Vesicular Transporter Inhibitor Research Articles

Hepatic extracellular ATP/adenosine dynamics in zebrafish models of alcoholic and metabolic steatotic liver disease

Steatotic liver disease (SLD) is a burgeoning health problem predominantly associated with excessive alcohol consumption, which causes alcohol-related liver disease (ALD), and high caloric intake, which results in metabolic dysfunction-associated SLD (MASLD). The pathogenesis of ALD and MASLD, which can progress from steatohepatitis to more severe conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, is complicated by several factors. Recently, extracellular ATP and adenosine (Ado), as damage-associated molecular patterns, were reported to promote inflammation and liver fibrosis, contributing to SLD pathogenesis. Here, we explored the in vivo dynamics of hepatic extracellular ATP and Ado during the progression of steatohepatitis using a genetically encoded GPCR-activation-based sensor (GRAB) in zebrafish models. We established hepatocyte-specific GRABATP and GRABAdo in zebrafish and investigated the changes in in vivo hepatic extracellular ATP and Ado levels under ALD or MASLD conditions. Disease-specific changes in hepatocyte extracellular ATP and Ado levels were observed, clearly indicating a correlation between hepatocyte extracellular ATP/Ado dynamics and disease progression. Furthermore, clodronate, a vesicular nucleotide transporter inhibitor, alleviated the MASLD phenotype by reducing the hepatic extracellular ATP and Ado content. These findings provide deep insights into extracellular ATP/Ado dynamics in disease progression, suggesting therapeutic potential for ALD and MASLD.

A novel class of insecticidal alkylsulfones are potent inhibitors of vesicular acetylcholine transport

Pyridine alkylsulfone derivatives typified by oxazosulfyl (Sumitomo Chemical Company Ltd.) and compound A2 (Syngenta) represent a new class of insecticides, with potent activity against several insect orders. Whilst the MOA of this class has been attributed to interaction with the voltage-gated sodium channel (VGSC), here we present strong evidence that their toxicity to insects is mediated primarily through inhibition of the vesicular acetylcholine transporter (VAChT). Alkylsulfone intoxication in insects is characterised by (i) a reduction in cholinergic synaptic transmission efficiency demonstrated by a depression of cercal afferent activity in giant-interneurone preparations of American cockroach (Periplaneta americana), (ii) selective block of cholinergic-transmission dependent post-synaptic potentials in the Drosophila giant-fibre pathway and (iii) abolition of miniature excitatory post-synaptic currents (mEPSCs) in an identified synapse in Drosophila larvae.Ligand-binding studies using a tritiated example compound ([3H]-A1) revealed a single saturable binding-site, with low nanomolar Kd value, in membrane fractions of green bottle fly (Lucilia sericata). Binding is inhibited by vesamicol and by several examples of a previously identified class of insecticidal compounds known to target VAChT, the spiroindolines. Displacement of this binding by analogues of the radioligand reveals a strong correlation with insecticidal potency. No specific binding was detected in untransformed PC12 cells but a PC12 line stably expressing Drosophila VAChT showed similar affinity for [3H]-A1 as that seen in fly head membrane preparations. Previously identified VAChT point mutations confer resistance to the spiroindoline class of insecticides in Drosophila by Gal-4/UAS directed expression in cholinergic neurones and by CRISPR gene-editing of VAChT, but none of these flies show detectable cross-resistance to this new chemical class.Oxazosulfyl was previously shown to stabilise voltage-gated sodium channels in their slow-inactivated conformation with an IC50 value of 12.3μM but inhibits binding of [3H]-A1 with approximately 5000 times greater potency. We believe this chemistry class represents a novel mode-of-action with high potential for invertebrate selectivity.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 μg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

Azetidines with All-Carbon Quaternary Centers: Merging Relay Catalysis with Strain Release Functionalization.

