scholarly journals Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels

2021 ◽  
Vol 12 ◽  
Author(s):  
Christopher Katnik ◽  
Javier Cuevas
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Loop Diuretics ◽  
Clinical Effects ◽  
Voltage Gated ◽  
Membrane Transport Proteins ◽  
Acid Sensing Ion Channels ◽  
Cultured Cortical Neurons ◽  
Increased Activity

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Abstract P747: Gpr68 Play a Protective Role in Ischemic Stroke in Mice

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
tao wang ◽  
Guokun Zhou ◽  
mingdi he ◽  
yuanyuan xu ◽  
w.g. Rusyniak ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Brain Injury ◽  
Neuronal Injury ◽  
Protective Role ◽  
Functional Screening ◽  
Rt Pcr ◽  
Brain Neurons ◽  
Acid Sensing Ion Channels ◽  
The Brain

Introduction: Acidosis is one prevalent phenomenon in ischemic stroke. The literature has shown that protons directly gate acid-sensing ion channels (ASICs) and proton-activated chloride channel, both lead to neuronal injury However, it is unclear whether protons activate metabotropic pathways in brain neurons. There are four proton-sensitive G-protein coupled receptors (GPCRs): GPR4, GPR65, GPR68 and GPR132. It remains unknown whether any of these GPCRs mediate acid-induced signals in brain neurons or whether they contribute to ischemia-induced brain injury. Methods: Total RNA from human cortical tissue or mouse brain was isolated using TRIzol and an RNase Kit. Standard RT-PCR was performed to determine the expression of these GPCRs in the brain. An in vitro slice injury model was used for functional screening. To determine the effect of ischemia, WT and knockout male mice were subjected to MCAO. To study brain injury, brains were sectioned coronally at 1 mm intervals and stained by vital dye immersion: (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC). Locomotor analysis and corner test were used to assess behavior outcome. Adeno-associated virus (AAV) -mediated gene delivery was used to determine the outcome of GPR68 overexpression. Results: RT-PCR showed that brain tissue expressed GPR4, -65, and -68. The expression of GPR68 was evident at 30 cycles. In organotypic slices, compared to the WT, deleting GPR4 or GPR65 had no effect while deleting GPR68 significantly increased acidosis-induced neuronal injury. At both 24 hour and 72 hour after 45 minutes MCAO, GPR68 deletion increased brain injury (p=0.0020 for 24hour, p=0.0392 for 72hour, Mann-Whitney U test). WT and GPR68-/- mice did not differ in baseline locomotor activities or corner test. On the third day following MCAO, GPR68-/- exhibited significantly more left rotations (p=0.0287, Mann-Whitney U test) than WT animals. Lastly, mice receiving AAV-GPR68 exhibited an average infarct of 21.97 ± 12.4%, significantly (p = 0.0022, Mann-Whitney U test) smaller than those receiving AAV-GFP (37.2 ± 6.8%). Conclusion: These data showed that GPR68 functions as a neuroprotective proton receptor in the brain.

scholarly journals Glucose Deprivation Neuronal Injury in vitro is Modified by Withdrawal of Extracellular Glutamine

1990 ◽  
Vol 10 (3) ◽  
pp. 337-342 ◽  
Author(s):  
Hannelore Monyer ◽  
Dennis W. Choi
Keyword(s):  
Cortical Neurons ◽  
Excitatory Amino Acids ◽  
Neuronal Loss ◽  
Nmda Antagonist ◽  
Neuronal Injury ◽  
Glucose Deprivation ◽  
Glutamine Concentration ◽  
Energy Substrate ◽  
Cultured Cortical Neurons

