RESUMEN
Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Células Endoteliales , Óxido Nítrico , Células Receptoras Sensoriales , Transducción de Señal , Sustancia P , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Sustancia P/farmacología , Sustancia P/metabolismo , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Animales , Óxido Nítrico/metabolismo , Técnicas de Cocultivo , Comunicación Celular/fisiología , Comunicación Celular/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Células Cultivadas , Humanos , RatasRESUMEN
Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.
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Analgésicos Opioides , AMP Cíclico , Gabapentina , Neuralgia , Transducción de Señal , Traumatismos de la Médula Espinal , Animales , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , AMP Cíclico/metabolismo , Ratas , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Analgésicos Opioides/farmacología , Gabapentina/farmacología , Transducción de Señal/efectos de los fármacos , Ratas Sprague-Dawley , Masculino , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Pregabalina/farmacología , Pregabalina/uso terapéutico , Sinergismo Farmacológico , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
The development of noninvasive approaches to precisely control neural activity in mammals is highly desirable. Here, we used the ion channel transient receptor potential ankyrin-repeat 1 (TRPA1) as a proof of principle, demonstrating remote near-infrared (NIR) activation of endogenous neuronal channels in mice through an engineered nanoagonist. This achievement enables specific neurostimulation in nongenetically modified mice. Initially, target-based screening identified flavins as photopharmacological agonists, allowing for the photoactivation of TRPA1 in sensory neurons upon ultraviolet A/blue light illumination. Subsequently, upconversion nanoparticles (UCNPs) were customized with an emission spectrum aligned to flavin absorption and conjugated with flavin adenine dinucleotide, creating a nanoagonist capable of NIR activation of TRPA1. Following the intrathecal injection of the nanoagonist, noninvasive NIR stimulation allows precise bidirectional control of nociception in mice through remote activation of spinal TRPA1. This study demonstrates a noninvasive NIR neurostimulation method with the potential for adaptation to various endogenous ion channels and neural processes by combining photochemical toolboxes with customized UCNPs.
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Rayos Infrarrojos , Nanopartículas , Canal Catiónico TRPA1 , Animales , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/agonistas , Ratones , Nanopartículas/química , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Canales Iónicos/metabolismo , Nocicepción/efectos de los fármacosRESUMEN
Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis.
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Neoplasias de la Mama , Metástasis de la Neoplasia , ARN , Células Receptoras Sensoriales , Sustancia P , Receptor Toll-Like 7 , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metástasis Linfática , Invasividad Neoplásica , Proteínas del Tejido Nervioso/metabolismo , ARN/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Sustancia P/metabolismo , Análisis de Supervivencia , Receptor Toll-Like 7/metabolismo , Receptores de Neuroquinina-1/metabolismoRESUMEN
Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility, often due to a lack of optimization. Here, we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors, while cell line differences also contributed to variation. A replicable dose-response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally, a replicable dose-dependent reduction in neurite outgrowth was demonstrated, demonstrating the applicability of the model for the examination of additional phenotypes. Overall, we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity.
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Supervivencia Celular , Células Madre Pluripotentes Inducidas , Células Receptoras Sensoriales , Taxoides , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Taxoides/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Docetaxel/farmacología , Síndromes de Neurotoxicidad/etiología , Hidrocarburos Aromáticos con Puentes/farmacología , Diferenciación Celular/efectos de los fármacos , Paclitaxel/farmacología , Paclitaxel/toxicidad , Línea Celular , Células CultivadasRESUMEN
Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration-response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.
Asunto(s)
Canales Iónicos Sensibles al Ácido , Ganglios Espinales , Insulina , Receptor de Insulina , Células Receptoras Sensoriales , Animales , Canales Iónicos Sensibles al Ácido/metabolismo , Insulina/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Ratas , Receptor de Insulina/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Ratas Sprague-Dawley , Hiperalgesia/metabolismo , Células CultivadasRESUMEN
Manipulation of neural circuits targeted by morphine enables pain relief without opioids.
