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A 2-day closed workshop was held in Liverpool, United Kingdom, to discuss the results of research concerning symptom-based disorders (SBDs) caused by autoantibodies, share technical knowledge, and consider future plans. Twenty-two speakers and 14 additional participants attended. This workshop set out to consolidate knowledge about the contribution of autoantibodies to SBDs. Persuasive evidence for a causative role of autoantibodies in disease often derives from experimental "passive transfer" approaches, as first established in neurological research. Here, serum immunoglobulin (IgM or IgG) is purified from donated blood and transferred to rodents, either systemically or intrathecally. Rodents are then assessed for the expression of phenotypes resembling the human condition; successful phenotype transfer is considered supportive of or proof for autoimmune pathology. Workshop participants discussed passive transfer models and wider evidence for autoantibody contribution to a range of SBDs. Clinical trials testing autoantibody reduction were presented. Cornerstones of both experimental approaches and clinical trial parameters in this field were distilled and presented in this article. Mounting evidence suggests that immunoglobulin transfer from patient donors often induces the respective SBD phenotype in rodents. Understanding antibody binding epitopes and downstream mechanisms will require substantial research efforts, but treatments to reduce antibody titres can already now be evaluated.
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The platinum chemotherapeutic oxaliplatin produces dose-limiting pain, dysesthesia, and cold hypersensitivity in most patients immediately after infusion. An improved understanding of the mechanisms underlying these symptoms is urgently required to facilitate the development of symptomatic or preventative therapies. In this study, we have used skin-saphenous nerve recordings in vitro and behavioral experiments in mice to characterize the direct effects of oxaliplatin on different types of sensory afferent fibers. Our results confirmed that mice injected with oxaliplatin rapidly develop mechanical and cold hypersensitivities. We further noted profound changes to A fiber activity after the application of oxaliplatin to the receptive fields in the skin. Most oxaliplatin-treated Aδ- and rapidly adapting Aß-units lost mechanical sensitivity, but units that retained responsiveness additionally displayed a novel, aberrant cold sensitivity. Slowly adapting Aß-units did not display mechanical tachyphylaxis, and a subset of these fibers was sensitized to mechanical and cold stimulation after oxaliplatin treatment. C fiber afferents were less affected by acute applications of oxaliplatin, but a subset gained cold sensitivity. Taken together, our findings suggest that direct effects on peripheral A fibers play a dominant role in the development of acute oxaliplatin-induced cold hypersensitivity, numbness, and dysesthesia. PERSPECTIVE: The chemotherapeutic drug oxaliplatin rapidly gives rise to dose-limiting cold pain and dysesthesia. Here, we have used behavioral and electrophysiological studies of mice to characterize the responsible neurons. We show that oxaliplatin directly confers aberrant cold responsiveness to subsets of A-fibers while silencing other fibers of the same type.
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Antineoplásicos , Síndromes Periódicos Asociados a Criopirina , Humanos , Ratones , Animales , Oxaliplatino/efectos adversos , Parestesia , Síndromes Periódicos Asociados a Criopirina/inducido químicamente , Dolor , Hiperalgesia/inducido químicamente , Antineoplásicos/efectos adversosRESUMEN
Serum progesterone sulfates were evaluated in the etiology of gestational diabetes mellitus (GDM). Serum progesterone sulfates were measured using ultra-performance liquid chromatography-tandem mass spectrometry in four patient cohorts: 1) the Hyperglycemia and Adverse Pregnancy Outcomes study; 2) London-based women of mixed ancestry and 3) U.K.-based women of European ancestry with or without GDM; and 4) 11-13 weeks pregnant women with BMI ≤25 or BMI ≥35 kg/m2 with subsequent uncomplicated pregnancies or GDM. Glucose-stimulated insulin secretion (GSIS) was evaluated in response to progesterone sulfates in mouse islets and human islets. Calcium fluorescence was measured in HEK293 cells expressing transient receptor potential cation channel subfamily M member 3 (TRPM3). Computer modeling using Molecular Operating Environment generated three-dimensional structures of TRPM3. Epiallopregnanolone sulfate (PM5S) concentrations were reduced in GDM (P < 0.05), in women with higher fasting plasma glucose (P < 0.010), and in early pregnancy samples from women who subsequently developed GDM with BMI ≥35 kg/m2 (P < 0.05). In islets, 50 µmol/L PM5S increased GSIS by at least twofold (P < 0.001); isosakuranetin (TRPM3 inhibitor) abolished this effect. PM5S increased calcium influx in TRPM3-expressing HEK293 cells. Computer modeling and docking showed identical positioning of PM5S to the natural ligand in TRPM3. PM5S increases GSIS and is reduced in GDM serum. The activation of GSIS by PM5S is mediated by TRPM3 in both mouse and human islets.
