Activation Of Acid-sensing Ion Channel 1a Research Articles

Enhanced glycolysis causes extracellular acidification and activates acid-sensing ion channel 1a in hypoxic pulmonary hypertension.

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H+ transport/secretion through carbonic anhydrases, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMCs from CH rats. These data suggest that multiple H+ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H+ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN.NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H+ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

Acid-sensing ion channel 1a exacerbates renal ischemia–reperfusion injury through the NF-κB/NLRP3 inflammasome pathway

Ischemia-reperfusion injury (IRI) is the main cause of acute kidney injury (AKI), and there is no effective therapy. Microenvironmental acidification is generally observed in ischemic tissues. Acid-sensing ion channel 1a (ASIC1a) can be activated by a decrease in extracellular pH which mediates neuronal IRI. Our previous study demonstrated that, ASIC1a inhibition alleviates renal IRI. However, the underlying mechanisms have not been fully elucidated. In this study, we determined that renal tubule-specific deletion of ASIC1a in mice (ASIC1afl/fl/CDH16cre) attenuated renal IRI, and reduced the expression of NLRP3, ASC, cleaved-caspase-1, GSDMD-N, and IL-1β. Consistent with these in vivo results, inhibition of ASIC1a by the specific inhibitor PcTx-1 protected HK-2 cells from hypoxia/reoxygenation (H/R) injury, and suppressed H/R-induced NLRP3 inflammasome activation. Mechanistically, the activation of ASIC1a by either IRI or H/R induced the phosphorylation of NF-κB p65, which translocates to the nucleus and promotes the transcription of NLRP3 and pro-IL-1β. Blocking NF-κB by treatment with BAY 11-7082 validated the roles of H/R and acidosis in NLRP3 inflammasome activation. This further confirmed that ASIC1a promotes NLRP3 inflammasome activation, which requires the NF-κB pathway. In conclusion, our study suggests that ASIC1a contributes to renal IRI by affecting the NF-κB/NLRP3 inflammasome pathway. Therefore, ASIC1a may be a potential therapeutic target for AKI.Key messagesKnockout of ASIC1a attenuated renal ischemia-reperfusion injury.ASIC1a promoted the NF-κB pathway and NLRP3 inflammasome activation.Inhibition of the NF-κB mitigated the NLRP3 inflammasome activation induced by ASIC1a.

Functional modulation of a pH-sensitive ion channel by a transporter family.

The acid-sensing ion channel 1a (ASIC1a) is a trimeric ligand-gated ion channel. Its activation by protons results in influx of Na+ and, to a lesser extent, Ca2+ before the channel rapidly desensitizes. ASIC1a-mediated currents contribute to normal brain function, including long term potentiation, which is important for learning and memory. However, elevated activity of ASIC1a has been directly implicated in the pathogenesis of numerous disorders, such as ischemic brain injury, cancer, pain and Alzheimer's disease. Recent evidence suggests that during such pathological conditions, association with protein interaction partners can affect ASIC1a activity, thereby contributing to neuronal damage and cell death. Consequently, there is an increasing interest in understanding the regulation of ASIC1a by protein-interaction partners, which remain largely enigmatic. Here, we have used a combination of mass spectrometry, biochemistry and electrophysiology to identify a family of transporters that directly interacts with ASIC1a and regulates its function. Depending on the specific transporter isoform present in the channel-transporter complex, the activation of ASIC1a is either prolonged and potentiated or decreased. We anticipate this work to offer promising starting points for therapeutic targeting of ASIC1a-containing macromolecular complexes in various diseases.

Acid-sensing ion channel 1a activates IKCa/SKCa channels and contributes to endothelium-dependent dilation.

