Assay of 2‐Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography‐Mass Spectrometry

Noto, Tadashi; Hasegawa, Takeshi; Hashimoto, Hiromichi; Nakajima, Teruo

doi:10.1111/j.1471-4159.1988.tb02933.x

Cited by 3 publications

(3 citation statements)

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“…However, the precursor to GABOB, 2-hydroxyputrescine, is found throughout the brain, and Noto and his colleagues have quantified the amount of 2-hydroxyputrescine in rat cerebellum (2.44 ± 0.49 pg/μg protein) and hippocampus (1.52 ± 0.20 pg/μg protein). 4 Introducing a hydroxyl group at the C3 position of GABA generates a stereogenic center, thus producing the enantiomers R-(−)-GABOB and S-(+)-GABOB ( Figure 1). Of the two enantiomers, R-(−)-GABOB is the more potent anticonvulsant 2 and has been shown to have a greater inhibitory effect on induced seizures in cat brain and rabbit motor cortex.…”

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“…There are conflicting results concerning the concentration of GABOB in the CNS, with concentration ranging between <0.01 and 4.8 μmol/g for rat and bovine brain, respectively. However, the precursor to GABOB, 2-hydroxyputrescine, is found throughout the brain, and Noto and his colleagues have quantified the amount of 2-hydroxyputrescine in rat cerebellum (2.44 ± 0.49 pg/μg protein) and hippocampus (1.52 ± 0.20 pg/μg protein) …”

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Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors

Yamamoto

Absalom

Carland

et al. 2012

ACS Chem. Neurosci.

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Designing potent and subtype-selective ligands with therapeutic value requires knowledge about how endogenous ligands interact with their binding site. 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous ligand found in the central nervous system in mammals. It is a metabolic product of GABA, the major inhibitory neurotransmitter. Homology modeling of the GABA C ρ 1 receptor revealed a potential H-bond interaction between the hydroxyl group of GABOB and threonine 244 (T244) located on loop C of the ligand binding site of the ρ 1 subunit. Using sitedirected mutagenesis, we examined the effect of mutating T244 on the efficacy and pharmacology of GABOB and various ligands. It was found that mutating T244 to amino acids that lacked a hydroxyl group in their side chains produced GABA insensitive receptors. Only by mutating ρ 1 T244 to serine (ρ 1 T244S) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than ρ 1 wild-type. We also observed changes in the activities of the GABA C receptor partial agonists, muscimol and imidazole-4-acetic acid (I4AA). At the concentrations we tested, the partial agonists antagonized GABA-induced currents at ρ 1 T244S mutant receptors (Muscimol: ρ 1 wild-type, EC 50 = 1.4 μM; ρ 1 T244S, IC 50 = 32.8 μM. I4AA: ρ 1 wild-type, EC 50 = 8.6 μM; ρ 1 T244S, IC 50 = 21.4 μM). This indicates that T244 is predominantly involved in channel gating. R-(−)-GABOB and S-(+)-GABOB are full agonists at ρ 1 wild-type receptors. In contrast, R-(−)-GABOB was a weak partial agonist at ρ 1 T244S (1 mM activates 26% of the current produced by GABA EC 50 versus ρ 1 wild-type, EC 50 = 19 μM; I max 100%), and S-(+)-GABOB was a competitive antagonist at ρ 1 T244S receptors (ρ 1 wildtype, EC 50 = 45 μM versus ρ 1 T244S, IC 50 = 417.4 μM, K B = 204 μM). This highlights that the interaction of GABOB with T244 is enantioselective. In contrast, the potencies of a range of antagonists tested, 3-aminopropyl(methyl)phosphinic acid (3-APMPA), 3-aminopropylphosphonic acid (3-APA), S-and R-(3-amino-2-hydroxypropyl)methylphosphinic acid (S-(−)-CGP44532 and R-(+)-CGP44533), were not altered. This suggests that T244 is not critical for antagonist binding. Receptor gating is dynamic, and this study highlights the role of loop C in agonist-evoked receptor activation, coupling agonist binding to channel gating. KEYWORDS: T244, channel gating, enantioselective actions of GABOB, loop C, coupling agonist binding, channel gating 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous molecule found within the CNS, possessing anticonvulsant properties.1 It is formed by two metabolic pathways: either by the metabolism of putrescine to γ-aminobutyric acid (GABA) and then hydroxylation of GABA at the third carbon (C3), 2,3 or by the hydroxylation of putrescine to 2-hydroxyputrescine and then oxidative N-dealkylation to GABOB.3 There are conflicting results concerning the concentration of GABOB in the CNS, with concentration ranging between <0.01 and 4.8 μmol/g for rat and bovine brain, respectively. H...

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Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors

Yamamoto

Absalom

Carland

et al. 2012

ACS Chem. Neurosci.

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“…Therefore, if GABOB is synthesized only from 2-hydroxyputrescine, the level of GABOB in the CNS is probably <2 X lo-" mol/g wet tissue based on the level of 2-hydroxyputrescine (1 . O 1-2.55 pg/pg protein; Noto et al, 1988).…”

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confidence: 99%

Formation of γ‐Amino‐β‐Hydroxybutyric Acid from 2‐Hydroxyputrescine in Rat Brain

Noto¹,

Hasegawa²,

Nakao³

et al. 1988

Journal of Neurochemistry

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gamma-Amino-beta-[3H]hydroxybutyric acid ([3H]-GABOB) was formed in rat brain from 2-[3H]-hydroxyputrescine that had been chemically synthesized from 2-oxoputrescine and [3H]sodium borohydride. After the injection of 2-[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion-exchange chromatography, high-voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.

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Analysis of polyamines as markers of (patho)physiological conditions

Teti¹,

Visalli²,

McNair

2002

Journal of Chromatography B

115

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Assay of 2‐Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography‐Mass Spectrometry

Cited by 3 publications

References 10 publications

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors

Formation of γ‐Amino‐β‐Hydroxybutyric Acid from 2‐Hydroxyputrescine in Rat Brain

Analysis of polyamines as markers of (patho)physiological conditions

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Assay of 2‐Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography‐Mass Spectrometry

Cited by 3 publications

References 10 publications

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

Formation of γ‐Amino‐β‐Hydroxybutyric Acid from 2‐Hydroxyputrescine in Rat Brain

Analysis of polyamines as markers of (patho)physiological conditions

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Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABA_C ρ₁ Receptors