RESUMO
Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.
Assuntos
Hiperalgesia , Neuralgia , Nociceptores , Pele , Animais , Dor Crônica/fisiopatologia , Hiperalgesia/fisiopatologia , Mecanorreceptores/patologia , Camundongos , Neuralgia/fisiopatologia , Nociceptores/patologia , Pele/inervação , Pele/fisiopatologiaRESUMO
Piezo channels are mechanically activated ion channels that confer mechanosensitivity to a variety of different cell types. Piezos oligomerize as propeller-shaped homotrimers that are thought to locally curve the membrane into spherical domes that project into the cell. While several studies have identified domains and amino acids that control important properties such as ion permeability and selectivity as well as inactivation kinetics and voltage sensitivity, only little is known about intraprotein interactions that govern mechanosensitivity-the most unique feature of PIEZOs. Here we used site-directed mutagenesis and patch-clamp recordings to investigate the mechanogating mechanism of PIEZO2. We demonstrate that charged amino acids at the interface between the beam domain-i.e., a long α-helix that protrudes from the intracellular side of the "propeller" blade toward the inner vestibule of the channel-and the C-terminal domain (CTD) as well as hydrophobic interactions between the highly conserved Y2807 of the CTD and pore-lining helices are required to ensure normal mechanosensitivity of PIEZO2. Moreover, single-channel recordings indicate that a previously unrecognized intrinsically disordered domain located adjacent to the beam acts as a cytosolic plug that limits ion permeation possibly by clogging the inner vestibule of both PIEZO1 and PIEZO2. Thus, we have identified several intraprotein domain interfaces that control the mechanical activation of PIEZO1 and PIEZO2 and which might thus serve as promising targets for drugs that modulate the mechanosensitivity of Piezo channels.
RESUMO
Nerve growth factor is an inflammatory mediator that induces long-lasting hyperalgesia, which can partially be attributed to nerve growth factor-induced sensitization of primary afferent nociceptors. It was shown that nerve growth factor increases the excitability of polymodal C-fibre nociceptors by modulating tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated sodium channels, but hitherto only little is known about the effects of nerve growth factor on sodium currents in other nociceptor subtypes that express the nerve growth factor receptor TrkA. We previously characterized two reporter mouse lines that allow the unequivocal identification of two important subclasses of TrkA-expressing nociceptors - i.e. neuropeptide Y receptor type 2 (NPY2R+ ) Aδ-fibre nociceptors that mediate pinprick pain and nicotinic acetylcholine receptor alpha-3 subunit (CHRNA3+ ) silent nociceptors, which are the most abundant TrkA+ nociceptors in visceral organs and deep somatic tissues. Here, we utilized these mouse lines to investigate the expression patterns and the possible nerve growth factor-dependent modulation of sodium channels in these neurons using whole-cell patch-clamp recordings and quantitative real-time polymerase chain reaction. We demonstrate that NPY2R+ nociceptors, CHRNA3+ 'silent' nociceptors and polymodal C-fibre nociceptors express different combinations of sodium channel α- and ß-subunits and accordingly exhibit functionally different sodium currents. Moreover, we demonstrate that nerve growth factor produces robust hyperpolarizing shifts in the half-activation voltage of tetrodotoxin-resistant currents in NPY2R+ nociceptors and polymodal C-fibre nociceptors and also shifts the half-activation of tetrodotoxin-sensitive currents in polymodal C-fibre nociceptors. In silent nociceptors, however, nerve growth factor solely increases the current density of the tetrodotoxin-resistant current but does not alter other sodium channel properties. Considering the different peripheral target tissues and the previously reported roles in different forms of pain of the nociceptor subpopulations that were examined here, our results suggest that nerve growth factor differentially contributes to the development visceral and cutaneous pain hypersensitivity and highlights the importance of developing different therapeutic strategies for different forms of pain.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Nociceptores/metabolismo , Tetrodotoxina/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptores/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Transient receptor potential (TRP) proteins are a family of ion channels central for sensory signaling. These receptors and, in particular, those involved in thermal sensing are also involved in pain signaling. Noteworthy, thermosensory receptors are polymodal ion channels that respond to both physical and chemical stimuli, thus integrating different environmental clues. In addition, their activity is modulated by algesic agents and lipidergic substances that are primarily released in pathological states. Lipids and lipid-like molecules have been found that can directly activate some thermosensory channels or modulate their activity by either potentiating or inhibiting it. To date, more than 50 endogenous lipids that can regulate TRP channel activity in sensory neurons have been described, thus representing the majority of known endogenous TRP channel modulators. Lipid modulators of TRP channels comprise lipids from a variety of metabolic pathways, including metabolites of the cyclooxygenase, lipoxygenase and cytochrome-P450 pathways, phospholipids and lysophospholipids. Therefore, TRP-channels are able to integrate and interpret incoming signals from the different metabolic lipid pathways. Taken together, the large number of lipids that can activate, sensitize or inhibit neuronal TRP-channels highlights the pivotal role of these molecules in sensory biology as well as in pain transduction and perception. This article is part of a Special Issue entitled: Lipid-protein interactions. Guest Editors: Amitabha Chattopadhyay and Jean-Marie Ruysschaert.