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Diabetes Gestacional , Canales Catiónicos TRPM , Animales , Glucemia/metabolismo , Calcio/metabolismo , Femenino , Células HEK293 , Humanos , Insulina/metabolismo , Secreción de Insulina , Ratones , Embarazo , Progesterona , Sulfatos/metabolismoRESUMEN
BACKGROUND: Fibromyalgia syndrome (FMS) is the most common chronic widespread pain condition in rheumatology. Until recently, no clear pathophysiological mechanism for fibromyalgia had been established, resulting in management challenges. Recent research has indicated that serum immunoglobulin Gs (IgGs) may play a role in FMS. We undertook a research prioritisation exercise to identify the most pertinent research approaches that may lead to clinically implementable outputs. METHODS: Research priority setting was conducted in five phases: situation analysis; design; expert group consultation; interim recommendations; consultation and revision. A dialogue model was used, and an international multi-stakeholder expert group was invited. Clinical, patient, industry, funder, and scientific expertise was represented throughout. Recommendation-consensus was determined via a voluntary closed eSurvey. Reporting guideline for priority setting of health research were employed to support implementation and maximise impact. RESULTS: Arising from the expert group consultation (n = 29 participants), 39 interim recommendations were defined. A response rate of 81.5% was achieved in the consensus survey. Six recommendations were identified as high priority- and 15 as medium level priority. The recommendations range from aspects of fibromyalgia features that should be considered in future autoantibody research, to specific immunological investigations, suggestions for trial design in FMS, and therapeutic interventions that should be assessed in trials. CONCLUSIONS: By applying the principles of strategic priority setting we directed research towards that which is implementable, thereby expediating the benefit to the FMS patient population. These recommendations are intended for patients, international professionals and grant-giving bodies concerned with research into causes and management of patients with fibromyalgia syndrome.
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Dolor Crónico , Fibromialgia , Autoanticuerpos , Fibromialgia/terapia , Humanos , Inmunoglobulina G , Encuestas y CuestionariosRESUMEN
Fibromyalgia syndrome (FMS) is characterized by widespread pain and tenderness, and patients typically experience fatigue and emotional distress. The etiology and pathophysiology of fibromyalgia are not fully explained and there are no effective drug treatments. Here we show that IgG from FMS patients produced sensory hypersensitivity by sensitizing nociceptive neurons. Mice treated with IgG from FMS patients displayed increased sensitivity to noxious mechanical and cold stimulation, and nociceptive fibers in skin-nerve preparations from mice treated with FMS IgG displayed an increased responsiveness to cold and mechanical stimulation. These mice also displayed reduced locomotor activity, reduced paw grip strength, and a loss of intraepidermal innervation. In contrast, transfer of IgG-depleted serum from FMS patients or IgG from healthy control subjects had no effect. Patient IgG did not activate naive sensory neurons directly. IgG from FMS patients labeled satellite glial cells and neurons in vivo and in vitro, as well as myelinated fiber tracts and a small number of macrophages and endothelial cells in mouse dorsal root ganglia (DRG), but no cells in the spinal cord. Furthermore, FMS IgG bound to human DRG. Our results demonstrate that IgG from FMS patients produces painful sensory hypersensitivities by sensitizing peripheral nociceptive afferents and suggest that therapies reducing patient IgG titers may be effective for fibromyalgia.