Given the importance and beneficial characteristics of decorated azetidines in medicinal chemistry, efficient strategies for their synthesis are highly sought after. Herein, we report a facile synthesis of the elusive all-carbon quaternary-center-bearing azetidines. By adopting a well-orchestrated polar-radical relay strategy, ring strain release of bench-stable benzoylated 1-azabicyclo[1.1.0]butane (ABB) can be harnessed for nickel-catalyzed Suzuki Csp2-Csp3 cross-coupling with commercially available boronic acids in broad scope (>50 examples), excellent functional group tolerance, and gram-scale utility. Preliminary mechanistic studies provided insights into the underlying mechanism, wherein the ring opening of ABB with a catalytic quantity of bromide accounts for the conversion of ABB into a redox-active azetidine, which subsequently engages in the cross-coupling reaction through a radical pathway. The synergistic bromide and nickel catalysis could intriguingly be derived from a single nickel source (NiBr2). Application of the method to modify natural products, biologically relevant molecules, and pharmaceuticals has been successfully achieved as well as the synthesis of melanocortin-1 receptor (MC-1R) agonist and vesicular acetylcholine transporter (VAChT) inhibitor analogues through bioisosteric replacements of piperidine with azetidine moieties, highlighting the potential of the method in drug optimization studies. Aside from the synthesis of azetidines, we demonstrate the ancillary utility of our nickel catalytic system toward the restricted Suzuki cross-coupling of tertiary alkyl bromides with aryl boronic acids to construct all-carbon quaternary centers.

Reserpine and PCPA reduce heat tolerance in Drosophila melanogaster

Drosophila melanogaster is a model organism to study molecular mechanisms and the role of the genes and proteins involved in thermal nociception. Monoamines (i.e. dopamine) have been involved in temperature preference behavior in D. melanogaster. Therefore, we investigated whether the monoamines, particularly dopamine and serotonin, participate in the response to thermal nociceptive stimuli in D. melanogaster. Flies were treated with reserpine (an inhibitor of vesicular monoamines transporter, 3–300 μM), 3-Iodo-L-tyrosine (3-I-T, an inhibitor of tyrosine hydroxylase, 16.28–65.13 mM), and para-Chloro-DL-phenylalanine (PCPA, an inhibitor of tryptophan hydroxylase, 20–80 mM); then, the flies were subjected to tests of thermal tolerance and avoidance of noxious heat. Climbing behavior was used as a test to evaluate locomotor activity. Reserpine reduces the thermal tolerance profile of the D. melanogaster, as well as the avoidance of noxious heat and locomotor activity depending on the concentration. PCPA, but not 3-I-T, decreased heat tolerance and avoidance of noxious heat. These data suggest that monoamines, particularly serotonin, are associated with the impaired avoidance of noxious heat which could be related to the reduction of heat tolerance in D. melanogaster.

β-N-methylamino-l-alanine is a non-competitive inhibitor of vesicular monoamine transporter 2

The neurotoxic, non-proteinogenic amino acid β-N-methylamino-l-alanine (BMAA) has been implicated in the development of neurodegenerative diseases; however, the mechanism(s) and mode(s) of toxicity remain unclear. Similarities in the neuropathology and behavioural deficits of neonatal rats exposed to either BMAA or reserpine, a known vesicular monoamine transporter 2 (VMAT2) inhibitor, suggest a similar mode of action. The aims of this study were therefore to determine if BMAA could prevent the uptake of serotonin into dense granules via inhibition of VMAT2, and, if so, the type of inhibition caused by BMAA. Exposing platelet dense granules to BMAA resulted in a concentration-dependent reduction in serotonin uptake. The inhibition of VMAT2 was non-competitive. The findings from this study support previous reports that BMAA-associated neuropathologies in a neonatal rat model may be due to VMAT2 inhibition during critical periods of neurogenesis.

A new era in the diagnosis and treatment of tardive dyskinesia.

Tardive dyskinesia (TD) is a heterogeneous, hyperkinetic movement disorder induced by dopamine-receptor blocking agents that presents a unique challenge in the treatment of psychosis. Although acceptance of TD as a serious consequence of antipsychotic treatment was resisted initially, subsequent research by many investigators in psychopharmacology contributed to a rich store of knowledge on many aspects of the disorder. While basic neuroscience investigations continue to deepen our understanding of underlying motor circuitry, past trials of potential treatments of TD focusing on a range of theoretical targets were often inconclusive. Development of newer antipsychotics promised to reduce the risk of TD compared to older drugs, but their improved tolerability unexpectedly enabled an expanding market that paradoxically both increased the absolute number of patients at risk and diminished attention to TD which was relegated to legacy status. Fortunately, development and approval of novel vesicular monoamine transporter inhibitors offered evidence-based symptomatic treatment of TD for the first time and rekindled interest in the disorder. Despite recent progress, many questions remain for future research including the mechanisms underlying TD, genetic predisposition, phenomenological diversity, whether new cases are reversible, how to implement best practices to prevent and treat TD, and whether the development of novel antipsychotics free of the risk of TD is attainable. We owe our patients the aspirational goal of striving for zero prevalence of persistent symptoms of TD in anyone treated for psychosis.

Deutetrabenazine for the treatment of chorea associated with Huntington's disease.