Assuntos
Lipídeos de Membrana/metabolismo , Dor/fisiopatologia , Transdução de Sinais/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Humanos , Lipídeos de Membrana/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/metabolismo , Canais de Potencial de Receptor Transitório/químicaRESUMO
ThermoTRPs are unique channels that mediate Na(+) and Ca(2+) currents in response to changes in ambient temperature. In combination with their activation by other physical and chemical stimuli, they are considered key integrators of environmental cues into neuronal excitability. Furthermore, roles of thermoTRPs in non-neuronal tissues are currently emerging such as insulin secretion in pancreatic ß-cells, and links to cancer. Calcium permeability through thermoTRPs appears a central hallmark for their physiological and pathological activities. Moreover, it is currently being proposed that beyond working as a second messenger, Ca(2+) can function locally by acting on protein complexes near the membrane. Interestingly, thermoTRPs can enhance and expand the inherent plasticity of signalplexes by conferring them temperature, pH and lipid regulation through Ca(2+) signalling. Thus, unveiling the local role of Ca(2+) fluxes induced by thermoTRPs on the dynamics of membrane-attached signalling complexes as well as their significance in cellular processes, are central issues that will expand the opportunities for therapeutic intervention in disorders involving dysfunction of thermoTRP channels.
Assuntos
Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Humanos , Transporte de Íons , Permeabilidade , Conformação Proteica , Canais de Cátion TRPC/químicaRESUMO
ABSTRACT: Evidence from previous studies supports the concept that spinal cord injury (SCI)-induced neuropathic pain (NP) has its neural roots in the peripheral nervous system. There is uncertainty about how and to which degree mechanoreceptors contribute. Sensorimotor activation-based interventions (eg, treadmill training) have been shown to reduce NP after experimental SCI, suggesting transmission of pain-alleviating signals through mechanoreceptors. The aim of the present study was to understand the contribution of mechanoreceptors with respect to mechanical allodynia in a moderate mouse contusion SCI model. After genetic ablation of tropomyosin receptor kinase B expressing mechanoreceptors before SCI, mechanical allodynia was reduced. The identical genetic ablation after SCI did not yield any change in pain behavior. Peptidergic nociceptor sprouting into lamina III/IV below injury level as a consequence of SCI was not altered by either mechanoreceptor ablation. However, skin-nerve preparations of contusion SCI mice 7 days after injury yielded hyperexcitability in nociceptors, not in mechanoreceptors, which makes a substantial direct contribution of mechanoreceptors to NP maintenance unlikely. Complementing animal data, quantitative sensory testing in human SCI subjects indicated reduced mechanical pain thresholds, whereas the mechanical detection threshold was not altered. Taken together, early mechanoreceptor ablation modulates pain behavior, most likely through indirect mechanisms. Hyperexcitable nociceptors seem to be the main drivers of SCI-induced NP. Future studies need to focus on injury-derived factors triggering early-onset nociceptor hyperexcitability, which could serve as targets for more effective therapeutic interventions.
