Acid-sensing ion channel 2 (asic 2) and trkb interrelationships within the intervertebral disc.
ABSTRACT: The cells of the intervertebral disc (IVD) have an unusual acidic and hyperosmotic microenvironment. They express acid-sensing ion channels (ASICs), gated by extracellular protons and mechanical forces, as well as neurotrophins and their signalling receptors. In the nervous tissues some neurotrophins regulate the expression of ASICs. The expression of ASIC2 and TrkB in human normal and degenerated IVD was assessed using quantitative-PCR, Western blot, and immunohistochemistry. Moreover, we investigated immunohistochemically the expression of ASIC2 in the IVD of TrkB-deficient mice. ASIC2 and TrkB mRNAs were found in normal human IVD and both increased significantly in degenerated IVD. ASIC2 and TrkB proteins were also found co-localized in a variable percentage of cells, being significantly higher in degenerated IVD than in controls. The murine IVD displayed ASIC2 immunoreactivity which was absent in the IVD of TrkB-deficient mice. Present results demonstrate the occurrence of ASIC2 and TrkB in the human IVD, and the increased expression of both in pathological IVD suggest their involvement in IVD degeneration. These data also suggest that TrkB-ligands might be involved in the regulation of ASIC2 expression, and therefore in mechanisms by which the IVD cells accommodate to low pH and hypertonicity.
Project description:Intervertebral disc (IVD) degeneration is associated with local inflammation and increased expression of neurotrophins. Acidic microenvironment is believed to cause the progression of IVD degeneration. However, there is a paucity of information regarding the relationship between acidic microenvironment and the inflammation and expression of neurotrophins in IVD. G-protein-coupled receptor 4 (GPR4) is a pH-sensing receptor, which can activate the inflammation and increase the expression levels of nerve growth factor in acidic microenvironment. In this study, culture media with pH?7.2 (representing the normal IVD-like acidic condition) and pH?6.5 (degenerated IVD-like acidic condition) were prepared. The gene and protein expression levels of GPR4 in SD rat nucleus pulposus cells were determined under the acidic conditions. And cyclic AMP (cAMP), the second messenger of GPR4, was assayed. Furthermore, the expression levels of receptor activator of nuclear factor ? B (RANK), RANKL ligand (RANKL), osteoprotegerin (OPG), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were also determined. To clarify the involvement of GPR4 in the upregulation of the expression of RANK/RANKL/OPG system and neurotrophins, gene knockdown and forced expression of GPR4 and inhibiting its downstream cAMP accumulation and Ca2+ mobilization were performed. The alternation of the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, and aggrecanase-2 (ADAMTS-5) were evaluated by RT-PCR and western blot. The results showed that GPR4 was expressed in rat nucleus pulposus cells, and the expression was upregulated under the degenerated IVD-like acidic microenvironment. cAMP accumulation levels were increased under the degenerated IVD-like acidic culture conditions. The expression levels of RANK, RANKL, OPG, NGF, and BNDF were significantly upregulated under the degenerated IVD-like acidic microenvironment. GPR4 knockdown and reduction of cAMP by the inhibitor SQ22536 abolished the upregulation of the expression of RANK, RANKL, OPG, NGF, and BNDF under the degenerated IVD-like acidic microenvironment. On the opposite, acidosis-induced cAMP accumulation and upregulation of RANK, RANKL, OPG, NGF, and BNDF were further promoted by GPR4 overexpression. The expression levels of MMP-3, MMP-13, and ADAMTS-5 were upregulated under the degenerated IVD-like acidic condition, which can be promoted or attenuated by GPR4 overexpression or knockdown, respectively. We concluded that GPR4-mediated cAMP accumulation was involved in the increased expression of RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells under the degenerated IVD-like acidic microenvironment.
Project description:Many of the causes of low back pain are still unknown; sufficient evidence indicates that both degenerative and mechanical change within the intervertebral disk (IVD) is a relevant factor. This article reviews intracellular signaling pathways related to pain receptors in the degenerated IVD. Several reports have demonstrated the number of nerve fibers in the IVD was increased in degenerated disks. In recent years, some groups have reported that an increase in nerve fibers is associated with the presence of inflammatory mediators and/or neurotrophins in the IVD. Cell signaling events, which are regulated by inflammatory mediators and neurotrophins, must be identified to clarify the mechanism underlying low back pain. Major intracellular signaling pathways (nuclear factor kappa β, mitogen-activated protein kinases, and Wnts) potentially play vital roles in mediating the molecular events responsible for the initiation and progression of IVD degeneration. These signaling pathways may represent therapeutic targets for the treatment of IVD degeneration and its associated back pain.
