allodynia in mice is entirely dependent from TRPA1 activation. GTN directly activates TRPA1 in vitro and in vivo (only if given locally). However, systemic treatment with GTN indicated that NO, liberated from GTN by ALDH-2, activates TRPA1 to induce allodynia. By gating TRPA1 in trigeminal nociceptors, NO leads to activation of NOX1/NOX2, which generate ROS and the ensuing aldehyde endproducts. These molecules ultimately activate TRPA1 again, generating an autocrine pathway. TRPA1 antagonists currently in clinical development, or established antimigraine drugs identified as selective channel inhibitors, may be used to verify if a mechanism similar to the present one mediates the effects induced by GTN in migraineurs. Finally, we investigated the possibility that ibuprofen-acyl glucuronide (IAG), a metabolite of ibuprofen, could antagonize TRPA1. Ibuprofen is a classical NSAID, widely used to relieve inflammation and several types of pain, including headache. Its therapeutic effects are attributed to a non-selective, reversible inhibition of COX1 and COX2. 10-15% of ibuprofen is metabolized, through glucuronidation, to IAG, which may covalently bind various macromolecules. Our major finding is that IAG, but not ibuprofen, antagonizes the proalgesic TRPA1 channel. IAG was able, unlike ibuprofen, to selectively inhibit the human and rodent TRPA1 channel. IAG attenuated nociception evoked only by reactive TRPA1 agonists. A systemic administration of ibuprofen also produced a partial attenuation of TRPA1-mediated nociception, probably due to the formation of IAG through liver metabolism. Local IAG produced a more potent anti-inflammatory effect than ibuprofen, attributable to a combination of COX-inhibition and TRPA1-antagonism, since PGE2 levels were ablated by both compounds in the same way. Hence, the analgesic and anti-inflammatory activity of ibuprofen can be attributed to TRPA1 inhibition, in addition to COX inhibition, via IAG generation. The findings of this three years study indicate that TRPA1 expressed in neuronal and non-neuronal cells is a key mediator in inflammatory and neuropathic pain modulation. Our results suggest novel therapeutic approaches to treat pain, in particular migraine, involving TRPA1 antagonists and TRPA1-desensitizing agents.