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Fibromialgia/inmunología , Fibromialgia/fisiopatología , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Fibromialgia/etiología , Ganglios Espinales/fisiopatología , Humanos , Inmunización Pasiva , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Nociceptores/inmunología , Nociceptores/fisiología , Dolor/fisiopatología , Umbral del Dolor/fisiologíaRESUMEN
Animal models are important tools in diabetes research because ethical and logistical constraints limit access to human tissue. ß-Cell dysfunction is a common contributor to the pathogenesis of most types of diabetes. Spontaneous hyperglycemia was developed in a colony of C57BL/6J mice at King's College London (KCL). Sequencing identified a mutation in the Ins2 gene, causing a glycine-to-serine substitution at position 32 on the B chain of the preproinsulin 2 molecule. Mice with the Ins2 +/G32S mutation were named KCL Ins2 G32S (KINGS) mice. The same mutation in humans (rs80356664) causes dominantly inherited neonatal diabetes. Mice were characterized, and ß-cell function was investigated. Male mice became overtly diabetic at â¼5 weeks of age, whereas female mice had only slightly elevated nonfasting glycemia. Islets showed decreased insulin content and impaired glucose-induced insulin secretion, which was more severe in males. Transmission electron microscopy and studies of gene and protein expression showed ß-cell endoplasmic reticulum (ER) stress in both sexes. Despite this, ß-cell numbers were only slightly reduced in older animals. In conclusion, the KINGS mouse is a novel model of a human form of diabetes that may be useful to study ß-cell responses to ER stress.
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Diabetes Mellitus/genética , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/metabolismo , Animales , Ecosistema , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gßγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gßγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gßγ sink ßARK1-ct (C terminus of ß-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gßγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gßγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gßγ subunits and TRPM3. Since Gßγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gßγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gßγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gßγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.
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Subunidades beta de la Proteína de Unión al GTP/fisiología , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Receptores Acoplados a Proteínas G/fisiología , Canales Catiónicos TRPM/fisiología , Animales , Conducta Animal , Femenino , Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades gamma de la Proteína de Unión al GTP/antagonistas & inhibidores , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Células HEK293 , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Nociceptores/efectos de los fármacos , Toxina del Pertussis/farmacología , Receptor de Adenosina A2B/fisiología , Receptor Muscarínico M1/fisiología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/fisiología , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/genéticaRESUMEN
Complex regional pain syndrome (CRPS) is a posttraumatic pain condition with an incompletely understood pathophysiological basis. Here, we have examined the cellular basis of pain in CRPS using behavioral and electrophysiological methods in mice treated with IgG from CRPS patients, in combination with a paw incision. Mice were subjected to a hind paw skin-muscle incision alone, or in combination with administration of IgG purified from either healthy control subjects or patients with persistent CRPS. Nociceptive function was examined behaviorally in vivo, and electrophysiologically in vitro using skin-nerve preparations to study the major classes of mechanosensitive single units. Administration of IgG from CRPS patients exacerbated and prolonged the postsurgical hypersensitivity to noxious mechanical, cold, and heat stimulation, but did not influence tactile sensitivity after a paw incision. Studies of IgG preparations pooled from patient cohorts (n = 26-27) show that pathological autoantibodies are present in the wider population of patients with persistent CRPS, and that patients with more severe pain have higher effective autoantibody titres than patients with moderate pain intensity. Electrophysiological investigation of skin-nerve preparations from mice treated with CRPS IgG from a single patient identified both a significantly increased evoked impulse activity in A and C nociceptors, and an increased spontaneous impulse rate in the intact saphenous nerve. Our results show that painful hypersensitivity in persistent CRPS is maintained by autoantibodies, which act by sensitizing A and C nociceptors.