Cultured cortical neurons deprived of glucose in a defined solution containing 2 m M glutamine became acutely swollen and went on to degenerate over the next day; this neuronal loss could be substantially attenuated by an N-methyl-D-aspartate (NMDA) antagonist. Removal of extracellular glutamine produced two effects: an increase in overall neuronal injury and a decrease in the protective effect of an NMDA antagonist. Both effects of glutamine removal were glutamine concentration dependent (EC50 for both ∼300 μ M) and not reversed by substitution of equimolar concentrations of alanine or arginine. These observations suggest that glucose deprivation neuronal injury may be tonically regulated by the presence of extracellular glutamine. We speculate that glutamine may reduce overall injury by serving as an energy substrate in the absence of glucose, but may increase NMDA receptor-mediated injury by serving as a precursor for transmitter excitatory amino acids.

scholarly journals Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

2021 ◽  
Vol 118 (32) ◽  
pp. e2018850118
Author(s):  
Hiroo Takahashi ◽  
Ryo Asahina ◽  
Masayuki Fujioka ◽  
Takeshi K. Matsui ◽  
Shigeki Kato ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
Small Gtpase ◽  
Neuroprotective Effects ◽  
Necessary And Sufficient ◽  
Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

The AMPAR antagonist perampanel regulates neuronal necroptosis via Akt/GSK3β signaling after acute traumatic injury in cortical neurons

2020 ◽  
Vol 19 ◽  
Author(s):  
Tao Chen ◽  
Li-Kun Yang ◽  
Jie Zhu ◽  
Chun-Hua Hang ◽  
Yu-Hai Wang
Keyword(s):  
Traumatic Brain Injury ◽  
Protective Effect ◽  
Neuronal Death ◽  
Cortical Neurons ◽  
Therapeutic Potential ◽  
Traumatic Injury ◽  
Neuroprotective Effects ◽  
Stroke Models ◽  
Cultured Cortical Neurons

Background: Perampanel is a highly selective and non-competitive α-amino-3-hydroxy-5 -methyl-4-isoxazole propionate (AMPA) receptor (AMPAR) antagonist, which has been licensed as an orally administered antiepileptic drug in more than 55 countries. Recently, perampanel was found to exert neuroprotective effects in hemorrhagic and ischemic stroke models. Objective: In this study, the protective effect of perampanel was investigated. Method: The protective effect of perampanel was investigated in an in vitro traumatic neuronal injury (TNI) model in primary cultured cortical neurons. Conclusion: Our present data suggest that necroptosis plays a key role in the pathogenesis of neuronal death after TNI, and that perampanel might have therapeutic potential for patients with traumatic brain injury (TBI).

scholarly journals Nigella sativaL. Seed Extract Modulates the Neurotransmitter Amino Acids Release in Cultured NeuronsIn Vitro

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Tarek El-Naggar ◽  
María Pilar Gómez-Serranillos ◽  
Olga María Palomino ◽  
Carmen Arce ◽  
María Emilia Carretero
Keyword(s):  
Amino Acids ◽  
Cortical Neurons ◽  
High Performance ◽  
Methanolic Extract ◽  
Nigella Sativa ◽  
Colorimetric Method ◽  
Gamma Aminobutyric Acid ◽  
Cultured Cortical Neurons

Nigella sativaL. (NS) has been used for medicinal purposes since ancient times. This study aimed to investigate the cytotoxicity of NS dry methanolic extract on cultured cortical neurons and its influence on neurotransmitter release, as well as the presence of excitatory (glutamate and aspartate) and inhibitory amino acids (gamma-aminobutyric acid—GABA—and glycine) in NS extract. Cultured rat cortical neurons were exposed to different times and concentrations of NS dry methanolic extract and cell viability was then determined by a quantitative colorimetric method. NS did not induce any toxicity. The secretion of different amino acids was studied in primary cultured cortical neurons by high-performance liquid chromatography (HPLC) using a derivation before injection with dansyl chloride. NS modulated amino acid release in cultured neurons; GABA was significantly increased whereas secretion of glutamate, aspartate, and glycine were decreased. Thein vitrofindings support the hypothesis that the sedative and depressive effects of NS observedin vivocould be based on changes of inhibitory/excitatory amino acids levels.

scholarly journals Effect of Conantokin G on NMDA Receptor–Mediated Spontaneous EPSCs in Cultured Cortical Neurons

