RESUMO
KEY POINTS: TRESK background K+ channel is expressed in sensory neurons and acts as a brake to reduce neuronal activation. Deletion of the channel enhances the excitability of nociceptors. Skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation in TRESK knockout animals. Channel deletion selectively enhances mechanical and cold sensitivity in mice, without altering sensitivity to heat. These results indicate that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing. ABSTRACT: Background potassium-permeable ion channels play a critical role in tuning the excitability of nociceptors, yet the precise role played by different subsets of channels is not fully understood. Decreases in TRESK (TWIK-related spinal cord K+ channel) expression/function enhance excitability of sensory neurons, but its role in somatosensory perception and nociception is poorly understood. Here, we used a TRESK knockout (KO) mouse to address these questions. We show that TRESK regulates the sensitivity of sensory neurons in a modality-specific manner, contributing to mechanical and cold sensitivity but without any effect on heat sensitivity. Nociceptive neurons isolated from TRESK KO mice show a decreased threshold for activation and skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation that was also observed in behavioural tests in vivo. TRESK is also involved in osmotic pain and in early phases of formalin-induced inflammatory pain, but not in the development of mechanical and heat hyperalgesia during chronic pain. In contrast, mice lacking TRESK present cold allodynia that is not further enhanced by oxaliplatin. In summary, genetic removal of TRESK uncovers enhanced mechanical and cold sensitivity, indicating that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing, acting as a brake to prevent activation by innocuous stimuli.
Assuntos
Nociceptores , Canais de Potássio , Animais , Hiperalgesia/genética , Camundongos , Nociceptividade , Células Receptoras SensoriaisRESUMO
TRESK belongs to the K2P family of potassium channels, also known as background or leak potassium channels due to their biophysical properties and their role regulating membrane potential of cells. Several studies to date have highlighted the role of TRESK in regulating the excitability of specific subtypes of sensory neurons. These findings suggest TRESK could be involved in pain sensitivity. Here, we review the different evidence available that involves the channel in pain and sensory perception, from studies knocking out the channel or overexpressing it to identified mutations that link the channel to migraine pain. In addition, the therapeutic possibilities are discussed, as targeting the channel seems an interesting therapeutic approach to reduce nociceptor activation and to decrease pain.
Assuntos
Potenciais da Membrana/genética , Mutação , Nociceptividade , Manejo da Dor , Dor , Canais de Potássio , Células Receptoras Sensoriais , Humanos , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/patologia , Transtornos de Enxaqueca/terapia , Dor/genética , Dor/metabolismo , Dor/patologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologiaRESUMO
OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. DESIGN: Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. RESULTS: Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. CONCLUSIONS: We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.
Assuntos
Colo/inervação , Células Receptoras Sensoriais/classificação , Análise de Sequência de RNA , Transcriptoma , Animais , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Animais , Membrana Celular/genética , Guanidinas/farmacologia , Ratos-Toupeira , Subunidades Proteicas/genética , Quinazolinas/farmacologiaRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
Assuntos
Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Encefalopatias/patologia , Canais de Cloro CLC-2 , Canais de Cálcio Tipo L/genética , Canais de Cloreto , Cistos/genética , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Transporte Proteico/genéticaRESUMO
Arthritic conditions are a major source of chronic pain. Furthering our understanding of disease mechanisms creates the opportunity to develop more targeted therapeutics. In rheumatoid arthritis (RA), measurements of pH in human synovial fluid suggest that acidosis occurs, but that this is highly variable between individuals. Here we sought to determine if tissue acidosis occurs in a widely used rodent arthritis model: complete Freund's adjuvant (CFA)-induced inflammation. CFA robustly evoked paw and ankle swelling, concomitant with worsening clinical scores over time. We used magnetic resonance spectroscopic imaging of hyperpolarized [1-13 C]pyruvate metabolism to demonstrate that CFA induces an increase in the lactate-to-pyruvate ratio. This increase is indicative of enhanced glycolysis and an increased lactate concentration, as has been observed in the synovial fluid from RA patients, and which was correlated with acidosis. We also measured the 13 CO2 /H13 CO3- ratio, in animals injected with hyperpolarized H13 CO3- , to estimate extracellular tissue pH and showed that despite the apparent increase in glycolytic activity in CFA-induced inflammation there was no accompanying decrease in extracellular pH. The pH was 7.23 ± 0.06 in control paws and 7.32 ± 0.09 in inflamed paws. These results could explain why mice lacking acid-sensing ion channel subunits 1, 2 and 3 do not display any changes in mechanical or thermal hyperalgesia in CFA-induced inflammation.