Assuntos
Modelos Animais de Doenças , Hiperalgesia , Mecanorreceptores , Camundongos Endogâmicos C57BL , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Camundongos , Hiperalgesia/fisiopatologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Mecanorreceptores/metabolismo , Mecanorreceptores/fisiologia , Masculino , Humanos , Limiar da Dor/fisiologia , Feminino , Medição da Dor , Camundongos Transgênicos , Neuralgia/etiologia , Neuralgia/metabolismo , Neuralgia/fisiopatologiaRESUMO
Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.
Assuntos
Canais de Cálcio , Cálcio , Camundongos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Pâncreas/metabolismo , Exocitose/fisiologia , Vesículas Secretórias/genéticaRESUMO
Mechanically silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors in mice. Moreover, we show that mice lacking TMEM100 do not develop secondary mechanical hypersensitivity-i.e., pain hypersensitivity that spreads beyond the site of inflammation-during knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce mechanical hypersensitivity in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary mechanical hypersensitivity during inflammation.
Assuntos
Nociceptores , Dor , Animais , Camundongos , Inflamação/metabolismo , Articulação do Joelho , Nociceptores/metabolismo , Dor/metabolismo , Pele/metabolismoRESUMO
A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.
Assuntos
Canais Iônicos , Mecanotransdução Celular , Citoesqueleto/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologiaRESUMO
The vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons, marked by tropomyosin receptor kinase C (TrkC) and tyrosine hydroxylase in the dorsal root ganglia, which projects to blood vessels. Local stimulation of TrkC neurons decreases vessel diameter and blood flow, whereas systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Ablation of the neurons provokes variability in local blood flow, leading to a reduction in systolic blood pressure, increased heart rate variability, and ultimately lethality within 48 h. Thus, a population of TrkC+ sensory neurons forms part of a sensory-feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system.
Assuntos
Pressão Sanguínea/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Comportamento Animal , Fluoresceína/metabolismo , Gânglios Espinais/fisiologia , Frequência Cardíaca/fisiologia , Camundongos Transgênicos , Receptor trkC/metabolismoRESUMO
Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner's corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner's corpuscles. Pathogenic USH2A mutations cause Usher's syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner's corpuscles, recorded from Ush2a-/- mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.
Assuntos
Proteínas da Matriz Extracelular/genética , Mecanorreceptores/metabolismo , Sensação/fisiologia , Vibração , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Mutação/genética , Células de Schwann/fisiologia , Pele/inervação , Tato/fisiologia , Síndromes de Usher/genéticaRESUMO
Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.
Assuntos
Neuropatias Diabéticas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Nociceptividade , Células Receptoras Sensoriais/metabolismo , Sumoilação , Canais de Cátion TRPV/metabolismo , Animais , Células Cultivadas , Ciclo do Ácido Cítrico , Neuropatias Diabéticas/fisiopatologia , Feminino , Gânglios Espinais/citologia , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The yeast ß-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher rate of protein degradation but to a defect in Cln2 synthesis. In fact, analysis of polysome profiles indicated that Msn5 inactivation causes a shift of CLN2 and SWI5 mRNAs from heavy-polysomal to light-polysomal and non-polysomal fractions, supporting a defect in Cln2 and Swi5 protein synthesis in the msn5 mutant. The analysis of truncated versions of Cln2 and of chimeric cyclins combining distinct domains from Cln2 and the related Cln1 cyclin identified an internal region in Cln2 from 181 to 225 residues that when fused to GFP is able to confer Msn5-dependent regulation of protein cellular content. Finally, we showed that a high level of Cln2 is toxic in the absence of Msn5. In summary, we described that Msn5 is required for the proper protein synthesis of specific proteins, introducing a new level of control of cell cycle regulators.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Carioferinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Ciclo Celular/genética , Ciclinas/genética , Regulação Fúngica da Expressão Gênica , Carioferinas/genética , Mutagênese , Polirribossomos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.
