RESUMO
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.
Assuntos
Anoctamina-1 , Axônios , Dinoprostona , Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Animais , Dinoprostona/farmacologia , Dinoprostona/metabolismo , Axônios/metabolismo , Axônios/efeitos dos fármacos , Axônios/fisiologia , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Ratos , Anoctamina-1/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , AMP Cíclico/metabolismoRESUMO
Voltage gated sodium channels are key players in aberrant pain signaling and sensitization of nociceptors after peripheral nerve injury. The extent to which sodium channel activity after injury contributes to synaptic transmission at the first pain synapse however remains unclear. To investigate the effect of axotomy on synaptic transmission between dorsal root ganglia neurons and dorsal horn neurons, we reconstructed the first pain synapse in a novel microfluidic based compartmentalized cell culture system, which recapitulates the connectivity of peripheral pain signaling. We show that following axotomy of the distal axons, inhibition of NaV1.7 and NaV1.8 sodium channels in incoming presynaptic DRG axons is no longer sufficient to block activation of these axons and the resulting synaptic transmission to dorsal horn neurons. We found that blockade of NaV1.6 activity is highly effective in reducing activation of incoming axons contributing to synaptic transmission after axotomy of DRG neurons. The microfluidic culture system described here offers an in vitro platform to recapitulate and study the first pain synapse.
Assuntos
Microfluídica , Sinapses/metabolismo , Transmissão Sináptica , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Axotomia , Biomarcadores , Técnicas de Cocultura , Imunofluorescência , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Ratos , Células Receptoras Sensoriais/metabolismoRESUMO
Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain.
Assuntos
Anexina A6/fisiologia , Conotoxinas/metabolismo , Mecanotransdução Celular , Dor/prevenção & controle , Fragmentos de Peptídeos/metabolismo , Células Receptoras Sensoriais/fisiologia , Animais , Artrite Experimental/etiologia , Artrite Experimental/fisiopatologia , Biotinilação , Células Cultivadas , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/etiologia , Osteoartrite/fisiopatologia , Dor/metabolismo , Dor/patologiaRESUMO
Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.
Assuntos
Axônios/fisiologia , Microfluídica/métodos , Nociceptividade , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axotomia , Bioensaio , Cálcio/metabolismo , Capsaicina/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Estimulação Elétrica , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/lesões , Gânglios Espinais/patologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Microfluídica/instrumentação , Modelos Biológicos , Fator de Crescimento Neural/farmacologia , Nociceptividade/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos Wistar , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/patologia , Bloqueadores dos Canais de Sódio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Canais de Cátion TRPV/metabolismoRESUMO
Polycystin 2 (Pkd2), which belongs to the transient receptor potential family, plays a critical role in development. Pkd2 is mainly localized in the primary cilia, which also function as mechanoreceptors in many cells that influence multiple biological processes including Ca(2+) influx, chemical activity and signalling pathways. Mutations in many cilia proteins result in craniofacial abnormalities. Orofacial tissues constantly receive mechanical forces and are known to develop and grow through intricate signalling pathways. Here we investigate the role of Pkd2, whose role remains unclear in craniofacial development and growth. In order to determine the role of Pkd2 in craniofacial development, we located expression in craniofacial tissues and analysed mice with conditional deletion of Pkd2 in neural crest-derived cells, using Wnt1Cre mice. Pkd2 mutants showed many signs of mechanical trauma such as fractured molar roots, distorted incisors, alveolar bone loss and compressed temporomandibular joints, in addition to abnormal skull shapes. Significantly, mutants showed no indication of any of these phenotypes at embryonic stages when heads perceive no significant mechanical stress in utero. The results suggest that Pkd2 is likely to play a critical role in craniofacial growth as a mechanoreceptor. Pkd2 is also identified as one of the genes responsible for autosomal dominant polycystic kidney disease (ADPKD). Since facial anomalies have never been identified in ADPKD patients, we carried out three-dimensional photography of patient faces and analysed these using dense surface modelling. This analysis revealed specific characteristics of ADPKD patient faces, some of which correlated with those of the mutant mice.