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Autoanticuerpos , Síndromes de Dolor Regional Complejo/fisiopatología , Hiperalgesia/fisiopatología , Nociceptores/fisiología , Umbral del Dolor/fisiología , Animales , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G , Ratones , Dimensión del Dolor , Piel/inervaciónRESUMEN
Increasing evidence suggests that nerve fibers responding to noxious stimuli (nociceptors) modulate immunity in a variety of tissues, including the skin. Yet, the role of nociceptors in regulating sterile cutaneous inflammation remains unexplored. To address this question, we have developed a detailed description of the sterile inflammation caused by overexposure to UVB irradiation (i.e., sunburn) in the mouse plantar skin. Using this model, we observed that chemical depletion of nociceptor terminals did not alter the early phase of the inflammatory response to UVB, but it caused a significant increase in the number of dendritic cells and αß+ T cells as well as enhanced extravasation during the later stages of inflammation. Finally, we showed that such regulation was driven by the nociceptive neuropeptide calcitonin gene-related peptide. In conclusion, we propose that nociceptors not only play a crucial role in inflammation through avoidance reflexes and behaviors, but can also regulate sterile cutaneous immunity in vivo.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Dermatitis/inmunología , Nociceptores/inmunología , Piel/efectos de la radiación , Quemadura Solar/inmunología , Animales , Péptido Relacionado con Gen de Calcitonina/genética , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Diterpenos/toxicidad , Femenino , Humanos , Ratones , Ratones Noqueados , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/inmunología , Fibras Nerviosas/metabolismo , Neurotoxinas/toxicidad , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Piel/citología , Piel/inmunología , Piel/inervación , Canal Catiónico TRPA1/genética , Canal Catiónico TRPA1/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
The mechanisms responsible for painful and insensate diabetic neuropathy are not completely understood. Here, we have investigated sensory neuropathy in the Ins2+/Akita mouse, a hereditary model of diabetes. Akita mice become diabetic soon after weaning, and we show that this is accompanied by an impaired mechanical and thermal nociception and a significant loss of intraepidermal nerve fibers. Electrophysiological investigations of skin-nerve preparations identified a reduced rate of action potential discharge in Ins2+/Akita mechanonociceptors compared with wild-type littermates, whereas the function of low-threshold A-fibers was essentially intact. Studies of isolated sensory neurons demonstrated a markedly reduced heat responsiveness in Ins2+/Akita dorsal root ganglion (DRG) neurons, but a mostly unchanged function of cold-sensitive neurons. Restoration of normal glucose control by islet transplantation produced a rapid recovery of nociception, which occurred before normoglycemia had been achieved. Islet transplantation also restored Ins2+/Akita intraepidermal nerve fiber density to the same level as wild-type mice, indicating that restored insulin production can reverse both sensory and anatomical abnormalities of diabetic neuropathy in mice. The reduced rate of action potential discharge in nociceptive fibers and the impaired heat responsiveness of Ins2+/Akita DRG neurons suggest that ionic sensory transduction and transmission mechanisms are modified by diabetes.