Project description:Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%-95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65-75%). ASIC1, ASIC2, and ASIC3 were expressed in 65-75%, 55-70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression.
Project description:The role of acid-sensing ion channels (ASICs) in the ventrolateral medulla (VLM) remains uncertain. Here, we found that ASIC1a and ASIC2 are widely expressed in rat medulla, and the expression level is higher at neonatal stage as compared to adult stage. The two ASIC subunits co-localized in medualla neurons. Furthermore, pH reduction triggered typical ASIC-type currents in the medulla, including the VLM. These currents showed a pH50 value of 6.6 and were blocked by amiloride. Based on their sensitivity to psalmotoxin 1 (PcTx1) and zinc, homomeric ASIC1a and heteromeric ASIC1a/2 channels were likely responsible for acid-mediated currents in the mouse medulla. ASIC currents triggered by pH 5 disappeared in the VLM neurons from ASIC1-/-, but not ASIC2-/- mice. Activation of ASICs in the medulla also triggered neuronal excitation. Moreover, microinjection of artificial cerebrospinal fluid at a pH of 6.5 into the VLM increased integrated phrenic nerve discharge, inspiratory time and respiratory drive in rats. Both amiloride and PcTx1 inhibited the acid-induced stimulating effect on respiration. Collectively, our data suggest that ASICs are highly expressed in the medulla including the VLM, and activation of ASICs in the VLM contributes to central chemoreception.
Project description:Acid-sensing ion channels (ASICs) generate H(+) -gated Na(+) currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid-evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1(-/-) mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO2 inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans.
Project description:Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1-/- and Asic2-/- mice and observed a significant reduction of clinical score in Asic1-/- mice and a significant reduction in the clinical score in Asic2-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.
Project description:Intervertebral disc (IVD) degeneration and consequent low back pain (LBP) are common and costly pathological processes that require improved treatment strategies. Transient Receptor Potential (TRP) channels constitute a family of multimodal ion channels that have recently emerged as contributors to disc pathologies and were thus proposed as potential therapeutic targets, although limited data on their presence and function in the IVD exist. The purpose of this study was to determine the mRNA and protein expression of TRP channels in non-degenerated and degenerated human IVD tissue (with different pain intensity and chronicity) using gene array, conventional qPCR and immunohistochemistry. We could demonstrate that 26 out of 28 currently known TRP channels are expressed in the IVD on the mRNA level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared to non-degenerated IVDs. Additionally, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity influenced the gene and/or protein expression of TRPC6, TRPM2 and TRML1. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This study supports the importance of TRP channels in IVD homeostasis and pathology and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. However, the exact function and activation of the highlighted TRP channels will have to be determined in future studies.
Project description:The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization.
Project description:Intervertebral disc (IVD) degeneration is a remodeling process mediated by several growth factors and cytokines. This process has been extensively studied in vitro and with pathologic specimens obtained during surgery for scoliosis or back pain. However, the occurrence and temporal evolution of these molecules during normal aging, particularly in the cervical segment, is not known. Our objective was to study and compare the presence of putative mediators in the IVD of young (<35 years, G1) and elderly (>65 years, G2) presumably asymptomatic individuals. Thirty C4-5 and C5-6 discs and thirty L4-5 and L5-S1 discs per group were collected during the autopsy of individuals whose family members denied a history of neck or back pain. Discs were divided into anterior, central (lumbar only) and posterior sectors for analysis. Immunohistochemistry for TNF-?, IL-1?, VEGF, NGF-?, BDNF, TIMP-1, MMP-1, -2 and -3 was performed and reactivity compared between groups and sectors. All of these molecules were detected in every disc sector of both G1 and G2. Most statistical comparisons (25/45, 55.6%) revealed an increase in mediator expression in G2 in relation to G1. Regional differences in the expression of remodeling enzymes were rare; NGF-? and BDNF had slightly higher expression in the cervical segment of elderly individuals. A senescent profile with elevated VEGF, MMP-2 and MMP-3 was observed across most G2 disc regions and were generally elevated from G1. In conclusion, the mere presence of any of the studied molecules inside the IVD cannot be considered pathologic. Expression of remodeling enzymes and inflammatory mediators is relatively similar across different vertebral segments and disc regions leading to a common degenerated pattern, while neurotrophins have slightly higher expression in cervical discs. These findings support the concept that disc remodeling in different segments follows a similar pathway that can be potentially mediated to avoid structural failure.