Asunto(s)
Analgésicos Opioides , Morfina , Vías Nerviosas , Nocicepción , Manejo del Dolor , Células Receptoras Sensoriales , Animales , Humanos , Ratones , Analgésicos Opioides/farmacología , Morfina/farmacología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Nocicepción/efectos de los fármacos , Nocicepción/fisiología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiologíaRESUMEN
BACKGROUND: Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in peripheral sensory neurons and serve as sensors for various irritants. METHODS: We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from wild-type, TRPA1(-/-) and TRPV1(-/-) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection model was employed to assess econazole-induced itch and pain in vivo. RESULTS: Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(-/-) mouse DRG neurons but was not significant in the TRPV1(-/-) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole. Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole induced itch and pain in wild-type mice, with reduced responses in TRPA1(-/-) mice. CONCLUSIONS: These findings suggested that the imidazole derivatives econazole and miconazole may induce skin irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the adverse effects of econazole.
Asunto(s)
Antifúngicos , Calcio , Econazol , Ganglios Espinales , Células Receptoras Sensoriales , Canal Catiónico TRPA1 , Canales Catiónicos TRPV , Canales de Potencial de Receptor Transitorio , Animales , Econazol/farmacología , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Antifúngicos/toxicidad , Antifúngicos/farmacología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Humanos , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Células HEK293 , Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Ratones , Masculino , Ratones Noqueados , Ratones Endogámicos C57BL , Prurito/inducido químicamente , Dolor/tratamiento farmacológicoRESUMEN
Cadaverine is an endogenous metabolite produced by the gut microbiome with various activity in physiological and pathological conditions. However, whether cadaverine regulates pain or itch remains unclear. In this study, we first found that cadaverine may bind to histamine 4 receptor (H4R) with higher docking energy score using molecular docking simulations, suggesting cadaverine may act as an endogenous ligand for H4R. We subsequently found intradermal injection of cadaverine into the nape or cheek of mice induces a dose-dependent scratching response in mice, which was suppressed by a selective H4R antagonist JNJ-7777120, transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine and PLC inhibitor U73122, but not H1R antagonist or TRPA1 antagonist or TRPV4 antagonist. Consistently, cadaverine-induced itch was abolished in Trpv1-/- but not Trpa1-/- mice. Pharmacological analysis indicated that mast cells and opioid receptors were also involved in cadaverine-induced itch in mice. scRNA-Seq data analysis showed that H4R and TRPV1 are mainly co-expressed on NP2, NP3 and PEP1 DRG neurons. Calcium imaging analysis showed that cadaverine perfusion enhanced calcium influx in the dissociated dorsal root ganglion (DRG) neurons, which was suppressed by JNJ-7777120 and capsazepine, as well as in the DRG neurons from Trpv1-/- mice. Patch-clamp recordings found that cadaverine perfusion significantly increased the excitability of small diameter DRG neurons, and JNJ-7777120 abolished this effect, indicating involvement of H4R. Together, these results provide evidences that cadaverine is a novel endogenous pruritogens, which activates H4R/TRPV1 signaling pathways in the primary sensory neurons.
Asunto(s)
Cadaverina , Ganglios Espinales , Ratones Endogámicos C57BL , Prurito , Canales Catiónicos TRPV , Animales , Prurito/metabolismo , Prurito/inducido químicamente , Canales Catiónicos TRPV/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Masculino , Cadaverina/análogos & derivados , Cadaverina/farmacología , Cadaverina/metabolismo , Ratones , Ratones Noqueados , Humanos , Mastocitos/metabolismo , Mastocitos/efectos de los fármacos , Canal Catiónico TRPA1/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Capsaicina/análogos & derivadosRESUMEN
Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.
Asunto(s)
Anoctamina-1 , Axones , Dinoprostona , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Animales , Dinoprostona/farmacología , Dinoprostona/metabolismo , Axones/metabolismo , Axones/efectos de los fármacos , Axones/fisiología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ratas , Anoctamina-1/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Ratas Sprague-Dawley , Células Cultivadas , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , AMP Cíclico/metabolismoRESUMEN
Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in patients suffering from this largely untreated disease. While many intracellular signalling mechanisms have been examined in preclinical models that drive spontaneous activity, none have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we showed that inhibition of mitogen-activated protein kinase interacting kinase (MNK) with tomivosertib (eFT508, 25 nM) reversibly suppresses spontaneous activity in human sensory neurons that are likely nociceptors based on size and action potential characteristics associated with painful dermatomes within minutes of treatment. Tomivosertib treatment also decreased action potential amplitude and produced alterations in the magnitude of after hyperpolarizing currents, suggesting modification of Na+ and K+ channel activity as a consequence of drug treatment. Parallel to the effects on electrophysiology, eFT508 treatment led to a profound loss of eIF4E serine 209 phosphorylation in primary sensory neurons, a specific substrate of MNK, within 2 min of drug treatment. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain.