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Neuropatías Diabéticas/metabolismo , Epidermis/inervación , Ganglios Espinales/metabolismo , Insulina/metabolismo , Fibras Nerviosas Amielínicas/metabolismo , Trastornos Somatosensoriales/metabolismo , Termorreceptores/metabolismo , Potenciales de Acción , Sustitución de Aminoácidos , Animales , Conducta Animal , Células Cultivadas , Diabetes Mellitus/sangre , Diabetes Mellitus/cirugía , Neuropatías Diabéticas/patología , Neuropatías Diabéticas/fisiopatología , Neuropatías Diabéticas/prevención & control , Epidermis/metabolismo , Epidermis/patología , Epidermis/fisiopatología , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Heterocigoto , Insulina/genética , Trasplante de Islotes Pancreáticos , Riñón , Masculino , Mecanorreceptores/metabolismo , Mecanorreceptores/patología , Ratones Endogámicos C57BL , Ratones Mutantes , Fibras Nerviosas Amielínicas/patología , Dimensión del Dolor , Trastornos Somatosensoriales/complicaciones , Trastornos Somatosensoriales/fisiopatología , Trastornos Somatosensoriales/prevención & control , Termorreceptores/patología , Termorreceptores/fisiopatología , Trasplante HeterotópicoRESUMEN
Transient receptor potential (TRP) ion channels in peripheral sensory neurons are functionally regulated by hydrolysis of the phosphoinositide PI(4,5)P2 and changes in the level of protein kinase mediated phosphorylation following activation of various G protein coupled receptors. We now show that the activity of TRPM3 expressed in mouse dorsal root ganglion (DRG) neurons is inhibited by agonists of the Gi-coupled µ opioid, GABA-B and NPY receptors. These agonist effects are mediated by direct inhibition of TRPM3 by Gßγ subunits, rather than by a canonical cAMP mediated mechanism. The activity of TRPM3 in DRG neurons is also negatively modulated by tonic, constitutive GPCR activity as TRPM3 responses can be potentiated by GPCR inverse agonists. GPCR regulation of TRPM3 is also seen in vivo where Gi/o GPCRs agonists inhibited and inverse agonists potentiated TRPM3 mediated nociceptive behavioural responses.
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Subunidades beta de la Proteína de Unión al GTP/antagonistas & inhibidores , Subunidades gamma de la Proteína de Unión al GTP/antagonistas & inhibidores , Canales Iónicos/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Canales Catiónicos TRPM/efectos de los fármacos , Analgésicos Opioides/antagonistas & inhibidores , Animales , Baclofeno/antagonistas & inhibidores , Células CHO , Calcio/análisis , Capsaicina , Cricetulus , Electrofisiología/métodos , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfina/antagonistas & inhibidores , Dolor/metabolismo , Dimensión del Dolor , Fosfatidilinositoles/metabolismo , Receptor Cannabinoide CB1/agonistas , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
Sensory-guided fractionation of extracts of Tasmanian pepper berries revealed 20 drimane sesquiterpens, among which polygodial, warburganal, and 1ß-acetoxy-9-deoxy-isomuzigadial exhibited the lowest pungency threshold concentrations on the tongue surface (0.6-2.8 nmol/cm2) and elicited a dose-dependent calcium influx into mTRPA1 expressing CHO cells with the lowest EC50 values (4.5 ± 1.0 to 16.7 ± 7.5 µmol/L) and a good correlation to oral pungency thresholds (R2 = 0.986, linear regression). Calcium imaging assays demonstrated these chemosensates to induce a calcium influx into cultured trigeminal neurons prepared from wildtype (TRPA1+/+) mice, whereas no calcium influx was observed in neurons from TRPA1 knockout mice (TRPA1-/-), thus confirming the α,ß-unsaturated 1,4-dialdehyde structure to be the required structural motif for a low oral puncency thresholds and activation of the Transient Receptor Potential Channel A1 (TRPA1). Time-resolved NMR experiments confirmed the pungency mediating mechanism for electrophilic drimane sesquiterpene dialdehydes to be different from that found for other electrophilic pungent agents like isothiocyanates, which have been shown to undergo a covalent binding with cysteine residues in TRPA1. Instead, the high-impact chemosensates polygodial, warburganal, and 1ß-acetoxy-9-deoxy-isomuzigadial showed immediate reactivity with the ε-amino group of lysine side chains to give pyrrole-type conjugates, thus showing evidence for TRPA1 activation by covalent lysine modification.
