An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats.

Cheng-Wei Lu,Tzu-Yu Lin,Chen-Jung Lin,Kuan-Ming Chiu,Su-Jane Wang,Ming-Yi Lee,Pei-Wen Hsieh

doi:10.3390/ijms23052641

Abstract

The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebro-cortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca2+ level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca2+ channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine-rich C kinase substrate (MARCKS) in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q-type Ca2+ channels and PKC/MARCKS pathways.

Highlights

Glutamate is a key excitatory neurotransmitter in brain development, synaptic transmission, synaptic plasticity, and learning and memory processes [1,2]
This study aimed to investigate the effect of HFP034 on glutamate release from nerve terminals in the rat cerebral cortex and evaluate its neuroprotective effect in a rat model of excitotoxicity induced by systemic administration of kainic acid (KA), a glutamate analog [18]
To analyze whether HFP034 affected synaptic glutamate release, isolated nerve terminals were stimulated with 4-aminopyridine (4-AP) (1 mM), which led to the activation of voltage-dependent Ca2+ channels (VDCCs) and glutamate release [19]

Summary

Introduction

Glutamate is a key excitatory neurotransmitter in brain development, synaptic transmission, synaptic plasticity, and learning and memory processes [1,2]. Glutamate leads to excessive activation of glutamate receptors, which increases the concentration of intracellular calcium and results in the activation of proteases, production of free radicals, induction of mitochondrial dysfunction, and activation of proapoptotic factors. These effects lead to neurodegeneration and neuronal death. Anthranilate derivatives were reported to have antidepressant and anticonvulsant effects in different animal models [14,15], indicating that they are potential candidates for drug therapy for related diseases such as anxiety, depression, and epilepsy. This study aimed to investigate the effect of HFP034 on glutamate release from nerve terminals in the rat cerebral cortex and evaluate its neuroprotective effect in a rat model of excitotoxicity induced by systemic administration of kainic acid (KA), a glutamate analog [18]

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