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Potenciales de Acción , Ganglios Espinales , Radiculopatía , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Humanos , Masculino , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Radiculopatía/tratamiento farmacológico , Células Cultivadas , Persona de Mediana Edad , Femenino , Anciano , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismoRESUMEN
In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.
Asunto(s)
Receptores ErbB , Interleucina-6 , Células Receptoras Sensoriales , Médula Espinal , Animales , Femenino , Ratones , Ratas , Artritis/metabolismo , Artritis Experimental/metabolismo , Línea Celular , Receptores ErbB/metabolismo , Ganglios Espinales/metabolismo , Gefitinib/farmacología , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal , Médula Espinal/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: and Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) constitutes a significant health problem due to the increasing prevalence and lack of therapies for treatment and prevention. While pivotal for routine cancer treatment, paclitaxel and vincristine frequently cause CIPN and impact the quality of life among cancer patients and survivors. Here, we investigate molecular mechanisms and drug transport in CIPN. EXPERIMENTAL APPROACH: Human sensory neurons were derived from induced pluripotent stem cells (iPSC-SNs), which were characterized using flow cytometry and immunolabeling. These iPSC-SNs were exposed to different concentrations of the two microtubule-targeting agents, paclitaxel and vincristine, with and without pre-exposure to inhibitors and inducers of efflux transporters. Neuronal networks were quantified via fluorescent staining against sensory neuron markers. Transcriptional effects of the chemotherapeutics were examined using quantitative polymerase chain reactions (qPCR). KEY RESULTS: Paclitaxel exposure resulted in axonal retraction and thickening, while vincristine caused fragmentation and abolishment of axons. Both agents increased the mRNA expression of the pain receptor, transient receptor potential vanilloid (TRPV1), and highly induced neuronal damage, as measured by activating transcription factor 3 (ATF3) mRNA. iPSC-SNs express the efflux transporters, P-glycoprotein (P-gp, encoded by ABCB1) and multidrug resistance-associated protein 1 (MPR1, encoded by ABCC1). Modulation of efflux transporters indicate that P-gp and MRP1 play a role in modulating neuronal accumulation and neurotoxicity in preliminary experiments. CONCLUSION: and Implications: iPSC-SNs are a valuable and robust model to study the role of efflux transporters and other mechanistic targets in CIPN. Efflux transporters may play a role in CIPN pathogenesis as they regulate the disposition of chemotherapy to the peripheral nervous system, and they may present potential therapeutic targets for CIPN.
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Células Madre Pluripotentes Inducidas , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Paclitaxel , Enfermedades del Sistema Nervioso Periférico , Células Receptoras Sensoriales , Vincristina , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Paclitaxel/toxicidad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Canales Catiónicos TRPV/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Células CultivadasRESUMEN
Clary sage essential oil (CSEO) is utilized in perfumery, aromatherapy, and skincare. Linalyl acetate (LA), a primary component of CSEO, possesses sedative, anxiolytic, and analgesic properties. However, the mechanism of its analgesic action is not clearly understood. Transient receptor potential ankyrin 1 (TRPA1) channel, a non-selective cation channel, is mainly expressed in sensory neurons and serves as a sensor of various irritants. In this study, we investigated the effects of LA on TRPA1 channel using heterologous expression system and isolated sensory neurons. To detect channel activity, we employed Ca2+ imaging and the whole-cell patch-clamp technique. The analgesic action of LA was measured in a pain-related behavioral mouse model. In cells that heterologously expressed TRPA1, LA diminished [Ca2+]i and current responses to allylisothiocyanate (AITC) and carvacrol: exogenous TRPA1 agonists, and the inhibitory effects were more pronounced for the former than for the latter. Moreover, LA suppressed [Ca2+] i and current responses to PGJ2: an endogenous TRPA1 agonist. Similar inhibitory actions were observed in native TRPA1 channels expressed in mouse sensory neurons. Furthermore, LA diminished PGJ2-induced nociceptive behaviors in mice. These findings suggest that analgesic effects of LA exert through inhibition of nociceptive TRPA1, making it a potential candidate for novel analgesic development.