Assuntos
Acidose/patologia , Bicarbonatos/metabolismo , Isótopos de Carbono/química , Inflamação/patologia , Articulações/patologia , Ácido Láctico/metabolismo , Animais , Dióxido de Carbono/química , Modelos Animais de Doenças , Feminino , Adjuvante de Freund , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos C57BLAssuntos
Sistema Nervoso Periférico/metabolismo , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiologia , Animais , Bradicinina/farmacologia , Bradicinina/uso terapêutico , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Camundongos , Ratos-Toupeira , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/patologia , Ratos , Dor Visceral/tratamento farmacológico , Dor Visceral/patologiaRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.
Assuntos
Astrócitos/fisiologia , Canais de Cloreto/fisiologia , Cistos/fisiopatologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Proteínas/genética , Proteínas/metabolismo , Animais , Astrócitos/ultraestrutura , Canais de Cloro CLC-2 , Proteínas de Ciclo Celular , Cistos/genética , Variação Genética , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Fenótipo , Estabilidade Proteica , Interferência de RNA , Ratos , Vacúolos/fisiologiaRESUMO
ABSTRACT: The pressing need for safer, more efficacious analgesics is felt worldwide. Preclinical tests in animal models of painful conditions represent one of the earliest checkpoints novel therapeutics must negotiate before consideration for human use. Traditionally, the pain status of laboratory animals has been inferred from evoked nociceptive assays that measure their responses to noxious stimuli. The disconnect between how pain is tested in laboratory animals and how it is experienced by humans may in part explain the shortcomings of current pain medications and highlights a need for refinement. Here, we survey human patients with chronic pain who assert that everyday aspects of life, such as cleaning and leaving the house, are affected by their ongoing level of pain. Accordingly, we test the impact of painful conditions on an ethological behavior of mice, digging. Stable digging behavior was observed over time in naive mice of both sexes. By contrast, deficits in digging were seen after acute knee inflammation. The analgesia conferred by meloxicam and gabapentin was compared in the monosodium iodoacetate knee osteoarthritis model, with meloxicam more effectively ameliorating digging deficits, in line with human patients finding meloxicam more effective. Finally, in a visceral pain model, the decrease in digging behavior correlated with the extent of disease. Ultimately, we make a case for adopting ethological assays, such as digging, in studies of pain in laboratory animals, which we believe to be more representative of the human experience of pain and thus valuable in assessing clinical potential of novel analgesics in animals.
Assuntos
Comportamento Animal , Animais , Camundongos , Humanos , Masculino , Feminino , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Dor/tratamento farmacológico , Dor/psicologia , Dor/fisiopatologia , Analgésicos/uso terapêutico , Analgésicos/farmacologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Pessoa de Meia-Idade , Medição da Dor/métodos , Idoso , Dor Crônica/psicologia , Dor Crônica/tratamento farmacológico , Dor Crônica/fisiopatologia , Gabapentina/uso terapêutico , Gabapentina/farmacologia , Adulto , Meloxicam/uso terapêuticoRESUMO
By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.
