RESUMO
The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.
Assuntos
Inflamação/genética , Neurônios Aferentes/metabolismo , Canais de Cátion TRPM/genética , Quinases da Família src/genética , Animais , Transporte Biológico/genética , Temperatura Baixa , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neurônios Aferentes/patologia , Dor/tratamento farmacológico , Dor/genética , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ratos , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
Voltage-gated sodium channels are responsible not only for the fast upstroke of the action potential, but they also modify cellular excitability via persistent and resurgent currents. Insecticides act via permanently opening sodium channels to immobilize the animals. Cellular recordings performed decades ago revealed distinctly hooked tail currents induced by these compounds. Here, we applied the classical type-II pyrethroid deltamethrin on human cardiac Nav1.5 and observed resurgent-like currents at very negative potentials in the absence of any pore-blocker, which resemble those hooked tail currents. We show that deltamethrin dramatically slows both fast inactivation and deactivation of Nav1.5 and thereby induces large persistent currents. Using the sea anemone toxin ATx-II as a tool to prevent all inactivation-related processes, resurgent-like currents were eliminated while persistent currents were preserved. Our experiments suggest that, in deltamethrin-modified channels, recovery from inactivation occurs faster than delayed deactivation, opening a brief window for sodium influx and leading to hooked, resurgent-like currents, in the absence of an open channel blocker. Thus, we now explain with pharmacological methods the biophysical gating changes underlying the deltamethrin induced hooked tail currents. SUMMARY: The pyrethroid deltamethrin induces hooked resurgent-like tail currents in human cardiac voltage-gated Nav1.5 channels. Using deltamethrin and ATx-II, we identify additional conducting channel states caused by a faster recovery from inactivation compared to the deltamethrin-induced delayed deactivation.
RESUMO
UNLABELLED: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. SIGNIFICANCE STATEMENT: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirias/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Ácido Aminolevulínico/farmacologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Porfirias/terapia , Protoporfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células Receptoras Sensoriais/metabolismo , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Canal de Cátion TRPA1RESUMO
Matrilin-1 is the prototypical member of the matrilin protein family and is highly expressed in cartilage. However, gene targeting of matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of matrilin-1 deficiency. The results show that matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.
Assuntos
Cartilagem/embriologia , Colágeno Tipo II/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Matrilinas/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Cartilagem/citologia , Colágeno Tipo II/genética , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas Matrilinas/genética , Camundongos , Morfolinos/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-ZebraRESUMO
Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited "paroxysmal extreme pain disorder" (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079-11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.
Assuntos
Substituição de Aminoácidos , Eritromelalgia/metabolismo , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/metabolismo , Reto/anormalidades , Eritromelalgia/genética , Eritromelalgia/patologia , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Células HEK293 , Humanos , Transporte de Íons/genética , Masculino , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/genética , Dor/patologia , Reto/metabolismo , Reto/patologia , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologiaRESUMO
Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response.
Assuntos
Infecções Bacterianas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos/imunologia , Esfingomielina Fosfodiesterase/metabolismo , Cicatrização/fisiologia , Sequência de Aminoácidos , Animais , Infecções Bacterianas/metabolismo , Quimiotaxia de Leucócito , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Larva , Leucócitos/citologia , Microscopia Confocal , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingomielina Fosfodiesterase/imunologia , Peixe-ZebraRESUMO
BACKGROUND: PUVA (psoralen UVA) therapy is used to treat a variety of skin conditions, such as vitiligo psoriasis, eczema and mycosis fungoides, but it is frequently accompanied by phototoxicity leading to burning pain, itch and erythema. METHODS: We used a combination of calcium and reactive oxygen species (ROS) imaging, patch clamp and neuropeptide release measurement to investigate whether certain ion channels involved in pain and itch signalling could be responsible for these adverese effects of PUVA. RESULTS: Clinically used psoralen derivatives 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen at physiologically relevant concentrations were able to activate and photosensitize two recombinant thermoTRP (temperature-gated Transient Receptor Potential) ion channels, TRPA1 (Transient Receptor Potential Ankyrin type 1) and TRPV1 (Transient Receptor Potential Vanilloid type 1). 8-MOP enhanced ROS production by UVA light, and the effect of 8-MOP on TRPA1 could be abolished by the antioxidant N-acetyl cysteine and by removal of critical cysteine residues from the N-terminus domain of the channel. Natively expressed mouse TRPA1 and TRPV1 both contribute to photosensitization of cultured primary afferent neurons by 8-MOP, while direct neuronal activation by this psoralen-derivative is mainly dependent on TRPV1. Both TRPA1 and TRPV1 are to a large extent involved in controlling 8-MOP-induced neuropeptide release from mouse trachea. CONCLUSIONS: Taken together our results provide a better understanding of the phototoxicity reported by PUVA patients and indicate a possible therapeutic approach to alleviate the adverse effects associated with this therapy. SIGNIFICANCE: Our work provides evidence for the involvement of thermoTRP channels TRPA1 and TRPV1 in the activation and photosensitization of peripheral nociceptors during PUVA (Psoralen UVA) therapy.
