RESUMO
Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.
Assuntos
Trifosfato de Adenosina/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/inervação , Receptores Purinérgicos P2X5/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Acidose Láctica/metabolismo , Acidose Láctica/fisiopatologia , Trifosfato de Adenosina/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Isquemia/fisiopatologia , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X5/fisiologia , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/fisiologia , Canais de Sódio/fisiologiaRESUMO
The reflex that provides rapid neural control of blood pressure is triggered by an unknown molecular pressure sensor. ASIC2, an ion channel in a family that includes a mechanosensor from C. elegans, is shown by Lu et al. in this issue of Neuron to be critical for this reflex in mice, perhaps because ASIC2 is the elusive pressure sensor.
Assuntos
Sistema Nervoso Autônomo/metabolismo , Barorreflexo/fisiologia , Pressão Sanguínea/fisiologia , Fenômenos Fisiológicos Cardiovasculares , Proteínas do Tecido Nervoso/metabolismo , Pressorreceptores/metabolismo , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Humanos , Mecanotransdução Celular/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Disautonomias Primárias/genética , Disautonomias Primárias/metabolismo , Disautonomias Primárias/fisiopatologia , Células Receptoras Sensoriais/metabolismo , Canais de Sódio/genéticaAssuntos
Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Cátion TRPV/metabolismo , Anestesia Local/métodos , Anestésicos Locais/farmacologia , Animais , Capsaicina/farmacologia , Permeabilidade da Membrana Celular , Condutividade Elétrica , Humanos , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Nociceptores/citologia , Dor/metabolismo , Sódio/metabolismoRESUMO
Acid-sensing ion channels (ASICs) are thought to trigger some forms of acid-induced pain and taste, and to contribute to stroke-induced neural damage. After activation by low extracellular pH, different ASICs undergo desensitization on time scales from 0.1 to 10 s. Consistent with a substantial conformation change, desensitization slows dramatically when temperature drops (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. PNAS. 98:6459-6463). The nature of this conformation change is unknown, but two studies showed that desensitization rate is altered by mutations on or near the first transmembrane domain (TM1) (Coric, T., P. Zhang, N. Todorovic, and C.M. Canessa. 2003. J. Biol. Chem. 278:45240-45247; Pfister, Y., I. Gautschi, A.-N. Takeda, M. van Bemmelen, S. Kellenberger, and L. Schild. 2006. J. Biol. Chem. 281:11787-11791). Here we show evidence of a specific conformation change associated with desensitization. When mutated from glutamate to cysteine, residue 79, which is some 20 amino acids extracellular to TM1, can be altered by cysteine-modifying reagents when the channel is closed, but not when it is desensitized; thus, desensitization appears to conceal the residue from the extracellular medium. D78 and E79 are a pair of adjacent acidic amino acids that are highly conserved in ASICs yet absent from epithelial Na(+) channels, their acid-insensitive relatives. Despite large effects on desensitization by mutations at positions 78 and 79-including a shift to 10-fold lower proton concentration with the E79A mutant-there are not significant effects on activation.
Assuntos
Ácidos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Ratos , Canais de Sódio/genética , Relação Estrutura-AtividadeRESUMO
The majority of patients in pain clinics are treated for muscle pain yet methods to study it in animals are relatively weak compared to methods to study skin pain. Here we describe an in vitro muscle-nerve preparation and model of muscle ischemia and contractile fatigue in mice. Timed muscle contraction is electrically evoked, while single unit activity of muscle sensory neurons and muscle contractile force are simultaneously recorded. The muscle is placed in a small (<1 mL) chamber where oxygen levels can be manipulated, drugs can be applied, and the extracellular milieu can be highly controlled. We demonstrate that we can record from sensory afferents that have the properties expected of ischemic nociceptors. This method serves for studying the neuronal and molecular mechanisms underlying ischemic pains such as angina, intermittent claudication, and sickle cell crisis.