Acid-sensing ion channel 1a (ASIC1a) belongs to a novel family of proton-gated cation channels that are permeable to both Na+ and Ca2+. ASIC1a is expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds, yet little is known regarding the potential impact of ASIC1a to regulate local vascular reactivity. Our previous studies in rat mesenteric arteries suggest ASIC1a does not contribute to agonist-induced vasoconstriction but may mediate a vasodilatory response. The objective of the current study is to determine the role of ASIC1a in systemic vasodilatory responses by testing the hypothesis that the activation of endothelial ASIC1a mediates vasodilation of mesenteric resistance arteries through an endothelium-dependent hyperpolarization (EDH)-related pathway. The selective ASIC1a antagonist psalmotoxin 1 (PcTX1) largely attenuated the sustained vasodilatory response to acetylcholine (ACh) in isolated, pressurized mesenteric resistance arteries and ACh-mediated Ca2+ influx in freshly isolated mesenteric endothelial tubes. Similarly, basal tone was enhanced and ACh-induced vasodilation blunted in mesenteric arteries from Asic1a knockout mice. ASIC1a colocalizes with intermediate- and small-conductance Ca2+-activated K+ channels (IKCa and SKCa, respectively), and the IKCa/SKCa-sensitive component of the ACh-mediated vasodilation was blocked by ASIC1a inhibition. To determine the role of ASIC1a to activate IKCa/SKCa channels, we measured whole-cell K+ currents using the perforated-patch clamp technique in freshly isolated mesenteric endothelial cells. Inhibition of ASIC1a prevented ACh-induced activation of IKCa/SKCa channels. The ASIC1 agonist, α/β-MitTx, activated IKCa/SKCa channels and induced an IKCa/SKCa-dependent vasodilation. Together, the present study demonstrates that ASIC1a couples to IKCa/SKCa channels in mesenteric resistance arteries to mediate endothelium-dependent vasodilation.

Acid sensor ASIC1a induces synovial fibroblast proliferation via Wnt/β-catenin/c-Myc pathway in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and progressive joint destruction in the middle and late stages. Notably, activated rheumatoid arthritis synovial fibroblasts (RASFs) exhibit tumor-like features, including an increased proliferation rate that largely contributes to pannus formation and joint destruction. Our previous studies have demonstrated that acid-sensing ion channel 1a (ASIC1a) was highly expressed in RASFs, and acidic microenvironment of synovial fluid in patients with RA can activate ASIC1a to promote synovial inflammation, leading to the progression of RA. However, the role and possible mechanism of ASIC1a in RASF proliferation remains unclear. The present study aimed to investigate the effect of ASIC1a activation upon acidosis on RASF proliferation and its molecular mechanism in vivo and in vitro. The results of in vitro experiments showed that activation of ASIC1a upon acidosis promoted the proliferation of RASFs, which could be attenuated by the specific ASIC1a inhibitor Psalmotoxin-1 (PcTx-1) or specific siRNA for ASIC1a. Mechanistically, Wnt/β-catenin/c-Myc signaling pathway was involved in ASIC1a-induced RASF proliferation. The results of in vivo experiments indicated that intra-articular injection of PcTx-1 reduced synovial hyperplasia and ameliorated cartilage degradation in rats with adjuvant arthritis (AA). Collectively, these results suggest that activation of ASIC1a upon acidosis promotes RASF proliferation, and the mechanism may be related to Wnt/β-catenin/c-Myc pathway.

Smooth muscle Acid-sensing ion channel 1a as a therapeutic target to reverse hypoxic pulmonary hypertension.

Acid-sensing ion channel 1a (ASIC1a) is a voltage-independent, non-selective cation channel that conducts both Na+ and Ca2+. Activation of ASIC1a elicits plasma membrane depolarization and stimulates intracellular Ca2+-dependent signaling pathways in multiple cell types, including vascular smooth muscle (SM) and endothelial cells (ECs). Previous studies have shown that increases in pulmonary vascular resistance accompanying chronic hypoxia (CH)-induced pulmonary hypertension requires ASIC1a to elicit enhanced pulmonary vasoconstriction and vascular remodeling. Both SM and EC dysfunction drive these processes; however, the involvement of ASIC1a within these different cell types is unknown. Using the Cre-LoxP system to generate cell-type-specific Asic1a knockout mice, we tested the hypothesis that SM-Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling, whereas EC-Asic1a opposes the development of CH-induced pulmonary hypertension. The severity of pulmonary hypertension was not altered in mice with specific deletion of EC-Asic1a (TekCre-Asic1a fl/fl). However, similar to global Asic1a knockout (Asic1a −/-) mice, mice with specific deletion of SM-Asic1a (MHCCreER-Asic1a fl/fl) were protected from the development of CH-induced pulmonary hypertension and right heart hypertrophy. Furthermore, pulmonary hypertension was reversed when deletion of SM-Asic1a was initiated in conditional MHCCreER-Asic1a fl/fl mice with established pulmonary hypertension. CH-induced vascular remodeling was also significantly attenuated in pulmonary arteries from MHCCreER-Asic1a fl/fl mice. These findings were additionally supported by decreased CH-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) from Asic1a −/- mice. Together these data demonstrate that SM-, but not EC-Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1a inhibition to reverse pulmonary hypertension.