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Canales de Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Extractos Vegetales/química , Sesquiterpenos/química , Gusto , Canales de Potencial de Receptor Transitorio/metabolismo , Winteraceae/química , Adulto , Animales , Células CHO , Calcio/metabolismo , Canales de Calcio/genética , Cricetulus , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Extractos Vegetales/metabolismo , Sesquiterpenos Policíclicos , Sesquiterpenos/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/genética , Winteraceae/metabolismo , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Transient receptor potential ankyrin-1 (TRPA1) activation is known to mediate neurogenic vasodilatation. We investigated the mechanisms involved in TRPA1-mediated peripheral vasodilatation in vivo using the TRPA1 agonist cinnamaldehyde. EXPERIMENTAL APPROACH: Changes in vascular ear blood flow were measured in anaesthetized mice using laser Doppler flowmetry. KEY RESULTS: Topical application of cinnamaldehyde to the mouse ear caused a significant increase in blood flow in the skin of anaesthetized wild-type (WT) mice but not in TRPA1 knockout (KO) mice. Cinnamaldehyde-induced vasodilatation was inhibited by the pharmacological blockade of the potent microvascular vasodilator neuropeptide CGRP and neuronal NOS-derived NO pathways. Cinnamaldehyde-mediated vasodilatation was significantly reduced by treatment with reactive oxygen nitrogen species (RONS) scavenger such as catalase and the SOD mimetic TEMPOL, supporting a role of RONS in the downstream vasodilator TRPA1-mediated response. Co-treatment with a non-selective NOS inhibitor L-NAME and antioxidant apocynin further inhibited the TRPA1-mediated vasodilatation. Cinnamaldehyde treatment induced the generation of peroxynitrite that was blocked by the peroxynitrite scavenger FeTPPS and shown to be dependent on TRPA1, as reflected by an increase in protein tyrosine nitration in the skin of WT, but not in TRPA1 KO mice. CONCLUSION AND IMPLICATIONS: This study provides in vivo evidence that TRPA1-induced vasodilatation mediated by cinnamaldehyde requires neuronal NOS-derived NO, in addition to the traditional neuropeptide component. A novel role of peroxynitrite is revealed, which is generated downstream of TRPA1 activation by cinnamaldehyde. This mechanistic pathway underlying TRPA1-mediated vasodilatation may be important in understanding the role of TRPA1 in pathophysiological situations.
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Péptido Relacionado con Gen de Calcitonina/metabolismo , Neurogénesis , Óxidos de Nitrógeno/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Vasodilatación , Acroleína/análogos & derivados , Acroleína/farmacología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis/efectos de los fármacos , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/deficiencia , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. METHODS: Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. RESULTS: Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. CONCLUSIONS: We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.
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Artritis Experimental/metabolismo , Velocidad del Flujo Sanguíneo/fisiología , Frío/efectos adversos , Adyuvante de Freund/toxicidad , Articulaciones/metabolismo , Canales de Potencial de Receptor Transitorio/biosíntesis , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/patología , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Adyuvante de Freund/administración & dosificación , Miembro Posterior/efectos de los fármacos , Miembro Posterior/metabolismo , Miembro Posterior/patología , Inyecciones Intraarticulares , Articulaciones/efectos de los fármacos , Articulaciones/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/deficienciaRESUMEN
OBJECTIVE: Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain-sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA-related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation. METHODS: Ten-week-old mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Pain-related behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once pain-related behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcription-polymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wild-type or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbent assay. RESULTS: Mice developed pain-related behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying pain-related behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor ß-activated kinase 1-, fibroblast growth factor 2-, and Src kinase-dependent manner. CONCLUSION: Damaged joint tissues produce proalgesic molecules, including NGF, in murine OA.