Assuntos
Anormalidades Craniofaciais/genética , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Adulto , Animais , Cílios/genética , Cílios/metabolismo , Anormalidades Craniofaciais/patologia , Face , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Masculino , Mecanorreceptores/metabolismo , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fenótipo , Rim Policístico Autossômico Dominante/patologia , Transdução de Sinais , Canais de Cátion TRPP/metabolismoRESUMO
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
Assuntos
Regulação para Baixo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axotomia , Comportamento Animal/fisiologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Fibras Nervosas Mielinizadas/metabolismo , Neuralgia/etiologia , Neuralgia/fisiopatologia , Medição da Dor , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Interferente Pequeno , Ratos , Ratos WistarRESUMO
Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.
Assuntos
Células Ciliadas Auditivas/fisiologia , Mecanotransdução Celular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPC/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Tamanho Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico , Gânglios Espinais/citologia , Células Ciliadas Auditivas/classificação , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/fisiopatologia , Hipestesia/genética , Hipestesia/fisiopatologia , Imidazóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Cultura Primária de Células , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/ultraestrutura , Canais de Cátion TRPC/biossíntese , Canais de Cátion TRPC/deficiência , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6 , Doenças Vestibulares/genética , Doenças Vestibulares/fisiopatologiaRESUMO
Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.
Assuntos
Cálcio/fisiologia , Canais de Cloreto/metabolismo , Temperatura Alta , Nociceptores/metabolismo , Animais , Anoctamina-1 , Células Cultivadas , Agonistas dos Canais de Cloreto , Canais de Cloreto/deficiência , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Nociceptores/fisiologia , Medição da Dor/métodos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ß and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear. RESULTS: Here we show that deleting ß and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro. CONCLUSION: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.
Assuntos
Mecanotransdução Celular/fisiologia , Células Receptoras Sensoriais/metabolismo , Canais de Sódio/metabolismo , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.8 , Canais de Sódio/genéticaRESUMO
Mendelian heritable pain disorders have provided insights into human pain mechanisms and suggested new analgesic drug targets. Interestingly, many of the heritable monogenic pain disorders have been mapped to mutations in genes encoding ion channels. Studies in transgenic mice have also implicated many ion channels in damage sensing and pain modulation. It seems likely that aberrant peripheral or central ion channel activity underlies or initiates many pathological pain conditions. Understanding the mechanistic basis of ion channel malfunction in terms of trafficking, localization, biophysics, and consequences for neurotransmission is a potential route to new pain therapies.
Assuntos
Canalopatias , Canais Iônicos/genética , Dor , Animais , Canalopatias/genética , Canalopatias/metabolismo , Canalopatias/fisiopatologia , Humanos , Canais Iônicos/metabolismo , Camundongos , Camundongos Transgênicos , Dor/genética , Dor/metabolismo , Dor/fisiopatologiaRESUMO
Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anoctamina-1 , Cálcio/farmacologia , Canais de Cloreto/química , Canais de Cloreto/deficiência , Canais de Cloreto/genética , Condutividade Elétrica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Camundongos , Oócitos/metabolismo , Pilocarpina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Salivação/efeitos dos fármacos , XenopusRESUMO
Activation of microglia has been implicated in many neurological conditions including Alzheimer's disease and neuropathic pain. Recent studies provide evidence that P2X ATP receptors on the surface of microglia play a crucial role in initiation of inflammatory cascades. We investigated changes in surface P2X receptors in BV-2 murine microglial cells following their activation by pro-inflammatory bacterial lipopolysaccharides (LPS). mRNA analysis using RT-PCR confirmed the presence of P2X4 and P2X7 as the main P2X subunits. Application of ATP at low (< or =100 microM) and high (> or =1 mM) concentrations, as well as BzATP, activated inward currents in BV-2 cells. Current responses of P2X4 and P2X7 subtypes could be distinguished based on their respective sensitivity to the positive modulator ivermectin and to the antagonist Brilliant Blue G. Treatment of BV-2 cells with LPS leads to a transient increase in ivermectin-sensitive P2X4 currents, while dominant P2X7 currents remain largely unaffected. This increase in P2X4 function was concomitant with higher receptor protein expression, itself related to an upregulation of P2X4 mRNA levels that peaked at 48 h post-LPS treatment. Our data demonstrate that although LPS activation has a minor impact on P2X7 receptors that remain the major ionotropic ATP receptors in microglia, it specifically enhances responses to low ATP concentrations mediated by P2X4 receptors, highlighting the significant contribution of both subtypes to neuroinflammatory mechanisms and pathologies.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Microglia/fisiologia , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Potenciais Evocados/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Rim , Camundongos , Microglia/efeitos dos fármacos , Técnicas de Patch-Clamp , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