Acidosis induces RIPK1-dependent death of glioblastoma stem cells via acid-sensing ion channel 1a

Eliciting regulated cell death, like necroptosis, is a potential cancer treatment. However, pathways eliciting necroptosis are poorly understood. It has been reported that prolonged activation of acid-sensing ion channel 1a (ASIC1a) induces necroptosis in mouse neurons. Glioblastoma stem cells (GSCs) also express functional ASIC1a, but whether prolonged activation of ASIC1a induces necroptosis in GSCs is unknown. Here we used a tumorsphere formation assay to show that slight acidosis (pH 6.6) induces necrotic cell death in a manner that was sensitive to the necroptosis inhibitor Nec-1 and to the ASIC1a antagonist PcTx1. In addition, genetic knockout of ASIC1a rendered GSCs resistant to acid-induced reduction in tumorsphere formation, while the ASIC1 agonist MitTx1 reduced tumorsphere formation also at neutral pH. Finally, a 20 amino acid fragment of the ASIC1 C-terminus, thought to interact with the necroptosis kinase RIPK1, was sufficient to reduce the formation of tumorspheres. Meanwhile, the genetic knockout of MLKL, the executive protein in the necroptosis cascade, did not prevent a reduction in tumor sphere formation, suggesting that ASIC1a induced an alternative cell death pathway. These findings demonstrate that ASIC1a is a death receptor on GSCs that induces cell death during prolonged acidosis. We propose that this pathway shapes the evolution of a tumor in its acidic microenvironment and that pharmacological activation of ASIC1a might be a potential new strategy in tumor therapy.

Upregulation of acid sensing ion channel 1a (ASIC1a) by hydrogen peroxide through the JNK pathway.

Oxidative stress is intimately tied to neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis, and acute injuries, such as ischemic stroke and traumatic brain injury. Acid sensing ion channel 1a (ASIC1a), a proton-gated ion channel, has been shown to be involved in the pathogenesis of these diseases. However, whether oxidative stress affects the expression of ASIC1a remains elusive. In the current study, we examined the effect of hydrogen peroxide (H2O2), a major reactive oxygen species (ROS), on ASIC1a protein expression and channel function in NS20Y cells and primary cultured mouse cortical neurons. We found that treatment of the cells with H2O2 (20 µM) for 6 h or longer increased ASIC1a protein expression and ASIC currents without causing significant cell injury. H2O2 incubation activated mitogen-activated protein kinases (MAPKs) pathways, including the extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 pathways. We found that neither inhibition of the MEK/ERK pathway by U0126 nor inhibition of the p38 pathway by SB203580 affected H2O2-induced ASIC1a expression, whereas inhibition of the JNK pathway by SP600125 potently decreased ASIC1a expression and abolished the H2O2-mediated increase in ASIC1a expression and ASIC currents. Furthermore, we found that H2O2 pretreatment increased the sensitivity of ASIC currents to the ASIC1a inhibitor PcTx1, providing additional evidence that H2O2 increases the expression of functional ASIC1a channels. Together, our data demonstrate that H2O2 increases ASIC1a expression/activation through the JNK signaling pathway, which may provide insight into the pathogenesis of neurological disorders that involve both ROS and activation of ASIC1a.

Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1β level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

ASIC1a channels regulate mitochondrial ion signaling and energy homeostasis in neurons.

Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na+ and Ca2+ sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na+ and Ca2+ signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na+ and Ca2+ signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na+ and Ca2+ waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na+ and Ca2+ signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na+ signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.

ASIC1a induces synovial inflammation via the Ca2+/NFATc3/ RANTES pathway.