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Conducta Animal , Huesos/metabolismo , Cartílago Articular/metabolismo , Meniscos Tibiales/metabolismo , Dolor Nociceptivo/genética , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica , Quinasas Quinasa Quinasa PAM , Ratones , Factor de Crecimiento Nervioso/genética , Dolor Nociceptivo/metabolismo , Osteoartritis de la Rodilla , Dolor/genética , Dolor/metabolismo , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B2/genética , Receptores de Neuroquinina-1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Taquicininas/genética , Lesiones de Menisco Tibial , Familia-src QuinasasRESUMEN
Acetaminophen (APAP) is an effective antipyretic and one of the most commonly used analgesic drugs. Unlike antipyretic non-steroidal anti-inflammatory drugs, APAP elicits hypothermia in addition to its antipyretic effect. Here we have examined the mechanisms responsible for the hypothermic activity of APAP. Subcutaneous, but not intrathecal, administration of APAP elicited a dose dependent decrease in body temperature in wildtype mice. Hypothermia was abolished in mice pre-treated with resiniferatoxin to destroy or defunctionalize peripheral TRPV1-expressing terminals, but resistant to inhibition of cyclo-oxygenases. The hypothermic activity was independent of TRPV1 since APAP evoked hypothermia was identical in wildtype and Trpv1(-/-) mice, and not reduced by administration of a maximally effective dose of a TRPV1 antagonist. In contrast, a TRPA1 antagonist inhibited APAP induced hypothermia and APAP was without effect on body temperature in Trpa1(-/-) mice. In a model of yeast induced pyrexia, administration of APAP evoked a marked hypothermia in wildtype and Trpv1(-/-) mice, but only restored normal body temperature in Trpa1(-/-) and Trpa1(-/-)/Trpv1(-/-) mice. We conclude that TRPA1 mediates APAP evoked hypothermia.
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Acetaminofén/farmacología , Hipotermia Inducida , Canales de Potencial de Receptor Transitorio/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacología , Animales , Antipiréticos/farmacología , Benzoquinonas/farmacocinética , Diterpenos/farmacología , Femenino , Hipotermia/metabolismo , Iminas/farmacocinética , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Células Receptoras Sensoriales/efectos de los fármacos , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales de Potencial de Receptor Transitorio/agonistas , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Specific peripheral sensory neurons respond to increases in extracellular osmolality but the mechanism responsible for excitation is unknown. Here we show that small increases in osmolality excite isolated mouse dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons expressing the cold-sensitive TRPM8 channel (transient receptor potential channel, subfamily M, member 8). Hyperosmotic responses were abolished by TRPM8 antagonists, and were absent in DRG and TG neurons isolated from Trpm8(-/-) mice. Heterologously expressed TRPM8 was activated by increased osmolality around physiological levels and inhibited by reduced osmolality. Electrophysiological studies in a mouse corneal preparation demonstrated that osmolality regulated the electrical activity of TRPM8-expressing corneal afferent neurons. Finally, the frequency of eye blinks was reduced in Trpm8(-/-) compared with wild-type mice and topical administration of a TRPM8 antagonist reduced blinking in wild-type mice. Our findings identify TRPM8 as a peripheral osmosensor responsible for the regulation of normal eye-blinking in mice.
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Parpadeo , Células Receptoras Sensoriales/fisiología , Canales Catiónicos TRPM/fisiología , Potenciales de Acción , Animales , Células CHO , Frío , Córnea/fisiología , Cricetinae , Cricetulus , Femenino , Masculino , Ratones , Ratones Noqueados , Concentración OsmolarRESUMEN
A role for proteinase-activated receptor-4 (PAR-4) was recently suggested in itch sensation. Here, we investigated the mechanisms underlying the pruriceptive actions of the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice. Dorsal intradermal (i.d.) administration of AYP elicited intense scratching behavior in mice, which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10). PAR-4 was found to be coexpressed in 32% of tryptase-positive skin mast cells, and AYP caused a 2-fold increase in mast cell degranulation. However, neither the treatment with cromolyn nor the deficiency of mast cells (WBB6F1-Kit(W/Wv) mice) was able to affect AYP-induced itch. PAR-4 was also found on gastrin-releasing peptide (GRP)-positive neurons (pruriceptive fibers), and AYP-induced itch was reduced by the selective GRP receptor antagonist RC-3095. In addition, AYP evoked calcium influx in â¼1.5% of cultured DRG neurons also sensitive to TRPV1 (capsaicin) and/or TRPA1 (AITC) agonists. Importantly, AYP-induced itch was reduced by treatment with either the selective TRPV1 (SB366791), TRPA1 (HC-030031), or NK1 (FK888) receptor antagonists. However, genetic loss of TRPV1, but not of TRPA1, diminished AYP-induced calcium influx in DRG neurons and the scratching behavior in mice. These findings provide evidence that PAR-4 activation by AYP causes pruriceptive itch in mice via a TRPV1/TRPA1-dependent mechanism.