2006 ◽  
Vol 96 (3) ◽  
pp. 1084-1092 ◽  
Author(s):  
Anitha B. Alex ◽  
Anthony J. Baucum ◽  
Karen S. Wilcox
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Binding Site ◽  
Cortical Neurons ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Cultured Cortical Neurons

Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to N-methyl-d-aspartate (NMDA) receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. This study was designed to evaluate the action of Con G on NMDA receptor–mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13–19 days in vitro) using the whole cell patch-clamp technique. Con G inhibited NMDA receptor–mediated sEPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed after application of Con G in the presence of high (10 μM) and nominal (no added) concentrations of glycine was not different at 13 days in vitro (DIV). Furthermore, similar results were obtained with experiments on Con G–induced inhibition of NMDA-evoked whole cell currents. These results indicate that glycine concentrations do not have a direct effect on Con G–induced inhibition of NMDA currents. In addition, age dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.

An MD2-perturbing peptide has therapeutic effects in rodent and rhesus monkey models of stroke

2021 ◽  
Vol 13 (597) ◽  
pp. eabb6716
Author(s):  
Zongping Fang ◽  
Di Wu ◽  
Jiao Deng ◽  
Qianzi Yang ◽  
Xijing Zhang ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cortical Neurons ◽  
Hemorrhagic Stroke ◽  
Rna Binding ◽  
Infarct Volume ◽  
Therapeutic Index ◽  
Therapeutic Effects ◽  
Normal Brain ◽  
Experimental Treatments

Studies have failed to translate more than 1000 experimental treatments from bench to bedside, leaving stroke as the second leading cause of death in the world. Thrombolysis within 4.5 hours is the recommended therapy for stroke and cannot be performed until neuroimaging is used to distinguish ischemic stroke from hemorrhagic stroke. Therefore, finding a common and critical therapeutic target for both ischemic and hemorrhagic stroke is appealing. Here, we report that the expression of myeloid differentiation protein 2 (MD2), which is traditionally regarded to be expressed only in microglia in the normal brain, was markedly increased in cortical neurons after stroke. We synthesized a small peptide, Trans-trans-activating (Tat)–cold-inducible RNA binding protein (Tat-CIRP), which perturbed the function of MD2 and strongly protected neurons against excitotoxic injury in vitro. In addition, systemic administration of Tat-CIRP or genetic deletion of MD2 induced robust neuroprotection against ischemic and hemorrhagic stroke in mice. Tat-CIRP reduced the brain infarct volume and preserved neurological function in rhesus monkeys 30 days after ischemic stroke. Tat-CIRP efficiently crossed the blood-brain barrier and showed a wide therapeutic index for stroke because no toxicity was detected when high doses were administered to the mice. Furthermore, we demonstrated that MD2 elicited neuronal apoptosis and necroptosis via a TLR4-independent, Sam68-related cascade. In summary, Tat-CIRP provides robust neuroprotection against stroke in rodents and gyrencephalic nonhuman primates. Further efforts should be made to translate these findings to treat both ischemic and hemorrhagic stroke in patients.

NR2B-Containing NMDA Autoreceptors at Synapses on Entorhinal Cortical Neurons

2001 ◽  
Vol 86 (4) ◽  
pp. 1644-1651 ◽  
Author(s):  
Gavin Woodhall ◽  
D. Ieuan Evans ◽  
Mark O. Cunningham ◽  
Roland S. G. Jones
Keyword(s):  
Cortical Neurons ◽  
Glutamate Release ◽  
Nr2b Subunit ◽  
Voltage Gated ◽  
B Subunit ◽  
Selective Agonist ◽  
Bath Application ◽  
Mepsc Frequency ◽  
Layer V

We have previously shown that presynaptic N-methyl-d-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit–selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca2+ entry through either NMDARs or voltage-gated Ca2+channels with Co2+ prevented the effects of HQA, confirming that Ca2+ entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.

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