Rationale: Synovial inflammation is one of the main pathological features of rheumatoid arthritis (RA) and is a key factor leading to the progression of RA. Understanding the regulatory mechanism of synovial inflammation is crucial for the treatment of RA. Acid-sensing ion channel 1a (ASIC1a) is an H+-gated cation channel that promotes the progression of RA, but the role of ASIC1a in synovial inflammation is unclear. This study aimed to investigate whether ASIC1a is involved in the synovial inflammation and explore the underlying mechanisms in vitro and in vivo.Methods: The expression of ASIC1a and nuclear factor of activated T cells (NFATs) were analyzed by Western blotting, immunofluorescence, and immunohistochemistry both in vitro and in vivo. The Ca2+ influx mediated by ASIC1a was detected by calcium imaging and flow cytometry. The role of ASIC1a in inflammation was studied in rats with adjuvant-induced arthritis (AA). Inflammatory cytokine profile was analyzed by protein chip in RA synovial fibroblasts (RASF) and verified by a magnetic multi-cytokine assay and ELISA. The NFATc3-regulated RANTES (Regulated upon activation, normal T cell expressed and secreted) gene transcription was investigated by ChIP-qPCR and dual-luciferase reporter assay.Results: The expression of ASIC1a was significantly increased in human RA synovial tissues and primary human RASF as well as in ankle synovium of AA rats. Activated ASIC1a mediated Ca2+ influx to increase [Ca2+]i in RASF. The activation/overexpression of ASIC1a in RASF up-regulated the expression of inflammatory cytokines RANTES, sTNF RI, MIP-1a, IL-8, sTNF RII, and ICAM-1 among which RANTES was increased most remarkably. In vivo, ASIC1a promoted inflammation, synovial hyperplasia, articular cartilage, and bone destruction, leading to the progression of AA. Furthermore, activation of ASIC1a upregulated the nuclear translocation of NFATc3, which bound to RANTES promoter and directly regulated gene transcription to enhance RANTES expression.Conclusion: ASIC1a induces synovial inflammation, which leads to the progression of RA. Our study reveals a novel RA inflammation regulatory mechanism and indicates that ASIC1a might be a potential therapeutic target for RA.

ASIC1a activation induces calcium-dependent apoptosis of BMSCs under conditions that mimic the acidic microenvironment of the degenerated intervertebral disc.

Purpose: In the degenerated intervertebral disc (IVD), matrix acidity challenges transplanted bone marrow mesenchymal stem cells (BMSCs). The Ca2+-permeable acid-sensing ion channel 1a (ASIC1a) is responsible for acidosis-mediated tissue injury. The aim of our study was to confirm whether ASIC1a activation induces BMSC apoptosis under conditions that mimic the acidic microenvironment of the degenerated IVD.Methods: ASIC1a expression in rat BMSCs was investigated by real time-PCR, Western blot (WB) and immunofluorescence. The proliferation and apoptosis of BMSCs under acidic conditions were analyzed by MTT and TUNEL assays. Ca2+-imaging was used to assess the acid-induced increase in the intracellular Ca2+ concentration ([Ca2+]i). The activation of calpain and calcineurin was analyzed using specific kits, and WB analysis was performed to detect apoptosis-related proteins. Ultrastructural changes in BMSCs were observed using transmission electron microscopy (TEM).Results: Acid exposure led to the activation of ASIC1a and increased BMSC apoptosis. The Ca2+ imaging assay showed a significant increase in the [Ca2+]i in response to a solution at pH 6.0. However, BMSC apoptosis and [Ca2+]i elevation were alleviated in the presence of an ASIC1a inhibitor. Moreover, ASIC1a mediated the Ca2+ influx-induced activation of calpain and calcineurin in BMSCs. WB analysis and TEM revealed mitochondrial apoptosis, which was inhibited by an ASIC1a inhibitor, in BMSCs under acidic conditions.Conclusions: The mimical acidic microenvironment of the degenerated IVD can induce BMSC apoptosis by activating Ca2+-permeable ASIC1a. An acid-induced elevation of [Ca2+]i in BMSCs leads to the subsequent activation of calpain and calcineurin, further resulting in increased mitochondrial permeability and mitochondrial-mediated apoptosis.

Acid-Sensing Ion Channels: Novel Mediators of Cerebral Vascular Responses.

Precise regulation of cerebral blood flow is critical for normal brain function. Insufficient cerebral blood flow contributes to brain dysfunction and neurodegeneration. Carbon dioxide (CO2), via effects on local acidosis, is one of the most potent regulators of cerebral blood flow. Although a role for nitric oxide in intermediate signaling has been implicated, mechanisms that initiate CO2-induced vasodilation remain unclear. Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work that implicated ASIC1A in the amygdala and bed nucleus of the stria terminalis in CO2-evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO2. To test this hypothesis, we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO2-induced dilation of cerebral arterioles in vivo. Effects of inhalation of 5% or 10% CO2 on arteriolar diameter were greatly attenuated in mice with global deficiency in ASIC1A (Asic1a-/-) or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial nitric oxide synthase were unaffected, suggesting a nonvascular source of nitric oxide may be key for CO2 responses. Thus, we tested whether neurons may be the cell type through which ASIC1A influences microvessels. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO2 on arteriolar diameter were also attenuated. Together, these data are consistent with a model wherein activation of ASIC1A, particularly in neurons, is critical for CO2-induced nitric oxide production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone.

