An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats

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.

Akkermansia muciniphilaMucT attenuates sodium valproate‐induced hepatotoxicity and upregulation of Akkermansia muciniphila in rats

“… 41 The Chop, Grp‐78 and caspase‐12 are important endoplasmic reticulum stress biomarkers. 42 SVP administration significantly elevated the hepatic Chop, Grp‐78 and caspase‐12 protein levels, which was markedly inhibited by A. muciniphila MucT. These results indicated that anti‐endoplasmic reticulum stress is also an important mechanism for the protective effect of A. muciniphila MucT against SVP‐induced liver injury.…”

“…Endoplasmic reticulum stress is tightly linked to the liver injury of acetaminophen, isoniazid, SVP, etc 41 . The Chop, Grp‐78 and caspase‐12 are important endoplasmic reticulum stress biomarkers 42 . SVP administration significantly elevated the hepatic Chop, Grp‐78 and caspase‐12 protein levels, which was markedly inhibited by A. muciniphila MucT.…”

The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

“…Glutamate release from synaptosomes was detected through a glutamate dehydrogenase reaction, as described previously [ 48 , 49 ]. Briefly, synaptosomes (0.5 mg/mL of final protein concentration) were incubated in HEPES-buffered medium containing glutamate dehydrogenase (20 U/mL) (Sigma, St. Louis, MO, USA), β-nicotinamide adenine dinucleotide (NAD + , 1 mM), and CaCl 2 (1.2 mM), at 37 °C for 5 min.…”

“…As described previously [ 49 , 50 ], synaptosomes were resuspended in HEPES-buffered medium containing Fura-2-acetoxymethyl ester (5 μM) and CaCl 2 (0.1 mM) for 30 min at 37 °C. The synaptosome suspension was centrifuged (3000× g for 1 min), and the pellet was resuspended in HEPES-buffered medium.…”

“…As described previously [ 28 , 49 ], synaptosomes were re-suspended in HEPES-buffered medium containing DiSC 3 (5) (5 μM) and CaCl 2 (1.2 mM) for 10 min at 37 °C. DiSC 3 (5) fluorescence was measured in a Perkin-Elmer LS55 spectrofluorimeter at excitation and emission wavelengths of 646 nm and 674 nm, respectively.…”