This is a comprehensive review of the literature regarding the use of Deutetrabenazine in treating chorea associated with Huntington's disease. Unfortunately, treatment has been limited for many aspects of this neurodegenerative disease. The present investigation presents the background, evidence, and indications for the use Deutetrabenazine in the setting of Huntington's disease. Huntington's disease is characterized by a variety of motor, psychiatric, and cognitive symptoms with chorea being one of the more notable ones. Chorea is a movement disorder present in multiple neurologic diseases that causes involuntary and irregular muscle movements theorized to be stemming from high dopamine levels. Deutetrabenazine is thought to function as an inhibitor of the VMAT2 vesicular monoamine transporter resulting in decreased monoamine release, including dopamine, in the synaptic cleft which has a therapeutic effect in management of chorea. This drug was approved by the FDA in 2017 with a specific indication for tardive dyskinesia and choreiform movement in Huntington's disease. Currently, there is no definitive treatment for Huntington's disease. Thus, management is primarily focused on symptom management with the use of a variety of pharmaceutical agents. Chorea is one of the many manifestations that significantly alter the quality of life of many patients. Deutetrabenazine is a promising new option for the treatment of chorea in the setting of Huntington's disease. Although studies so far have displayed mixed results, further research, including head-to-head studies, is necessary to elucidate the true potential of this drug.

Lappaconitine inhibits glutamate release from rat cerebrocortical nerve terminals by suppressing Ca2+ influx and protein kinase A cascade

The inhibition of the excessive release of glutamate in the brain has emerged as a promising new option for developing therapeutic strategies for neurodegenerative disorders. This study investigated the effect and mechanism of lappaconitine, a diterpenoid alkaloid found in species of Aconitum, on glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Here, we report that in the rat cortical synaptosomal preparation, lappaconitine reduced the K+ channel blocker 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate. The inhibitory effect of lappaconitine on the evoked glutamate release was blocked by the vesicular transporter inhibitor bafilomycin A1 and calcium-chelating agent ethylene glycol tetraacetic acid (EGTA), but was unaffected by exposure to the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA). The depolarization-induced elevation of cytosolic calcium concentration ([Ca2+]c) was inhibited by lappaconitine, while the 4-AP-mediated depolarization of the synaptosomal membrane potential was not affected. The inhibition of glutamate release by lappaconitine was markedly decreased in synaptosomes pretreated with the Cav2.3 (R-type) channel blocker SNX-482 or the protein kinase A inhibitor H89. Nevertheless, the lappaconitine-mediated inhibition of glutamate release was not abolished by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Lappaconitine also significantly decreased the 4-AP-induced phosphorylation of PKA and SNAP-25, a presynaptic substrate for PKA. Our data suggest that lappaconitine reduces Ca2+ influx through R-type Ca2+ channels, subsequently reducing the protein kinase A cascade to inhibit the evoked glutamate release from rat cerebral cortex nerve terminals.

Using complex behavior to understand brain mechanisms in health and disease.

Using complex behavior to understand brain mechanisms in health and disease.

Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline, an Alkaloid from Corydalis yanhusuo.

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca2+ release dantrolene or the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca2+ entry and the MAPK/ERK/synapsin I signaling pathway.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Carrier-mediated serotonin efflux induced by pharmacological anoxia in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischaemia and induces deleterious effects on the cardiomyocytes. We aimed to investigate whether carrier-mediated 5-HT efflux contributed to the increase in interstitial 5-HT level during ischaemia. Using microdialysis technique applied to the heart of anaesthetised Wistar rats, myocardial interstitial concentration of 5-HT was measured by electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD) while simultaneously various pharmacological agents, which create a similar condition to ischaemia, were locally administered by reverse-microdialysis. Sodium cyanide-induced chemical anoxia increased dialysate 5-HT concentration. A similar increase in dialysate 5-HT concentration was induced by ouabain, an inhibitor of sodium-potassium ATPase and reserpine, an inhibitor of vesicular monoamine transporter. Fluoxetine, a selective serotonin reuptake inhibitor raised the baseline level of 5-HT, and neither sodium cyanide nor the combination of ouabain and reserpine induced further increase in 5-HT in the presence of fluoxetine. The results indicate that reverse transport of 5-HT via SERT, which is caused by an impaired ion gradient, contributes to the rise in interstitial level of 5-HT during ischaemia suggesting carrier-mediated 5-HT efflux occurs in the heart in vivo.

Valbenazine for the Treatment of Adults with Tardive Dyskinesia.