Assuntos
Dor , Canais de Potássio de Domínios Poros em Tandem , Animais , Camundongos , Avaliação Pré-Clínica de Medicamentos , Nociceptores , Nematoides , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidoresRESUMO
ABSTRACT: Prdm12 is a conserved epigenetic transcriptional regulator that displays restricted expression in nociceptors of the developing peripheral nervous system. In mice, Prdm12 is required for the development of the entire nociceptive lineage. In humans, PRDM12 mutations cause congenital insensitivity to pain, likely because of the loss of nociceptors. Prdm12 expression is maintained in mature nociceptors suggesting a yet-to-be explored functional role in adults. Using Prdm12 inducible conditional knockout mouse models, we report that in adult nociceptors Prdm12 is no longer required for cell survival but continues to play a role in the transcriptional control of a network of genes, many of them encoding ion channels and receptors. We found that disruption of Prdm12 alters the excitability of dorsal root ganglion neurons in culture. Phenotypically, we observed that mice lacking Prdm12 exhibit normal responses to thermal and mechanical nociceptive stimuli but a reduced response to capsaicin and hypersensitivity to formalin-induced inflammatory pain. Together, our data indicate that Prdm12 regulates pain-related behavior in a complex way by modulating gene expression in adult nociceptors and controlling their excitability. The results encourage further studies to assess the potential of Prdm12 as a target for analgesic development.
Assuntos
Proteínas de Transporte , Gânglios Espinais , Proteínas do Tecido Nervoso , Nociceptores , Animais , Proteínas de Transporte/genética , Gânglios Espinais/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Nociceptores/fisiologia , Dor/genética , Dor/metabolismoRESUMO
BACKGROUND: Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K(+) channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K(2P) channels after peripheral axotomy in mammals. RESULTS: Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo. CONCLUSIONS: In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.
Assuntos
Neuralgia/etiologia , Nociceptores/fisiologia , Canais de Potássio/metabolismo , Nervos Espinhais/lesões , Animais , Axotomia , Células Cultivadas , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Nociceptores/metabolismo , Canais de Potássio/genética , Ratos , Ratos Sprague-DawleyRESUMO
Protons reaching the eyeball from exogenous acidic substances or released from damaged cells during inflammation, immune cells, after tissue injury or during chronic ophthalmic conditions, activate or modulate ion channels present in sensory nerve fibers that innervate the ocular anterior surface. Their identification as well as their role during disease is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies. Among the ion channels expressed in trigeminal nociceptors innervating the anterior surface of the eye (cornea and conjunctiva) and annex ocular structures (eyelids), members of the TRP and ASIC families play a critical role in ocular acidic pain. Low pH (pH 6) activates TRPV1, a polymodal ion channel also activated by heat, capsaicin and hyperosmolar conditions. ASIC1, ASIC3 and heteromeric ASIC1/ASIC3 channels present in ocular nerve terminals are activated at pH 7.2-6.5, inducing pain by moderate acidifications of the ocular surface. These channels, together with TRPA1, are involved in acute ocular pain, as well as in painful sensations during allergic keratoconjunctivitis or other ophthalmic conditions, as blocking or reducing channel expression ameliorates ocular pain. TRPV1, TRPA1 and other ion channels are also present in corneal and conjunctival cells, promoting inflammation of the ocular surface after injury. In addition to the above-mentioned ion channels, members of the K2P and P2X ion channel families are also expressed in trigeminal neurons, however, their role in ocular pain remains unclear to date. In this report, these and other ion channels and receptors involved in acid sensing during ocular pathologies and pain are reviewed.