Assuntos
Furocumarinas , Canais de Potencial de Receptor Transitório , Animais , Anquirinas , Humanos , Camundongos , Canal de Cátion TRPA1 , Canais de Cátion TRPVRESUMO
Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.
Assuntos
Capsaicina/metabolismo , Gânglios Espinais/efeitos dos fármacos , Indóis/farmacologia , Canais de Potássio KCNQ/antagonistas & inibidores , Nociceptores/metabolismo , Piridinas/farmacologia , Canais de Cátion TRPV/agonistas , Animais , Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Células HEK293 , Temperatura Alta , Humanos , Indóis/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Piridinas/metabolismo , Ratos , Ratos Wistar , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismoRESUMO
The aminosteroid U73122 is frequently used as a phospholipase C (PLC) inhibitor and as such was used to investigate PLC-dependent activation and modulation of the transient receptor potential ankyrin type 1 (TRPA1) receptor channel. However, U73122 was recently shown to activate recombinant TRPA1 directly, albeit this interaction was not further explored. Our aim was to perform a detailed characterization of this agonistic action of U73122 on TRPA1. We used Fura-2 calcium microfluorimetry and the patch clamp technique to investigate the effect of U73122 on human and mouse wild type and mutant (C621S/C641S/C665S) TRPA1 expressed in HEK293t cells, as well as native TRPA1 in primary afferent neurons from wild type and TRPV1 and TRPA1 null mutant mice. In addition, we measured calcitonin gene-related peptide (CGRP) release from skin isolated from wild-type and TRPA1 null mutant mice. Human and mouse TRPA1 channels were activated by U73122 in the low nanomolar range. This activation was only partially dependent upon modification of the N-terminal cysteines 621, 641, and 665. U73122 also activated a subpopulation of neurons from wild-type and TRPV1 null mutant mice, but this effect was absent in mice deficient of TRPA1. In addition, U73122 evoked marked calcitonin gene-related peptide (CGRP) release from skin preparations of wild type but not TRPA1 null mutant mice. Our results indicate that U73122 is a potent and selective TRPA1 agonist. This effect should be taken into account when U73122 is used to inhibit PLC in TRPA1-expressing cells, such as primary nociceptors. In addition, U73122 may present a novel lead compound for the development of TRPA1-targeting drugs.
Assuntos
Estrenos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Canal de Cátion TRPA1/agonistas , Fosfolipases Tipo C/antagonistas & inibidores , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Cátion TRPA1/fisiologia , Fosfolipases Tipo C/fisiologiaRESUMO
This article describes the effect of the pyrethroid insecticide deltamethrin on the cardiac voltage-gated sodium channel Nav1.5. Two concentrations of deltamethrin were used and the effects were compared with those of the sea anemone toxin ATx-II and ß4-peptide, which is the C-terminus of the Nav channel ß-subunit. Activation, fast inactivation, deactivation, persistent currents and resurgent currents of Nav1.5 channels were assessed in the presence of these compounds. The data display not only the effect of separately applied compounds on Nav1.5 channels but also investigates how combinations of these substances affect Nav1.5 channel gating properties. The dataset presented in this article is related to the research article "Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5â³ (Sarah Thull, Cristian Neacsu, Andrias O. O'Reilly, Stefanie Bothe, Ralf Hausmann, Tobias Huth, Jannis Meents, Angelika Lampert, doi: 10.1016/j.taap.2020.11501), that investigates the effect of the pyrethroid insecticide deltamethrin on Nav channel gating properties and explains the mechanism underlying hooked, resurgent-like tail currents induced by deltamethrin in Nav1.5 channels.
RESUMO
Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.
Assuntos
Desidrocolesteróis/farmacologia , Síndrome de Smith-Lemli-Opitz/tratamento farmacológico , Canal de Cátion TRPA1/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Animais , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Masculino , Camundongos , Neurônios/efeitos dos fármacosRESUMO
Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.