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Analgésicos , Monoterpenos , Canal Catiónico TRPA1 , Animales , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Ratones , Analgésicos/farmacología , Monoterpenos/farmacología , Humanos , Masculino , Calcio/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células HEK293 , Modelos Animales de Enfermedad , Dolor/tratamiento farmacológico , Dolor/metabolismoRESUMEN
BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.
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Canales de Calcio Tipo T , Proteínas Quinasas Dependientes de AMP Cíclico , Receptores de Interleucina , Células Receptoras Sensoriales , Transducción de Señal , Ganglio del Trigémino , Familia-src Quinasas , Animales , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Familia-src Quinasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ganglio del Trigémino/metabolismo , Masculino , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Interleucinas/metabolismoRESUMEN
Activity-induced muscle pain increases interleukin-1ß (IL-1ß) release from muscle macrophages and the development of hyperalgesia is prevented by blockade of IL-1ß in muscle. Brain derived neurotrophic factor (BDNF) is released from sensory neurons in response to IL-1ß and mediates both inflammatory and neuropathic pain. Thus, we hypothesize that in activity-induced pain, fatigue metabolites combined with IL-1ß activate sensory neurons to increase BDNF release, peripherally in muscle and centrally in the spinal dorsal horn, to produce hyperalgesia. We tested the effect of intrathecal or intramuscular injection of BDNF-Tropomyosin receptor kinase B (TrkB) inhibitors, ANA-12 or TrkB-Fc, on development of activity-induced pain. Both inhibitors prevented the hyperalgesia when given before or 24hr after induction of the model in male but not female mice. BDNF messenger ribonucleic acid (mRNA) and protein were significantly increased in dorsal root ganglion (DRG) 24hr after induction of the model in both male and female mice. Blockade of IL-1ß in muscle had no effect on the increased BNDF mRNA observed in the activity-induced pain model, while IL-1ß applied to cultured DRG significantly induced BDNF expression, suggesting IL-1ß is sufficient but not necessary to induce BNDF. Thus, fatigue metabolites, combined with IL-1ß, upregulate BDNF in primary DRG neurons in both male and female mice, but contribute to activity-induced pain only in males.
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Factor Neurotrófico Derivado del Encéfalo , Ganglios Espinales , Hiperalgesia , Interleucina-1beta , Mialgia , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Masculino , Femenino , Ratones , Ganglios Espinales/metabolismo , Interleucina-1beta/metabolismo , Mialgia/metabolismo , Hiperalgesia/metabolismo , Ratones Endogámicos C57BL , Receptor trkB/metabolismo , Músculo Esquelético/metabolismo , Factores Sexuales , Caracteres Sexuales , Benzamidas/farmacología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , AzepinasRESUMEN
Cannabinoid CB2 agonists show therapeutic efficacy without unwanted CB1-mediated side effects. The G protein-biased CB2 receptor agonist LY2828360 attenuates the maintenance of chemotherapy-induced neuropathic nociception in male mice and blocks development of morphine tolerance in this model. However, the cell types involved in this phenomenon are unknown and whether this therapeutic profile is observed in female mice has never been investigated. We used conditional deletion of CB2 receptors to determine the cell population(s) mediating the anti-allodynic and morphine-sparing effects of CB2 agonists. Anti-allodynic effects of structurally distinct CB2 agonists (LY2828360 and AM1710) were present in paclitaxel-treated CB2f/f mice and in mice lacking CB2 receptors in CX3CR1 expressing microglia/macrophages (CX3CR1CRE/+; CB2f/f), but were absent in mice lacking CB2 receptors in peripheral sensory neurons (AdvillinCRE/+; CB2f/f). The morphine-sparing effect of LY28282360 occurred in a sexually-dimorphic manner, being present in male, but not female, mice. LY2828360 treatment (3â¯mg/kg per day i.p. x 12 days) blocked the development of morphine tolerance in male CB2f/f and CX3CR1CRE/+; CB2f/f mice with established paclitaxel-induced neuropathy but was absent in male (or female) AdvillinCRE/+; CB2f/f mice. Co-administration of morphine with a low dose of LY2828360 (0.1â¯mg/kg per day i.p. x 6 days) reversed morphine tolerance in paclitaxel-treated male CB2f/f mice, but not AdvillinCRE/+; CB2f/f mice of either sex. LY2828360 (3â¯mg/kg per day i.p. x 8 days) delayed, but did not prevent, the development of paclitaxel-induced mechanical or cold allodynia in either CB2f/f or CX3CR1CRE/+; CB2f/f mice of either sex. Our findings have potential clinical implications.