RESUMO
OBJECTIVE: Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee-innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. We undertook this study to evaluate whether the newly engineered adeno-associated virus (AAV) serotype, AAV-PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intraarticular knee injection. METHODS: The AAV-PHP.S virus, packaged with dTomato fluorescent protein and either excitatory (Gq ) or inhibitory (Gi ) designer receptors exclusively activated by designer drugs (DREADDs), was injected into the knee joints of adult mice. Labeling of DRG neurons with AAV-PHP.S from the knee was evaluated using immunohistochemistry. The functionality of Gq - and Gi -DREADDs was evaluated using whole-cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing, and rotarod performance, before and after intraperitoneal administration of the DREADD activator, Compound 21. RESULTS: We showed that AAV-PHP.S can deliver functional genes into ~7% of lumbar DRG neurons when injected into the knee joint in a similar manner to the well-established retrograde tracer, fast blue. Short-term activation of AAV-PHP.S-delivered Gq -DREADD increased excitability of knee neurons in vitro (P = 0.02 by unpaired t-test), without inducing overt pain in mice when activated in vivo. By contrast, in vivo Gi -DREADD activation alleviated digging deficits induced by Freund's complete adjuvant-mediated knee inflammation (P = 0.0002 by repeated-measures analysis of variance [ANOVA] followed by Holm-Sidak multiple comparisons test). A concomitant decrease in knee neuron excitability was observed in vitro (P = 0.005 by ANOVA followed by Holm-Sidak multiple comparisons test). CONCLUSION: We describe an AAV-mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.
Assuntos
Gânglios Espinais/metabolismo , Terapia Genética/métodos , Inflamação/terapia , Neurônios/metabolismo , Manejo da Dor/métodos , Dor/metabolismo , Animais , Dependovirus , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Articulação do Joelho/metabolismo , CamundongosRESUMO
ABSTRACT: Pain is a principal contributor to the global burden of arthritis with peripheral sensitization being a major cause of arthritis-related pain. Within the knee joint, distal endings of dorsal root ganglion neurons (knee neurons) interact with fibroblast-like synoviocytes (FLS) and the inflammatory mediators they secrete, which are thought to promote peripheral sensitization. Correspondingly, RNA sequencing has demonstrated detectable levels of proinflammatory genes in FLS derived from arthritis patients. This study confirms that stimulation with tumor necrosis factor (TNF-α) results in expression of proinflammatory genes in mouse and human FLS (derived from osteoarthritis and rheumatoid arthritis patients), as well as increased secretion of cytokines from mouse TNF-α-stimulated FLS (TNF-FLS). Electrophysiological recordings from retrograde labelled knee neurons cocultured with TNF-FLS, or supernatant derived from TNF-FLS, revealed a depolarized resting membrane potential, increased spontaneous action potential firing, and enhanced TRPV1 function, all consistent with a role for FLS in mediating the sensitization of pain-sensing nerves in arthritis. Therefore, data from this study demonstrate the ability of FLS activated by TNF-α to promote neuronal sensitization, results that highlight the importance of both nonneuronal and neuronal cells to the development of pain in arthritis.
Assuntos
Sinoviócitos , Animais , Células Cultivadas , Técnicas de Cocultura , Fibroblastos , Humanos , Articulação do Joelho , Camundongos , Dor , Células Receptoras Sensoriais , Membrana Sinovial , Fator de Necrose Tumoral alfaRESUMO
Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an α2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks α2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Simulação por Computador , Guanabenzo/análogos & derivados , Guanabenzo/metabolismo , Guanidinas/metabolismo , Quinazolinas/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Guanabenzo/química , Guanabenzo/farmacologia , Guanidinas/química , Guanidinas/farmacologia , Estrutura Secundária de Proteína , Quinazolinas/química , Quinazolinas/farmacologiaRESUMO
By studying healthy women who do not request analgesia during their first delivery, we investigate genetic effects on labor pain. Such women have normal sensory and psychometric test results, except for significantly higher cuff pressure pain. We find an excess of heterozygotes carrying the rare allele of SNP rs140124801 in KCNG4. The rare variant KV6.4-Met419 has a dominant-negative effect and cannot modulate the voltage dependence of KV2.1 inactivation because it fails to traffic to the plasma membrane. In vivo, Kcng4 (KV6.4) expression occurs in 40% of retrograde-labeled mouse uterine sensory neurons, all of which express KV2.1, and over 90% express the nociceptor genes Trpv1 and Scn10a. In neurons overexpressing KV6.4-Met419, the voltage dependence of inactivation for KV2.1 is more depolarized compared with neurons overexpressing KV6.4. Finally, KV6.4-Met419-overexpressing neurons have a higher action potential threshold. We conclude that KV6.4 can influence human labor pain by modulating the excitability of uterine nociceptors.
