Mirjam Eberhardt

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

TRPA1 and Substance P Mediate Colitis in Mice

BACKGROUND & AIMS The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Methylglyoxal Activates Nociceptors through Transient Receptor Potential Channel A1 (TRPA1) A POSSIBLE MECHANISM OF METABOLIC NEUROPATHIES

Background: Methylglyoxal is a reactive metabolite that modifies proteins and accumulates in diabetes and uremia. Results: Methylglyoxal excites nociceptors and releases neuropeptides via activation of TRPA1 channels by modifying their intracellular N-terminal cysteine and lysine residues. Conclusion: Methylglyoxal acting through TRPA1 is a possible cause of painful metabolic neuropathies. Significance: Methylglyoxal and its reaction with TRPA1 are promising targets for medicinal chemistry to fight neurotoxicity. Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.

Beyond H2S and NO interplay: hydrogen sulfide and nitroprusside react directly to give nitroxyl (HNO). A new pharmacological source of HNO.

Hydrogen sulfide (H2S) has been increasingly recognized as an important signaling molecule that regulates both blood pressure and neuronal activity. Attention has been drawn to its interactions with another gasotransmitter, nitric oxide (NO). Here, we provide evidence that the physiological effects observed upon the application of sodium nitroprusside (SNP) and H2S can be ascribed to the generation of nitroxyl (HNO), which is a direct product of the reaction between SNP and H2S, not a consequence of released NO subsequently reacting with H2S. Intracellular HNO formation has been confirmed, and the subsequent release of calcitonin gene-related peptide from a mouse heart has been demonstrated. Unlike with other thiols, SNP reacts with H2S in the same way as rhodanese, i.e., the cyanide transforms into a thiocyanate. These findings shed new light on how H2S is understood to interact with nitroprusside. Additionally, they offer a new and convenient pharmacological source of HNO for therapeutic purposes.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P‐CTX‐1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin‐resistant, TRPA1‐expressing peptidergic C‐fibres and tetrodotoxin‐sensitive A‐fibres. P‐CTX‐1 does not directly open heterologously expressed TRPA1, but when co‐expressed with Nav channels, sodium channel activation by P‐CTX‐1 is sufficient to drive TRPA1‐dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P‐CTX‐1 on TRPA1‐deficient nociceptive C‐fibres and of ciguatoxin‐induced cold allodynia in TRPA1‐null mutant mice. Functional MRI studies revealed that ciguatoxin‐induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1‐deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

Background: Extracellular acidosis mediates pain and inflammation by activating sensory afferent neurons. Results: Protons activate and sensitize human TRPA1 in a strongly species-specific manner encoded by transmembrane domains 5 and 6. Conclusion: Our data identify TRPA1 as an ion channel likely to mediate acid-induced pain in humans. Significance: Protons are the first known endogenous agonists of TRPA1 with species-specificity for human TRPA1. The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

The general anesthetic propofol excites nociceptors by activating TRPV1 and TRPA1 rather than GABAA receptors.

Anesthetic agents can induce a paradox activation and sensitization of nociceptive sensory neurons and, thus, potentially facilitate pain processing. Here we identify distinct molecular mechanisms that mediate an activation of sensory neurons by 2,6-diisopropylphenol (propofol), a commonly used intravenous anesthetic known to elicit intense pain upon injection. Clinically relevant concentrations of propofol activated the recombinant transient receptor potential (TRP) receptors TRPA1 and TRPV1 heterologously expressed in HEK293t cells. In dorsal root ganglion (DRG) neurons, propofol-induced activation correlated better to expression of TRPA1 than of TRPV1. However, pretreatment with the protein kinase C activator 4β-phorbol 12-myristate 13-acetate (PMA) resulted in a significantly sensitized propofol-induced activation of TRPV1 in DRG neurons as well as in HEK293t cells. Pharmacological and genetic silencing of both TRPA1 and TRPV1 only partially abrogated propofol-induced responses in DRG neurons. The remaining propofol-induced activation was abolished by the selective γ-aminobutyric acid, type A (GABAA) receptor antagonist picrotoxin. Propofol but not GABA evokes a release of calcitonin gene-related peptide, a key component of neurogenic inflammation, from isolated peripheral nerves of wild-type but not TRPV1 and TRPA1-deficient mice. Moreover, propofol but not GABA induced an intense pain upon intracutaneous injection. As both the release of calcitonin gene-related peptide and injection pain by propofol seem to be independent of GABAA receptors, our data identify TRPV1 and TRPA1 as key molecules for propofol-induced excitation of sensory neurons. This study warrants further investigations into the role of anesthetics to induce nociceptor sensitization and to foster postoperative pain.

Calcitonin gene-related peptide release from intact isolated dorsal root and trigeminal ganglia.

Neuropeptides like calcitonin gene-related peptide (CGRP) and substance P are found in significant proportions of primary afferent neurons. Release of these neuropeptides as well as prostaglandin E(2) is an approved index for the activation of these primary afferents. Previous studies have used cultures of enzyme-treated and mechanically dissociated primary afferent neurons, fresh tissue slices or cubes. In the present study we demonstrate CGRP and prostaglandin E(2) release from intact isolated dorsal root and trigeminal ganglia. Stimulation with noxious heat, low pH, inflammatory mediators and high potassium concentration increased CGRP release. In conclusion, neuropeptide release from intact isolated ganglia is a reliable method to study the responsiveness of sensory neurons in situ in comparison with neuronal cell cultures.

Inherited Pain SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION

Background: Mutations in the sodium channel Nav1.7 cause the inherited pain syndromes IEM and PEPD. Results: The new IEM mutation A1632T impairs channel inactivation, whereas an IEM/PEPD crossover mutation (A1632E) at the same position additionally increases resurgent sodium currents. Conclusion: Reduced inactivation without increased resurgent currents induces symptoms of IEM. Significance: Resurgent currents are likely to determine whether a mutation leads to IEM or PEPD. Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.