During muscarinic modulation, principal neurons from layer V of rat medial entorhinal cortex (mEC) respond to repeated applications of a brief stimulus with a graded change in persistent firing frequency. This pattern of discharge has been proposed to represent an intrinsic mechanism for short-term memory operations. To investigate the implementation of persistent activity in mEC during development, we characterized the electrophysiological properties of layer V principal neurons in the mEC over a range of postnatal stages. We observed significant differences in both passive (resistance, time constant, and resting membrane potential) and active properties (threshold, action potential, and adaptation) of principal neurons from rats aged 5-7, 10-13, 16-19, and 21-23 days. We also examined the properties of muscarinic-dependent persistent activity in EC slices from different age groups. Recordings were conducted using the perforated-patch whole cell technique because persistent activity runs down in the ruptured-patch configuration. Although no neuron in the youngest group exhibited graded persistent activity in response to muscarinic receptor activation, this activity was recorded in the 10- to 13-day-old group and its occurrence increased from 69% in the 16- to 19-day-old group to 76% in the 21- to 23-day-old group. This postnatal increase in neurons endowed with persistent firing properties in mEC was found to parallel the increase in density of ChAT-positive immunostaining of fibers and the developmental changes in M1 muscarinic receptor mRNA levels. All these data suggest that the implementation of mnemonic properties in mEC principal neurons matches the ontogenic development of afferent cholinergic circuits and their signaling components.
Assuntos
Acetilcolina/fisiologia , Condutividade Elétrica , Córtex Entorrinal/citologia , Córtex Entorrinal/crescimento & desenvolvimento , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Carbacol/farmacologia , Colina O-Acetiltransferase/metabolismo , Colinérgicos/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Postsynaptic P2X1 ATP-gated channels are expressed in smooth muscle cells of the vascular and genitourinary systems, where they mediate desensitizing neurogenic contractions. Using the model of the isolated rat tail artery, we show that the vasoactive mediator 5-hydroxytryptamine (5-HT), via the 5-HT2A metabotropic receptor, regulates the desensitization kinetics of P2X1 responses by increasing their rate of recovery. Reconstituting the potentiation of P2X1 ATP-gated currents by 5-HT2A receptors in the Xenopus oocyte expression system, we provide evidence that this modulation depends on the activation of novel protein kinase C isoforms and protein kinase D (also named PKCmu) downstream of phospholipase Cbeta. Other major kinases like Ca2+/calmodulin kinase II, protein kinase A, mitogen-activated protein kinases, and tyrosine kinases were found not to be involved. Moreover, we report that buffering intracellular Ca2+ ions with the chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) decreases the rate of recovery of P2X1 responses and increases their sensitivity to potentiation by 5-HT2A receptors or by the diacylglycerol analog phorbol ester 12-myristate 13-acetate. We conclude that intracellular Ca2+ and a subset of diacylglycerol-dependent protein kinases regulate the activity of P2X1 receptor channels by modulating their recovery from desensitization.
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Ativação do Canal Iônico , Proteína Quinase C/fisiologia , Receptor 5-HT2A de Serotonina/fisiologia , Receptores Purinérgicos P2/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Diglicerídeos/farmacologia , Receptores Purinérgicos P2X , Serotonina/farmacologia , Transdução de Sinais , Estaurosporina/farmacologia , Fosfolipases Tipo C/fisiologia , Vasoconstrição/efeitos dos fármacos , XenopusRESUMO
To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn2+ ions (IC50 = 0.4 microM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.
Assuntos
Receptores Purinérgicos P2/metabolismo , Zinco/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cinética , Dados de Sequência Molecular , Oócitos/metabolismo , Receptores Purinérgicos P2X , Schistosoma mansoni/metabolismo , XenopusRESUMO
Among the family of P2X ATP-gated cation channels, the P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X7-dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 +/- 9% and 25 +/- 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X7 receptors.