Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.
Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund’s adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.
Melanoma is the most dangerous form of skin cancer occurring in Caucasians with rising incidence. They are remarkably resistant to conventional anti-tumour therapies like chemotherapy and radiotherapy. Therefore, new treatment strategies are urgently needed. Anti-tumour effects of phytochemicals such as allyl isothiocyanate or cinnamaldehyde have been demonstrated in various melanoma models in vitro and in vivo. Considering their high potency as transient receptor potential A1 (TRPA1)-activating compounds, we examined the functional expression of TRPA1 channels in different melanoma cell lines as well as in non-malignantly transformed primary melanocytes. The presence of TRPA1 transcripts could be detected in most of the melanoma cell lines. Furthermore, single-cell calcium imaging and patch clamp electrophysiology confirmed the presence of functional TRPA1 channels in those cell lines. Proliferation assays revealed that allyl isothiocyanate and cinnamaldehyde clearly reduce the proliferation of melanoma cells, but this effect is independent of an activation of TRPA1 channels, making it unlikely that ionic currents through TRPA1 are responsible for the anti-tumour effects of mustard oil and cinnamaldehyde.
These authors contributed equally. BACKGROUND AND PURPOSEROS and their downstream molecules such as oxidized phospholipids (OxPL) and 4-hydroxynonenal activate TRPA1 and TRPV1 (vanilloid 1) cation channels in vivo and in vitro shaping thermal and mechanical hypersensitivity in inflammatory pain. E06/T15 is a monoclonal autoantibody against oxidized phosphatidylcholine (OxPC) used in diagnostics in arteriosclerosis. Recently, we provided evidence that E06 also ameliorates inflammatory pain. Here, we studied E06 for local treatment against hypersensitivity evoked by endogenous and exogenous agonists of TRPA1 or TRPV1 channels. EXPERIMENTAL APPROACHWe utilized a combination of reflexive and complex behavioural pain measurements, live-cell calcium imaging and OxPC-binding assays. The lipid peroxidation metabolite 4-hydroxynonenal, hydrogen peroxide as a source of ROS, allyl isothiocyanate and capsaicin were used to activate their respective receptors. KEY RESULTSAll irritants induced thermal and mechanical hypersensitivity, spontaneous nocifensive and affective-motivational behaviour, as well as calcium influx in HEK TRPA1 -or HEK TRPV1 -cells and dorsal root ganglion neurons. E06 prevented prolonged mechanical hypersensitivity induced by all irritants except for H 2 O 2 . E06 did not alter immediate irritant-induced nocifensive or affective motivational behaviour. In vitro, E06 blocked only 4-hydroxynonenal-induced calcium influx although this compound did not bind to E06. After 1-3 h, all tested irritants elicited formation of OxPC in paw tissue. CONCLUSIONS AND IMPLICATIONSE06 ameliorates not only inflammatory pain but also prolonged hypersensitivity due to formation of OxPC. This supports the view that neutralizing certain OxPL as endogenous activators of TRPA1 or TRPV1 channels may be valuable for pain therapy. Abbreviations
Apomorphine is a non-narcotic derivative of morphine, which acts as a dopamine agonist and is clinically used to treat "offstates" in patients suffering from Parkinson's disease. Adverse effects of apomorphine treatment include severe emesis and nausea, and ulceration and pain at the injection site. We wanted to test whether sensory transient receptor potential (TRP) channels are a molecular target for apomorphine. Here, we show that rTRPV1, rTRPV2, rTRPV3, and mTRPV4, as well as hTRPM8, and rTRPM3, which are expressed in dorsal root ganglion neurons, are insensitive toward apomorphine treatment. This also applied to the cellular redox sensor hTRPM2. On the contrary, human TRPA1 could be concentrationdependently modulated by apomorphine. Whereas the addition of apomorphine in the low micromolar range produced an irreversible activation of the channel, application of higher concentrations caused a reversible voltage-dependent inhibition of heterologously expressed TRPA1 channels, resulting from a reduction of single-channel open times. In addition, we provide evidence that apomorphine also acts on endogenous TRPA1 in cultured dorsal root ganglion neurons from rats and in the enterochromaffin model cell line QGP-1, from which serotonin is released upon activation of TRPA1. Our study shows that human TRPA1 is a target for apomorphine, suggesting that an activation of TRPA1 might contribute to adverse side effects such as nausea and painful injections, which can occur during treatment with apomorphine.
