Excitatory Amino Acids: Studies on the Biochemical and Chemical Stability of Ibotenic Acid and Related Compounds

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Abstract
The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA), (RS)-.alpha.-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-.alpha.-amino-3-hydroxy-4-bromo-5-isoxazolepropionic acid (4-Br-homoibotenic acid) were all inhibitors of (S)-glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal-5-phosphate (PALP) and was inhibited by (S)-glutamic acid, 3-mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allylglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min-1 mg-1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)-glutamic acid (3.2 nmol min-1 mg-1 of protein). At pH 7.4 and 37.degree. C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7-HPCA, but not AMPA and 4-Br-homoibotenic acid, decomposed, partially by decarboxylation, at 100.degree. C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose. Although muscimol was the only detectable reaction product, mechanisms other than decarboxylation may be involved. Under these conditions, the degradation reaction or reactions were partially dependent on PALP and were inhibited by AOAA and 3MPA but not by allylglycine. The present in vitro studies indicate that ibotenic acid is likely to undergo enzyme-catalyzed decomposition to give muscimol in brain tissue and after systemic administration to animals. These aspects must be taken into consideration in the interpretation of the pharmacological or neurotoxic effects of ibotenic acid after direct application near central neurons, after local injections into animal brains, or after systemic administration to animals.