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Capsaicina/farmacología , Prurito/fisiopatología , Receptores de Bombesina/metabolismo , Receptores de Trombina/metabolismo , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Animales , Conducta Animal , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/citología , Inmunohistoquímica , Inyecciones Intradérmicas , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prurito/inducido químicamente , Prurito/psicología , Distribución Aleatoria , Valores de Referencia , Transducción de Señal , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Streptozotocin (STZ)-induced diabetes is the most commonly used animal model of diabetes. Here, we have demonstrated that intraplantar injections of low dose STZ evoked acute polymodal hypersensitivities in mice. These hypersensitivities were inhibited by a TRPA1 antagonist and were absent in TRPA1-null mice. In wild type mice, systemic STZ treatment (180 mg/kg) evoked a loss of cold and mechanical sensitivity within an hour of injection, which lasted for at least 10 days. In contrast, Trpa1(-/-) mice developed mechanical, cold, and heat hypersensitivity 24 h after STZ. The TRPA1-dependent sensory loss produced by STZ occurs before the onset of diabetes and may thus not be readily distinguished from the similar sensory abnormalities produced by the ensuing diabetic neuropathy. In vitro, STZ activated TRPA1 in isolated sensory neurons, TRPA1 cell lines, and membrane patches. Mass spectrometry studies revealed that STZ oxidizes TRPA1 cysteines to disulfides and sulfenic acids. Furthermore, incubation of tyrosine with STZ resulted in formation of dityrosine, suggesting formation of peroxynitrite. Functional analysis of TRPA1 mutants showed that cysteine residues that were oxidized by STZ were important for TRPA1 responsiveness to STZ. Our results have identified oxidation of TRPA1 cysteine residues, most likely by peroxynitrite, as a novel mechanism of action of STZ. Direct stimulation of TRPA1 complicates the interpretation of results from STZ models of diabetic sensory neuropathy and strongly argues that more refined models of diabetic neuropathy should replace the use of STZ.
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Ácido Peroxinitroso/metabolismo , Estreptozocina/farmacología , Canales de Potencial de Receptor Transitorio/efectos de los fármacos , Analgésicos/farmacología , Animales , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a sensor of nociceptive stimuli, expressed predominantly in a subpopulation of peptidergic sensory neurons which co-express the noxious heat-sensor transient receptor potential vanilloid 1. In this study, we describe a spinal cord synaptosome-calcitonin gene-related peptide (CGRP) release assay for examining activation of TRPA1 natively expressed on the central terminals of dorsal root ganglion neurons. We have shown for the first time that activation of TRPA1 channels expressed on spinal cord synaptosomes by a selection of agonists evokes a concentration-dependent release of CGRP which is inhibited by TRPA1 antagonists. In addition, our results demonstrate that depolarization of spinal cord synaptosomes by a high concentration of KCl induces CGRP release via a T-type calcium channel-dependent mechanism whilst TRPA1-induced CGRP release functions independently of voltage-gated calcium channel activation. Finally, we have shown that pre-treatment of synaptosomes with the opioid agonist, morphine, results in a reduction of depolarization-induced CGRP release. This study has demonstrated the use of a dorsal spinal cord homogenate assay for investigation of natively expressed TRPA1 channels and for modulation of depolarizing stimuli at the level of the dorsal spinal cord.