β-Estradiol Protects Against Acidosis-Mediated and Ischemic Neuronal Injury by Promoting ASIC1a (Acid-Sensing Ion Channel 1a) Protein Degradation.

Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of β-estradiol. However, the detailed mechanisms underlying the neuroprotection by β-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of β-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with β-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca2+ were all attenuated by β-estradiol treatment. In addition, we showed that β-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- β-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.

Acid-Sensing Ion Channel 1a Modulates NMDA Receptor Function Through Targeting NR1/NR2A/NR2B Triheteromeric Receptors

The over-activation of N-methyl-D-aspartate receptors (NMDARs) is the main cause of neuronal death in brain ischemia. Both the NMDAR and the Acid-sensing ion channel 1a (ASIC1a) are present in the postsynaptic membrane of the central nervous system (CNS) and participate in physiological and pathological processes. However, the specific role played by ASIC1a in these processes remains elusive. We hypothesize that NMDARs are the primary mediators of normal synaptic transmission and excitatory neuronal death, while ASIC1a plays a modulatory role in facilitating NMDAR function. Using various experimental approaches including patch-clamp recordings on hippocampal slices and CHO cells, primary cultures of hippocampal neurons, calcium imaging, Western blot, cDNA transfection studies, and transient middle cerebral artery occlusion (tMCAO) mouse models, we demonstrate that stimulation of ASIC1a facilitates NMDAR function and inhibition of ASIC1a suppresses NMDAR over-activation. One of our key findings is that activation of ASIC1a selectively facilitates the NR1/NR2A/NR2B triheteromeric subtype of NMDAR currents. In accordance, inhibition of ASIC1a profoundly reduced the NMDAR-mediated EPSCs in older mouse brains, which are known to express much higher levels of triheteromeric NMDARs than younger brains. Furthermore, brain infarct sizes were reduced by a greater degree in older mice compared to younger ones when ASIC1a activity was suppressed. These data suggest that ASIC1a activity selectively enhances the function of triheteromeric NMDARs and exacerbates ischemic neuronal death especially in older animal brains. We propose ASIC1a as a novel therapeutic target for preventing and reducing the detrimental effect of brain ischemia in humans.

Na+] Increases in Body Fluids Sensed by Central Nax Induce Sympathetically Mediated Blood Pressure Elevations via H+-Dependent Activation of ASIC1a

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

Acid-Sensing Ion Channel 1a Regulates Fate of Rat Nucleus Pulposus Cells in Acid Stimulus Through Endoplasmic Reticulum Stress.

Acid-sensing ion channel 1a (ASIC1a) participates in human intervertebral disc degeneration (IVDD) and regulates the destiny of nucleus pulposus cells (NPCs) in acid stimulus. However, the mechanism of ASIC1a activation and its downstream pathway remain unclear. Endoplasmic reticulum (ER) stress also participates in the acid-induced apoptosis of NPCs. The main purpose of this study was to investigate whether there is any connection between ASIC1a and ER stress in an acid-induced nucleus pulposus degeneration model. The IVDs of Sprague-Dawley rats were stained by immunohistochemical staining to evaluate the expression of ASIC1a in normal and degenerated rat nucleus pulposus. ASIC1a expression was also quantified by quantitative real-time-polymerase chain reaction and Western blotting analysis. NPCs were exposed to the culture media with acidity at pH 7.2 and 6.5 for 24 h, with or without 4-phenylbutyrate (4-PBA, a blocker of the ER stress pathway). Cell apoptosis was examined by Annexin V/Propidium Iodide (PI) staining and was quantified using flow cytometry analysis. ASIC1a-mediated intracellular calcium was determined by Ca2+ imaging using Fura-2-AM. Acidity-induced changes in ER stress markers were studied using Western blotting analysis. In vivo, ASIC1a expression was upregulated in natural degeneration. In vitro, acid stimulus increased intracellular calcium levels, but this effect was blocked by knockdown of ASIC1a, and this reversal was partly inhibited by 4-PBA. In addition, blockade of ASIC1a reduced expression of ER stress markers, especially the proapoptotic markers. ASIC1a partly regulates ER stress and promotes apoptosis of NPCs under acid stimulus and may be a novel therapeutic target in IVDD.