Assuntos
Eletrofisiologia/métodos , Isquemia/fisiopatologia , Músculo Esquelético/fisiopatologia , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Potenciais de Ação/fisiologia , Animais , Eletrofisiologia/instrumentação , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/fisiologia , Músculo Esquelético/inervação , Condução Nervosa/fisiologiaRESUMO
Acid-sensing ion channel 3 (ASIC3) is highly expressed on sensory neurons that innervate heart and skeletal muscle and, therefore, is proposed to detect lactic acidosis and to transduce angina and muscle ischemic pain. A difficulty with this idea is that ASIC3 rapidly desensitizes. How can a desensitizing ion channel mediate a persisting sensation such as angina? Here, we show that rat ASIC3 produces a sustained current within the limited range of extracellular pH (7.3 to 6.7) that occurs during cardiac and skeletal muscle ischemia; experiments use patch clamp on transfected cell lines and on fluorescently tagged sensory neurons that innervate rat heart. No such sustained current occurs with ASIC1a (either as homomers or 1a/3 heteromers), whereas ASIC2a/3 heteromers give much larger currents than ASIC3 homomers. The sustained current persists even over tens of minutes because it is caused by a region of pH where there is overlap between inactivation and activation of the channel. Lactate, an anaerobic metabolite, allows the current to activate at slightly more basic pH. Surprisingly, amiloride, which blocks ASICs when they are activated at lower pH, increases ASIC3 current evoked at pH 7.0. Cardiac sensory neurons exhibit a small, perfectly sustained current when pH changes from 7.4 to 7.0. At least some of this current is carried by ASICs because the current is increased by both Zn(2+), an ASIC modulator, and amiloride. We suggest that this sustained mode is the most relevant form of ASIC3 gating for triggering angina and other ischemic pain.
Assuntos
Proteínas de Membrana/metabolismo , Isquemia Miocárdica/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Prótons , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Amilorida/farmacologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Condutividade Elétrica , Coração/inervação , Concentração de Íons de Hidrogênio , Ácido Láctico/farmacologia , Isquemia Miocárdica/metabolismo , Neurônios Aferentes , Ratos , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
BACKGROUND: ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. RESULTS: Less than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 microm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus. CONCLUSION: Our data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.
Assuntos
Ácidos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Animais , Vasos Sanguíneos/inervação , Vasos Sanguíneos/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Imunofluorescência , Músculos/inervação , Músculos/metabolismo , Nociceptores/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor trkA/metabolismo , Receptor trkC/metabolismo , Células Receptoras Sensoriais/enzimologia , Pele/inervação , Pele/metabolismoRESUMO
BACKGROUND: Despite the clinical significance of muscle pain, and the extensive investigation of the properties of muscle afferent fibers, there has been little study of the ion channels on sensory neurons that innervate muscle. In this study, we have fluorescently tagged sensory neurons that innervate the masseter muscle, which is unique because cell bodies for its muscle spindles are in a brainstem nucleus (mesencephalic nucleus of the 5th cranial nerve, MeV) while all its other sensory afferents are in the trigeminal ganglion (TG). We examine the hypothesis that certain molecules proposed to be used selectively by nociceptors fail to express on muscle spindles afferents but appear on other afferents from the same muscle. RESULTS: MeV muscle afferents perfectly fit expectations of cells with a non-nociceptive sensory modality: Opiates failed to inhibit calcium channel currents (I(Ca)) in 90% of MeV neurons, although ICa were inhibited by GABA(B) receptor activation. All MeV afferents had brief (1 msec) action potentials driven solely by tetrodotoxin (TTX)-sensitive Na channels and no MeV afferent expressed either of three ion channels (TRPV1, P2X3, and ASIC3) thought to be transducers for nociceptive stimuli, although they did express other ATP and acid-sensing channels. Trigeminal masseter afferents were much more diverse. Virtually all of them expressed at least one, and often several, of the three putative nociceptive transducer channels, but the mix varied from cell to cell. Calcium currents in 80% of the neurons were measurably inhibited by mu-opioids, but the extent of inhibition varied greatly. Almost all TG masseter afferents expressed some TTX-insensitive sodium currents, but the amount compared to TTX sensitive sodium current varied, as did the duration of action potentials. CONCLUSION: Most masseter muscle afferents that are not muscle spindle afferents express molecules that are considered characteristic of nociceptors, but these putative muscle nociceptors are molecularly diverse. This heterogeneity may reflect the mixture of metabosensitive afferents which can also signal noxious stimuli and purely nociceptive afferents characteristic of muscle.
Assuntos
Arcada Osseodentária/inervação , Músculos/inervação , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Potenciais de Ação , Animais , Tronco Encefálico/metabolismo , Cálcio/metabolismo , Corantes Fluorescentes/farmacologia , Masculino , Músculos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/metabolismoRESUMO
How would you make a drug that inhibits pain without side effects? The most obvious strategy for analgesia targets molecules that are expressed only on neurons used for pain. In this issue of Neuron, Bell et al. report a new splice variant of a calcium channel that controls neurotransmitter release and show that it is expressed primarily on nociceptors, the sensory neurons that trigger pain.