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Tolerancia a Medicamentos , Morfina , Neuralgia , Paclitaxel , Receptor Cannabinoide CB2 , Células Receptoras Sensoriales , Animales , Masculino , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/metabolismo , Receptor Cannabinoide CB2/genética , Femenino , Morfina/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Tolerancia a Medicamentos/fisiología , Ratones , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Nocicepción/efectos de los fármacos , Ratones Endogámicos C57BL , Caracteres Sexuales , Ratones Noqueados , Agonistas de Receptores de Cannabinoides/farmacologíaRESUMEN
Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.
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Senescencia Celular , Osteoclastos , Animales , Ratones , Osteoclastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Senescencia Celular/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Células Receptoras Sensoriales/metabolismo , Modelos Animales de Enfermedad , Masculino , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Netrina-1/metabolismo , Netrina-1/genética , Ratones Endogámicos C57BLRESUMEN
The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.
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Modelos Animales de Enfermedad , Prurito , Receptores Acoplados a Proteínas G , Animales , Prurito/metabolismo , Prurito/inducido químicamente , Prurito/patología , Prurito/tratamiento farmacológico , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Ratones , Células HEK293 , Fosforilación/efectos de los fármacos , Fosfatos/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Profármacos/farmacología , Microscopía por CrioelectrónRESUMEN
Skin denervation has been shown to cause remission of psoriatic lesions in patients, which can reappear if reinnervation occurs. This effect can be induced by the activation of dendritic cells through sensory innervation. However, a direct effect of nerves on the proliferation of keratinocytes involved in the formation of psoriatic plaques has not been investigated. We developed, by tissue engineering, a model of psoriatic skin made of patient skin cells that showed increased keratinocyte proliferation and epidermal thickness compared to healthy controls. When this model was treated with CGRP, a neuropeptide released by sensory neurons, an increased keratinocyte proliferation was observed in the psoriatic skin model, but not in the control. When a sensory nerve network was incorporated in the psoriatic model and treated with capsaicin to induce neuropeptide release, an increase of keratinocyte proliferation was confirmed, which was blocked by a CGRP antagonist while no difference was noticed in the innervated healthy control. We showed that sensory neurons can participate directly to keratinocyte hyperproliferation in the formation of psoriatic lesions through the release of CGRP, independently of the immune system. Our unique tissue-engineered innervated psoriatic skin model could be a valuable tool to better understand the mechanism by which nerves may modulate psoriatic lesion formation in humans. STATEMENT OF SIGNIFICANCE: This study shows that keratinocytes extracted from patients' psoriatic skin retain, at least in part, the disease phenotype. Indeed, when combined in a 3D model of tissue-engineered psoriatic skin, keratinocytes exhibited a higher proliferation rate, and produced a thicker epidermis than a healthy skin control. In addition, their hyperproliferation was aggravated by a treatment with CGRP, a neuropeptide released by sensory nerves. In a innervated model of tissue-engineered psoriatic skin, an increase in keratinocyte hyperproliferation was also observed after inducing neurons to release neuropeptides. This effect was prevented by concomitant treatment with an antagonist to CGRP. Thus, this study shows that sensory nerves can directly participate to affect keratinocyte hyperproliferation in psoriasis through CGRP release.