Abstract WMP115: Acid-Sensing Ion Channels: Novel Mediator of Cerebrovascular Responses

Carbon dioxide (CO 2 ), via effects on extracellular pH, is one of the most potent regulators of cerebral blood flow. Clinically, CO 2 inhalation or acetazolamide treatment (to produce tissue acidosis) are used commonly to estimate vascular reserve, the degree of vascular stenosis, and the effectiveness of collateral vessels. Despite the physiological importance and the clinical use of CO 2 , mechanisms that mediated these responses are still not defined. A role for NO produced by neuronal NO synthase (nNOS) in intermediate signaling has been described, but the molecular sensor that initiates vascular responses to CO 2 and acidosis has not been identified. Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work which implicated ASIC1A in CO 2 -evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO 2 . To test this hypothesis we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO 2 -induced dilation of cerebral arterioles. Effects of inhalation of 5 or 10% CO 2 on arteriolar diameter in vivo was greatly attenuated in Asic1a -/- mice or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial NOS were unaffected, consistent with nNOS playing a key role for CO 2 responses. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO 2 on arteriolar diameter were also attenuated. To further explore a role for ASIC1A in the regulation of NO production, we measured NO concentrations in whole brain lysates immediately after exposure to CO 2 . We found that inhalation of CO 2 induced NO production in wild-type mice, but not in mice lacking Asic1a globally or specifically in neurons. Together, these data are consistent with a model wherein activation of neuronal ASIC1A is critical for CO 2 -induced NO production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone in brain.

Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.

Acid-Sensing Ion Channels Activated by Evoked Released Protons Modulate Synaptic Transmission at the Mouse Calyx of Held Synapse.

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. We found that these channels can be activated in neurons of the medial nucleus of the trapezoid body (MNTB) of the auditory system in the CNS. A drop in extracellular pH induces transient inward ASIC currents (IASICs) in postsynaptic MNTB neurons from wild-type mice. The inhibition of IASICs by psalmotoxin-1 (PcTx1) and the absence of these currents in knock-out mice for ASIC-1a subunit (ASIC1a-/-) suggest that homomeric ASIC-1as are mediating these currents in MNTB neurons. Furthermore, we detect ASIC1a-dependent currents during synaptic transmission, suggesting an acidification of the synaptic cleft due to the corelease of neurotransmitter and H+ from synaptic vesicles. These currents are capable of eliciting action potentials in the absence of glutamatergic currents. A significant characteristic of these homomeric ASIC-1as is their permeability to Ca2+ Activation of ASIC-1a in MNTB neurons by exogenous H+ induces an increase in intracellular Ca2+ Furthermore, the activation of postsynaptic ASIC-1as during high-frequency stimulation (HFS) of the presynaptic nerve terminal leads to a PcTx1-sensitive increase in intracellular Ca2+ in MNTB neurons, which is independent of glutamate receptors and is absent in neurons from ASIC1a-/- mice. During HFS, the lack of functional ASICs in synaptic transmission results in an enhanced short-term depression of glutamatergic EPSCs. These results strongly support the hypothesis of protons as neurotransmitters and demonstrate that presynaptic released protons modulate synaptic transmission by activating ASIC-1as at the calyx of Held-MNTB synapse.SIGNIFICANCE STATEMENT The manuscript demonstrates that postsynaptic neurons of the medial nucleus of the trapezoid body at the mouse calyx of Held synapse express functional homomeric Acid-sensing ion channel-1a (ASIC-1as) that can be activated by protons (coreleased with neurotransmitter from acidified synaptic vesicles). These ASIC-1as contribute to the generation of postsynaptic currents and, more relevant, to calcium influx, which could be involved in the modulation of presynaptic transmitter release. Inhibition or deletion of ASIC-1a leads to enhanced short-term depression, demonstrating that they are concerned with short-term plasticity of the synapse. ASICs represent a widespread communication system with unique properties. We expect that our experiments will have an impact in the neurobiology field and will spread in areas related to neuronal plasticity.