Assuntos
Dor do Parto/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Analgésicos/farmacologia , Animais , Sequência de Bases , Membrana Celular/metabolismo , Cognição , Estudos de Coortes , Emoções , Feminino , Gânglios Espinais/metabolismo , Heterozigoto , Humanos , Ativação do Canal Iônico/genética , Dor do Parto/genética , Dor do Parto/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Nociceptores/metabolismo , Limiar da Dor , Polimorfismo de Nucleotídeo Único/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Gravidez , Multimerização Proteica , Células Receptoras Sensoriais/metabolismo , Canais de Potássio Shab/metabolismo , Frações Subcelulares/metabolismo , Útero/inervaçãoRESUMO
Nociceptors, i.e. sensory neurons tuned to detect noxious stimuli, are found in numerous phyla of the Animalia kingdom and are often polymodal, responding to a variety of stimuli, e.g. heat, cold, pressure and chemicals, such as acid. Owing to the ability of protons to have a profound effect on ionic homeostasis and damage macromolecular structures, it is no wonder that the ability to detect acid is conserved across many species. To detect changes in pH, nociceptors are equipped with an assortment of different acid sensors, some of which can detect mild changes in pH, such as the acid-sensing ion channels, proton-sensing G protein-coupled receptors and several two-pore potassium channels, whereas others, such as the transient receptor potential vanilloid 1 ion channel, require larger shifts in pH. This review will discuss the evolution of acid sensation and the different mechanisms by which nociceptors can detect acid. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Evolução Biológica , Nociceptividade , Nociceptores/metabolismo , Dor , Animais , Humanos , Dor/etiologia , Dor/fisiopatologiaRESUMO
Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.
Assuntos
Artralgia/metabolismo , Gânglios Espinais/metabolismo , Inflamação/metabolismo , Atividade Motora/fisiologia , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artralgia/tratamento farmacológico , Artralgia/patologia , Capsaicina , Células Cultivadas , Modelos Animais de Doenças , Feminino , Adjuvante de Freund , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Membro Posterior , Inflamação/tratamento farmacológico , Inflamação/patologia , Isoquinolinas/farmacologia , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Receptor trkA/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Pyrethroid insecticides are widely used for pest control in agriculture or in human public health commonly as a topical treatment for scabies and head lice. Exposure to pyrethroids such as permethrin or tetramethrin (TM) causes sensory alterations such as transient pain, burning, stinging sensations, and paraesthesias. Despite the well-known effects of pyrethroids on sodium channels, actions on other channels that control sensory neuron excitability are less studied. Given the role of 2-pore domain potassium (K2P) channels in modulating sensory neuron excitability and firing, both in physiological and pathological conditions, we examined the effect of pyrethroids on K2P channels mainly expressed in sensory neurons. Through electrophysiological and calcium imaging experiments, we show that a high percentage of TM-responding neurons were nociceptors, which were also activated by TRPA1 and/or TRPV1 agonists. This pyrethroid also activated and enhanced the excitability of peripheral saphenous nerve fibers. Pyrethroids produced a significant inhibition of native TRESK, TRAAK, TREK-1, and TREK-2 currents. Similar effects were found in transfected HEK293 cells. At the behavioral level, intradermal TM injection in the mouse paw produced nocifensive responses and caused mechanical allodynia, demonstrating that the effects seen on nociceptors in culture lead to pain-associated behaviors in vivo. In TRESK knockout mice, pain-associated behaviors elicited by TM were enhanced, providing further evidence for a role of this channel in preventing excessive neuronal activation. Our results indicate that inhibition of K2P channels facilitates sensory neuron activation and increases their excitability. These effects contribute to the generation of paraesthesias and pain after pyrethroid exposure.