Assuntos
Eritromelalgia/genética , Gânglios Espinais/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Dor/genética , Potenciais de Ação/genética , Estimulação Elétrica , Eritromelalgia/fisiopatologia , Gânglios Espinais/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fibras Nervosas Amielínicas , Dor/fisiopatologia , Técnicas de Patch-Clamp , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Tetrodotoxina/genéticaRESUMO
Human pluripotent stem cells (hPSCs) offer the opportunity to generate neuronal cells, including nociceptors. Using a chemical-based approach, we generated nociceptive sensory neurons from HUES6 embryonic stem cells and retrovirally reprogrammed induced hPSCs derived from fibroblasts. The nociceptive neurons expressed respective markers and showed tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) voltage-gated sodium currents in patch-clamp experiments. In contrast to their counterparts from rodent dorsal root ganglia, TTXr currents of hPSC-derived nociceptors unexpectedly displayed a significantly more hyperpolarized voltage dependence of activation and fast inactivation. This apparent discrepancy is most likely due to a substantial expression of the developmentally important sodium channel NAV1.5. In view of the obstacles to recapitulate neuropathic pain in animal models, our data advance hPSC-derived nociceptors as a better model to study developmental and pathogenetic processes in human nociceptive neurons and to develop more specific small molecules to attenuate pain.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Nociceptores/metabolismo , Animais , Linhagem Celular , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Ativação do Canal Iônico , Nociceptores/citologia , Ratos , TetrodotoxinaRESUMO
This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/fisiopatologia , Hiperalgesia/etiologia , Nociceptores/efeitos dos fármacos , Aldeído Pirúvico/farmacologia , Canais de Sódio/fisiologia , Animais , Circulação Cerebrovascular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.8 , Condução Nervosa/efeitos dos fármacos , Nociceptores/fisiologia , Estreptozocina , Tetrodotoxina/farmacologiaRESUMO
Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physical danger and choose optimal environments for survival. TRPM8 (Transient Receptor Potential Melastatin type 8) belongs to a select group of ion channels which are gated by changes in temperature, are expressed in sensory nerves and/or skin cells and may be involved in temperature sensing. This channel is activated by a moderate decrease in temperature, with a threshold of approximately 25 °C in heterologous expression systems, and by a variety of natural and synthetic compounds, including menthol. While the physiological role of TRPM8 as a transducer of gentle cooling is widely accepted, its involvement in acute noxious cold sensing in healthy tissues is still under debate. Although accumulating evidence indicates that TRPM8 is involved in neuropathic cold allodynia, in some animal models of nerve injury peripheral and central activation of TRPM8 is followed by analgesia. A variety of inflammatory mediators, including bradykinin and prostaglandin E(2), modulate TRPM8 by inhibiting the channel and shifting its activation threshold to colder temperatures, most likely counteracting the analgesic action of TRPM8. While important progress has been made in unraveling the biophysical features of TRPM8, including the revelation of its voltage dependence, the precise mechanism involved in temperature sensing by this channel is still not completely understood. This article will review the current status of knowledge regarding the (patho)physiological role(s) of TRPM8, its modulation by inflammatory mediators, the signaling pathways involved in this regulation, and the biophysical properties of the channel.
Assuntos
Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/fisiologia , Sensação Térmica , Animais , Temperatura Baixa , Humanos , Mamíferos , Mentol/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/químicaRESUMO
The matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in matrilin structure may lead to a modulation of extracellular matrix assembly. Some matrilins bind weakly to α1ß1 integrin and cell surface proteoglycans, but even though matrilins play a role in mechanotransduction and matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Animais , Doenças Ósseas/metabolismo , Comunicação Celular , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/química , Humanos , Especificidade de ÓrgãosRESUMO
UCMA (alternatively named GRP) is a novel member of the family of γ-carboxyglutamate (Gla) containing proteins that is mainly expressed in cartilage. We have used the zebrafish as a model organism to study UCMA function. Due to the whole genome duplication two Ucma genes are present in zebrafish, ucmaa and ucmab, located on chromosomes 25 and 4, respectively. UCMA gene structure, alternative splicing and protein sequence are highly conserved between mammals and zebrafish and Ucmaa and Ucmab are expressed in zebrafish skeletal tissues. Ucmaa is first detected in the notochord at 18 hpf and expression continues during notochord development. In addition, it is widely present in the developing craniofacial cartilage. In contrast, the weakly expressed Ucmab can be first detected at specific sites in the craniofacial cartilage at 96 hpf, but not in notochord. Knockdown of ucmaa leads to severe growth retardation and perturbance of skeletal development. The cartilage of the morphants has a decreased aggrecan and collagen II content. Similar malformations were observed when glutamate γ-carboxylation was inhibited by warfarin treatment, indicating that glutamate γ-carboxylation is crucial for Ucma function and pointing to a role of UCMA in the pathogenesis of "warfarin embryopathies" and other human skeletal diseases.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Cartilagem/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Processamento Alternativo , Sequência de Aminoácidos , Animais , Cartilagem/citologia , Cartilagem/embriologia , Cartilagem/metabolismo , Clonagem Molecular , Colágeno Tipo II/metabolismo , Biologia Computacional , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Proteínas da Matriz Extracelular , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Larva , Camundongos , Dados de Sequência Molecular , Notocorda/citologia , Notocorda/efeitos dos fármacos , Notocorda/embriologia , Notocorda/metabolismo , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Alinhamento de Sequência , Homologia de Sequência , Coloração e Rotulagem , Fatores de Tempo , Varfarina/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.