This a comprehensive review of the literature regarding the use of Valbenazine in treating tardive dyskinesia. A primarily oral movement disorder induced by chronic exposure to certain classes of medications, tardive dyskinesia is often resistant to many therapeutic approaches. This review presents the background, evidence, and indications for the use of Valbenazine as a treatment option for this condition. Tardive dyskinesia is a disorder arising from long-term exposure to medications that blocked dopamine receptors, primarily antipsychotics. It is characterized by abnormal movements of the oral-buccal-lingual structures as well as associated pain and hypertrophy. Simply stopping the use of the dopamine blocking agents effectively alleviates the symptoms but is not always reliable hence the need for another therapeutic approach.Valbenazine is thought to function as a highly selective inhibitor of the VMAT2 vesicular monoamine transporter resulting in decreased availability of dopamine in the presynaptic cleft. This leads to decreased dopaminergic activation of the striatal motor pathway. The FDA approved Valbenazine in 2017 to treat tardive dyskinesia in adults and needs to be evaluated with existing therapeutic approaches. The chronic use of dopamine receptor blocking agents, most commonly antipsychotics, can lead to a movement disorder called tardive dyskinesia. Once symptom onset has occurred, these movement abnormalities can persist for years to permanently, depending on the speed and effectiveness of treatment. Valbenazine is a relatively newer option for the treatment of tardive dyskinesia in adults. Compared to other pharmaceutical agents, it is more selective and has limited toxicities making it an effective treatment regimen. However, further research, including additional direct comparison studies, should be conducted to fully evaluate this drug's usefulness.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

Delayed increase of acetylcholine quantal size induced by the activity-dependent release of endogenous CGRP but not ATP in neuromuscular junctions.

In mouse motor synapses tetanic neuromuscular activity (30Hz, 2min) led to a delayed posttetanic potentiation of amplitude and duration of spontaneous miniature endplate potentials (MEPPs). Microelectrode recordings of MEPPs before and after nerve stimulation showed an increase in MEPP amplitude and time course by 30% and 15%, respectively, without changes in their frequency. Peak effect was detected 20min after tetanic activity and progressively faded throughout the next 40min of recording. The revealed potentiation of MEPPs was fully preserved in preparations from pannexin 1 knockout mice. It means, that myogenic ATP released via pannexin 1 channels from contracting muscle fibers is not likely to participate in the described phenomenon. But posttetanic potentiation of MEPPs was fully prevented by competitive antagonist of calcitonin gene-related peptide (CGRP) receptors CGRP8-37 , ryanodine receptors inhibitor ryanodine and by vesicular acetylcholine transporter inhibitor vesamicol. It is suggested that the combination of intensive synaptic and contractile activity in neuromuscular junctions is required to induce Ca2+ -dependent exocytosis of endogenous CGRP. The accumulation of CGRP in the synaptic cleft and its presynaptic activity may induce posttetanic potentiation of MEPP amplitude due to CGRP-stimulated acetylcholine loading into vesicles and subsequent increase of quantal size.

Risk of Neuroleptic Malignant Syndrome with Vesicular Monoamine Transporter Inhibitors.

ObjectiveVesicular monoamine transporter-2 (VMAT2) inhibitors have been proven to be effective for the treatment of tardive dyskinesia and their use is likely to increase. The evidence base of published clinical reports was reviewed to evaluate the possible risk of neuroleptic malignant syndrome (NMS) with these drugs.MethodsPubmed, Embase, Web of Science and PsycINFO databases were queried for all years using terms for “neuroleptic malignant syndrome”, “hyperthermia” AND “vesicular monoamine transporter inhibitors”, “reserpine”, “tetrabenazine”, “valbenazine” or “deutetrabenazine”ResultsThirteen clinical cases, ten of which involved tetrabenazine, were identified in which VMAT2 inhibitors were prescribed in patients with current or past NMS episodes. In most cases, the association was confounded by limited reporting of clinical data, variable temporal correlation with VMAT2 inhibitors, polypharmacy with antipsychotics, and uncertain differential diagnoses.ConclusionWhile rare cases of NMS meeting consensus criteria have been reported primarily with tetrabenazine, the risk with recently developed VMAT2 inhibitors may be even less. Evidence of causality of NMS with VMAT2 inhibitors is confounded by concomitant treatment with antipsychotics and diagnostic uncertainties in patients susceptible to basal ganglia dysfunction. Nevertheless, clinicians should remain vigilant for early signs of NMS in all patients treated with any drugs that affect brain dopamine activity.