Background: High doses of capsaicin are recommended for the treatment of neuropathic pain. However, low doses evoke mechanical hypersensitivity. Activation of the capsaicin chemosensor transient receptor potential vanilloid 1 (TRPV1) induces neurogenic inflammation. In addition to the release of pro-inflammatory mediators, reactive oxygen species are produced. These highly reactive molecules generate oxidised phospholipids and 4-hydroxynonenal (4-HNE) which then directly activate TRP ankyrin 1 (TRPA1). The apolipoprotein A-I mimetic peptide D-4F neutralises oxidised phospholipids. Here, we asked whether D-4F ameliorates neurogenic hypersensitivity in rodents by targeting reactive oxygen species and 4-HNE in the capsaicin-evoked pain model. Results: Co-application of D-4F ameliorated capsaicin-induced mechanical hypersensitivity and allodynia as well as persistent heat hypersensitivity measured by Randell-Selitto, von Frey and Hargreaves test, respectively. In addition, mechanical hypersensitivity was blocked after co-injection of D-4F with the reactive oxygen species analogue H 2 O 2 or 4-HNE. In vitro studies on dorsal root ganglion neurons and stably transfected cell lines revealed a TRPA1-dependent inhibition of the calcium influx when agonists were pre-incubated with D-4F. The capsaicin-induced calcium influx in TRPV1-expressing cell lines and dorsal root ganglion neurons sustained in the presence of D-4F. Conclusions: D-4F is a promising compound to ameliorate TRPA1-dependent hypersensitivity during neurogenic inflammation.
Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) were recently identified as novel proalgesic targets in acute and chronic inflammatory pain. These endogenous chemical irritants are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1 expressed in sensory neurons. Notably, prototypical therapeutics interfering with OxPL were shown to inhibit TRP channel activation and pain behavior. Here, we asked how OxPL excite primary sensory neurons of dorsal root ganglia (DRG neurons from mice of either sex). Acute stimulation of sensory neurons with the prototypical OxPL 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) evoked repetitive calcium spikes in small-diameter neurons. As NaV1.9, a voltage-gated sodium channel involved in nociceptor excitability, was previously shown to be essential for the generation of calcium spikes in motoneurons, we asked if this channel is also important for OxPL mediated calcium spike and action potential generation in nociceptors. In wild-type and NaV1.9-deficient neurons, the action potential firing rate and the calcium spike frequency to an acute PGPC stimulus was similar. When preincubated with inflammatory mediators, both, the action potential firing rate and the calcium spike frequency were markedly increased in response to an acute PGPC stimulus. However, this potentiating effect was completely lost in NaV1.9-deficient small-diameter neurons. After treatment with inflammatory mediators, the resting membrane potential of NaV1.9 KO neurons was slightly more negative than that of wild-type control neurons. This suggests that NaV1.9 channels are active under this condition and therefore increases the ease with which action potentials are elicited after OxPL stimulation. In summary, our data suggest that NaV1.9 has a switch function to potentiate the receptor potentials induced by OxPL under inflammatory conditions. Since human NaV1.9 has been shown to mediate painful and painless channelopathies, this study provides new insights into the mechanism by which NaV1.9 amplifies stimuli of endogenous irritants under inflammatory conditions.
Within the lipidome oxidized phospholipids (OxPL) form a class of chemically highly reactive metabolites. OxPL are acutely produced in inflamed tissue and act as endogenous, proalgesic (pain-inducing) metabolites. They excite sensory, nociceptive neurons by activating transient receptor potential ion channels, specifically TRPA1 and TRPV1. Under inflammatory conditions, OxPL-mediated receptor potentials even potentiate the action potential firing rate of nociceptors. Targeting OxPL with D-4F, an apolipoprotein A-I mimetic peptide or antibodies like E06, specifically binding oxidized headgroups of phospholipids, can be used to control acute, inflammatory pain syndromes, at least in rodents. With a focus on proalgesic specificities of OxPL, this article discusses, how targeting defined substances of the epilipidome can contribute to mechanism-based therapies against primary and secondary chronic inflammatory or possibly also neuropathic pain.
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