Assuntos
Canais Iônicos/genética , Mecanotransdução Celular/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Células Receptoras Sensoriais/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/fisiologia , Humanos , Canais Iônicos/química , Camundongos , Pressão Osmótica/fisiologia , Nervos Periféricos/metabolismo , Nervos Periféricos/fisiologia , Fosfoinositídeo Fosfolipase C/genética , Fosfolipídeos/química , Fosfolipídeos/genética , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Células Receptoras Sensoriais/fisiologiaRESUMO
Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hiperalgesia/terapia , Terapia a Laser , Glicoproteínas de Membrana/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , Proteínas Tirosina Quinases/metabolismo , Células Receptoras Sensoriais/efeitos da radiação , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Ligantes , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Neuralgia/genética , Neuralgia/fisiopatologia , Proteínas Tirosina Quinases/genética , Células Receptoras Sensoriais/metabolismo , Tato/efeitos da radiaçãoRESUMO
Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.
Assuntos
Peptídeos/química , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Portadores de Fármacos , Humanos , Cinética , Mamíferos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Proteína C Associada a Surfactante Pulmonar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Propriedades de SuperfícieRESUMO
Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for â¼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.
Assuntos
Hiperalgesia/genética , Canais Iônicos/genética , Mecanotransdução Celular , Fator de Crescimento Neural/farmacologia , Nociceptores/efeitos dos fármacos , Receptores Nicotínicos/genética , Animais , Fenômenos Biomecânicos , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Potenciais Somatossensoriais Evocados/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Canais Iônicos/metabolismo , Camundongos , Camundongos Transgênicos , Nociceptores/citologia , Nociceptores/metabolismo , Dor/genética , Dor/metabolismo , Dor/fisiopatologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Receptores Nicotínicos/metabolismoRESUMO
Painful mechanical stimuli activate multiple peripheral sensory afferent subtypes simultaneously, including nociceptors and low-threshold mechanoreceptors (LTMRs). Using an optogenetic approach, we demonstrate that LTMRs do not solely serve as touch receptors but also play an important role in acute pain signaling. We show that selective activation of neuropeptide Y receptor-2-expressing (Npy2r) myelinated A-fiber nociceptors evokes abnormally exacerbated pain, which is alleviated by concurrent activation of LTMRs in a frequency-dependent manner. We further show that spatial summation of single action potentials from multiple NPY2R-positive afferents is sufficient to trigger nocifensive paw withdrawal, but additional simultaneous sensory input from LTMRs is required for normal well-coordinated execution of this reflex. Thus, our results show that combinatorial coding of noxious and tactile sensory input is required for normal acute mechanical pain signaling. Additionally, we established a causal link between precisely defined neural activity in functionally identified sensory neuron subpopulations and nocifensive behavior and pain.
Assuntos
Potenciais de Ação , Dor Aguda/genética , Mecanorreceptores/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Neurônios/metabolismo , Nociceptividade/fisiologia , Nociceptores/metabolismo , Somação de Potenciais Pós-Sinápticos , Animais , Comportamento Animal , Gânglios Espinais/citologia , Imuno-Histoquímica , Camundongos , Fibras Nervosas Mielinizadas/fisiologia , Dor Nociceptiva , Optogenética , Dor , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Reflexo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tato/fisiologiaRESUMO
Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.
Assuntos
Adjuvantes Imunológicos/farmacologia , Ácido Hialurônico/farmacologia , Neurônios/efeitos dos fármacos , Dor Nociceptiva , Nociceptores/efeitos dos fármacos , Joelho de Quadrúpedes/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Bradicinina/farmacologia , Células CHO , Cálcio/metabolismo , Capsaicina/farmacologia , Linhagem Celular Tumoral , Cricetulus , Gânglios Espinais/citologia , Células HEK293 , Temperatura Alta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Fármacos do Sistema Sensorial/farmacologia , Joelho de Quadrúpedes/inervação , Canal de Cátion TRPA1 , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/metabolismo , Vasodilatadores/farmacologiaRESUMO
Transient receptor potential (TRP) proteins are sensory-related cation channels. TRPV subfamily responds to vanilloids, generating a Ca(2+) current. TRPV1, a thermal-sensitive non-selective ion channel, possesses six transmembrane helices and the intracellular N- and C-terminal domains. The latter contains the PIP(2) and calmodulin binding sites, the TRP domain and a temperature-responding flexible region. Although the function of C-TRPV1 is known, there are no experimental reports on its structural features. Here, we describe the conformational features of C-TRVP1, by using spectroscopic and biophysical approaches. Our results show that C-TRVP1 is an oligomeric protein, which shows features of natively unfolded proteins.