11-Keto-β-Boswellic Acid Attenuates Glutamate Release and Kainic Acid-Induced Excitotoxicity in the Rat Hippocampus

Excessive glutamate concentration induces neuronal death in acute brain injuries and chronic neurodegenerative diseases. Natural compounds from medicinal plants have attracted considerable attention for their use in the prevention and treatment of neurological disorders. 11-Keto-β-boswellic acid, a triterpenoid found in the medicinal plant Boswellia serrata, has neuroprotective potential. The present study investigated the effect of 11-keto-β-boswellic acid on glutamate release in vitro and kainic acid-induced glutamate excitotoxicity in vivo in the rat hippocampus. In rat hippocampal nerve terminals (synaptosomes), 11-keto-β-boswellic acid dose-dependently inhibited 4-aminopyridine-stimulated glutamate release. This effect was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-threo-β-benzyloxyaspartate, and was blocked by the vesicular transporter inhibitor bafilomycin A1. In addition, 11-keto-β-boswellic acid reduced the 4-aminopyridine-induced increase in intrasynaptosomal Ca2+ levels. The N- and P/Q-type channel blocker ω-conotoxin MVIIC and the protein kinase A inhibitor H89 significantly suppressed the 11-keto-β-boswellic acid-mediated inhibition of glutamate release, whereas the intracellular Ca2+-releasing inhibitors dantrolene, CGP37157, and xestospongin C, mitogen-activated protein kinase inhibitor PD98059, as well as protein kinase C inhibitor calphostin C had no effect. In a rat model of excitotoxicity induced by intraperitoneal kainic acid injection (15 mg/kg), intraperitoneal 11-keto-β-boswellic acid administration (10 or 50 mg/kg) 30 min before kainic acid injection considerably ameliorated kainic acid-induced glutamate concentration elevation and CA3 neuronal death. These data suggested that 11-keto-β-boswellic acid inhibits glutamate release from the rat hippocampal synaptosomes by suppressing N- and P/Q-type Ca2+ channels and protein kinase A activity, as well as exerts protective effects against kainic acid-induced excitotoxicity in vivo.

Cycloheterophyllin Inhibits the Release of Glutamate from Nerve Terminals of the Rat Hippocampus.

The effect of cycloheterophyllin, a prenylflavone isolated from Artocarpus heteophyllus, on glutamate release was studied in the rat hippocampus using synaptosome and slice preparations. In rat hippocampal synaptosomes, cycloheterophyllin inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevation of intrasynaptosomal calcium levels. The inhibitory effect of cycloheterophyllin on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor, the N- and P/Q-type calcium channel blocker, and the protein kinase C (PKC) inhibitor but was insensitive to the intracellular Ca2+ release inhibitors, the protein kinase A inhibitor, and the mitogen-activated/extracellular signal-regulated kinase inhibitor. Western blotting data in synaptosomes also showed that cycloheterophyllin significantly decreased the level of phosphorylation of PKC. In addition, cycloheterophyllin decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without influencing the amplitude of sEPSCs and glutamate-activated currents in hippocampal slices, supporting a presynaptic action. Together, these results suggest that cycloheterophyllin inhibits presynaptic glutamate release by suppressing N- and P/Q-type calcium channel and PKC activity in the rat hippocampus.

Allicin Inhibits Glutamate Release from Rat Cerebral Cortex Nerve Terminals Through Suppressing Ca2+ Influx and Protein Kinase C Activity.

Evidence indicates that indirect inhibitory regulation of glutamatergic transmission, via reducing glutamate release, may induce neuroprotection. The present work was designed to examine whether allicin, a major component of garlic with neuroprotective effects, affected the release of glutamate evoked by 4-aminopyridine in rat cerebrocortical nerve terminals (synaptosomes). Allicin caused a potent inhibition on the release of glutamate evoked by 4-aminopyridine, and this inhibitory effect was abolished in the presence of Ca2+-free medium and vesicular transporter inhibitor. Allicin decreased the 4-aminopyridine-evoked elevation of intrasynaptosomal Ca2+ levels, but had no effect on the synaptosomal plasma membrane potential. The allicin-mediated inhibition of glutamate release was prevented by the N- and P/Q-type channel blocker and the protein kinase C (PKC) inhibitor, but was not affected by the intracellular Ca2+-release inhibitors, mitogen-activated protein kinase inhibitor, and protein kinase A inhibitor. Western blotting data also showed that allicin significantly reduced the phosphorylation of PKC. Together, these data indicate that in rat cerebrocortical nerve terminals, allicin depresses glutamate release and appears to decrease N- and P/Q-type Ca2+ channel and PKC activity.