Assuntos
Canais de Cálcio Tipo N/genética , DNA Recombinante , Variação Genética , Dor/genética , AnimaisAssuntos
Microglia/metabolismo , Dor/fisiopatologia , Fosfato de Piridoxal/análogos & derivados , Receptores Purinérgicos P2/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Nervos Espinhais/lesões , Nervos Espinhais/metabolismo , Animais , Doença Crônica , Microglia/citologia , Microglia/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/farmacologia , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Tato/fisiologiaRESUMO
Acid-sensing ion channels (ASICs) open when extracellular pH drops and they are enhanced by lactate, making them specialized for detecting lactic acidosis. Highly expressed on cardiac nociceptors and some other sensory neurons, ASICs may help trigger pain caused by tissue ischemia. We report that H(+) opens ASIC3 by speeding release of Ca(2+) from a high-affinity binding site (K(Ca) = 150 nM) on the extracellular side of the pore. The bound Ca(2+) blocks permeation and the channel conducts when multiple H(+) ions relieve this block. Activation through Ca(2+) explains sensitivity to lactate, which decreases extracellular [Ca(2+)], and it may prove relevant in CNS pathologies (stroke, seizure) that simultaneously drop pH and Ca(2+).
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Quimiorreceptoras/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso , Neurônios Aferentes/metabolismo , Prótons , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células COS , Sinalização do Cálcio/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/fisiologia , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Células Quimiorreceptoras/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Cinética , Ácido Láctico/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , EstereoisomerismoRESUMO
A remarkable feature of opioids is that they inhibit pain that persists from previous injuries without eliminating either the initial pain of a new injury or the protective reflexes triggered by it. Here we ask whether selective expression of the mu-opioid receptor (MOR) gene in primary nociceptors (pain-sensing neurons) might contribute to this aspect of opioid specificity. We quantified single-cell levels of MOR mRNA and measured opioid inhibition of Ca channels on identified nociceptors and low-threshold mechanosensors (non-nociceptors) isolated from rats. Negligibly few non-nociceptors express MOR mRNA, thereby rendering nonpain sensations insensitive to opioids. Nearly half of nociceptors of all size classes also fail to express MOR mRNA or to respond to opioids. Among the opioid-responsive nociceptors, a gene dose-response relationship exists such that maximal opioid inhibition occurs when the MOR mRNA concentration of a cell is >15 pm. Almost all large, myelinated nociceptors express MOR mRNA below this level, whereas small, unmyelinated nociceptors are likely to express above it. Because myelinated nociceptors mediate anti-nociceptive reflexes, the data suggest that fine control of the MOR mRNA level contributes to a complex neural trait: the ability of opioids to suppress persistent pain without preventing response to a new injury.
Assuntos
Bainha de Mielina , Neurônios Aferentes/classificação , Nociceptores/química , RNA Mensageiro/análise , Receptores Opioides mu/genética , Analgésicos Opioides/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Células Cultivadas , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Mecanorreceptores/química , Mecanorreceptores/metabolismo , Mecanorreceptores/fisiologia , Modelos Neurológicos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Nociceptores/metabolismo , Nociceptores/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/agonistas , Receptores Opioides mu/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Recent advances-both experimental and theoretical-provide a tentative image of the structures in Ca channels that make them exceptionally selective. The image is very different from K channels, which obtain high selectivity with a rigid pore that tightly fits K(+) ions and is lined by carbonyl oxygens of the polypeptide backbone. Ca channels rely on four glutamate residues (the EEEE locus), whose carboxyl side chains likely reach into the pore lumen to interact with passing Ca(2+) ions. The structure is thought to be flexible, tightly binding a single Ca(2+) ion in order to block Na(+) flux but rearranging to interact with multiple Ca(2+) ions to allow Ca(2+) flux. The four glutamates are not equivalent, a fact that seems important for Ca(2+) permeation. This review describes the experimental evidence that leads to these conclusions and the attempts by theorists to explain the combination of high selectivity and high flux that characterizes Ca channels.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Dados de Sequência MolecularRESUMO
Extracellular ATP rapidly excites nociceptive sensory neurons by opening ATP-gated ion channels (P2X receptors). Here, we describe two actions of both ATP and UTP on rat sensory neurons that are relatively slow and sustained: phosphorylation of the transcription factor CREB and delayed action potential firing that persists for tens of seconds after removal of the ligand. The pharmacology of these responses indicates that they are mediated by the metabotropic receptor P2Y2, and not by P2X receptors. CREB phosphorylation occurred in a subset of small peripherin-positive neurons likely to be unmyelinated nociceptors. In situ hybridization analysis revealed widespread expression of P2Y2 mRNA in sensory neurons. CREB phosphorylation is mediated by both action-potential-evoked calcium influx and calcium release from intracellular stores. These findings suggest that P2Y2 contributes to the transduction of ATP-mediated sensory signalling, and may be involved in the activity-dependent regulation of nociceptor phenotype.