Imidazoleacetic Acid Research Articles

Effects of GABA receptor agonists on [ 3H] flunitrazepam binding to rat cerebellar and hippocampal membranes

The effects of GABA agonists on [ 3H] flunitrazepam binding were examined in membranes from CNS areas proposed to contain different populations of benzodiazepine binding site subtypes ( BZ 1 BZ 2 ). Since these effects were broadly similar in both cerebellar and hippocampal membranes, it seems unlikely that GABA receptors interacting with the proposed BZ 1 BZ 2 binding sites haue markedly different properties. Sodium chloride clearly facilitated the potentiating effect of muscimol on [ 3H] flunitrazepam binding but produced a complex interaction uith the effects of GABA itself and the partial agonist imidazoleacetic acid in the phosphate buffer system used.

Method for enzymatic determination of imidazole acetic acid

A method for enzymatic assay of imidazole acetic acid (ImAA) was developed, based on the strict substrate specificity of imidazole acetate monooxygenase from Pseudomonas sp. [Maki et al. (1969) J. Biol. Chem., 244., 2942–2950], which catalyzes concomitant conversion of NADH to NAD +. Thus, ImAA was determined by measuring decrease in absorbancy at 340 nm. Tissue extracts were partially purified and/or concentrated by column chromatography on Bio-Rad AG-1 before enzymatic assay. The lowest measurable level of ImAA by this method was 2 nmol.

Characteristics of gamma-aminobutyric acid (GABA) receptors in the rat central nervous system.

Characteristics of gamma-aminobutyric acid (GABA) were investigated in the rat central nervous system by radioreceptor assay (RRA). Scatchard analysis revealed that the rat brain had two distinct GABA binding sites with an apparent dissociation constant (Kd) of 11.7 nM and 34.7 nM. The highest level of specific [3H]-GABA binding was found in the rat cerebellum. Imidazole acetic acid, a potent GABA agonist, was effective in displacing [3H]-GABA binding but beta-alanine was slightly effective in inhibiting [3H]-GABA binding. Muscimol, the most potent GABA agonist, has been used as a ligand to characterize the postsynaptic GABA receptors. However, the maximal binding capacity (Bmax) of muscimol-RRA was about 3 times larger than that of GABA-RRA, suggesting that muscimol might label not only GABA receptors but other unknown receptors as well. An endogenous inhibitor of GABA receptor binding was purified from the P2 fraction of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA on GABA binding sites. The inhibition by the endogenous inhibitor of GABA receptor binding was blocked by the allosteric effect of diazepam. In the presence of diazepam, [3H]-GABA binding with the endogenous inhibitor was larger than that with GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself. The molecular weight of the endogenous inhibitor was estimate by gel filtration to be less than 3,000 daltons.

Pharmacological studies of ganglionic transmission: Antagonism by histamine and imidazole-4-acetic acid against the stimulant effect of angiotensin II on cardiac sympathetic agnglia in dogs

Histamine (HA), imidazole-4-acetic acid (IAA) and angiotensin II (AT II) were administered to the cardiac sympathetic ganglia through the right subclavian artery (i.a.) in spinal dogs. IAA, a metabolite of HA, at a dose of 500 ug, induced a marked inhibition of the rate increase produced by 1 and 2 μg AT II, 10 and 20 μg bethanechol (BCH), and 5 and 10 μg dimethylphenylpiperazinium (DMPP). This inhibition was strongly antagonized by i.a. injection of Picrotoxin, 5 mg, and partly inhibited by the’ same dose of bicuculline. Single i.a. injection of HA did not inhibit the positive chronotropism induced by these three ganglionic stimulants, but three repeated i.a. injections of 50 or 100 ug HA, at the interval of 5 min rendered the ganglion insensitive to the same dose of HA and also inhibited the positive chronotropism produced by AT II and BCH, but not by DMPP. The inhibition by the repeated HA administration of the AT II and BCH-induced rate increase was not antagonized by i.a. injection of Picrotoxin or i.v. injection of 10 mg/kg aminoguanidi ne, an inhibitor of histaminase and monoamine oxidase. The results suggest that IAA counteracts ganglionic stimulant effects of AT II, BCH and DMPP by acting on γ-aminobutyric acid (GABA) receptors located at the ganglia as similarly does GABA, whereas the repeated administration of HA inhibits the ganglionic stimulant effects of AT II and BCH through mechanisms other than the GABA-ergic one which is mediated via endogenous IAA formed by exogenous HA.

Biosynthesis and synaptic release of GABA and its regulation by presynaptic autoreceptor

It was found that 8-10 % of 3H-glutamic acid (GLU) taken up into striatal slices were converted to 3H-GABA (newly synthesized H-GABA) during the incubation in the presence of nipecotic acid (5×105M). The high potassium (60 mM)-evoked and Ca2 dependent release of newly synthesized 3H-GABA was found to be more prominent than that of accumulated 3H-GABA from the incubation medium. The evoked release of 3H-GABA from striatal slices was significantly suppressed by in vitro addition (10−5 M) of δ-aminolevulinic acid (DALA) as well as some GABA agonists such as imidazoleacetic acid and muscimol (MUS).

The effects of imidazole-4-acetic acid on cerebral carbohydrate metabolism.

The effects of intravenous administration of 50-400 mg/kg imidazole-4-acetic acid (IMA) on the carbohydrate metabolism of the rat brain were assessed by measurement of the cerebral hemisphere contents of energy phosphates and glycolytic--citric acid cycle metabolites. IMA (100-400 mg/kg) produced a spectrum of electroencephalographic (EEG) change ranging from desynchronization to electrical suppression which was associated with unchanged tissue contents of ATP, ADP, and AMP, increasing levels of phosphocreatine, glucose, and aspartate, and decreasing levels of pyruvate, lactate, alpha-ketoglutarate, and malate. The changes in glycolytic intermediates were present within 5 min of injecting IMA (200 mg/kg) and the pattern suggested a suppression of glycolysis. The EEG stage of electrical suppression with episodic spiking (400 mg/kg) was associated with a 30% reduction of cortical high-energy phosphate use. The lowest dose of IMA (50 mg/kg) resulted in episodic EEG desynchronization which was associated with no significant changes of the measured metabolites. The results indicate that IMA is associated with metabolite changes that are compatible with a state of cerebral depression and that the desynchronous EEG pattern is without a biochemical correlate of increased neuronal activity.

Presence and measurement of methylimidazoleacetic acids in brain and body fluids.

N tau-Methylimidazoleacetic acid, the histamine metabolite, and its isomer, N pi-methylimidazoleacetic acid, were demonstrated and measured in rat brain and in human cerebrospinal fluid, urine, and plasma by a gas chromatographic-mass spectrometric method that is simple and specific with a detection limit of about 7 pmol (i.e. 1 ng). The acids were separated in biological samples by ion exchange chromatography, derivatized as n-butyl esters with boron trifluoride-butanol, and extracted with chloroform. Complete chemical ionization mass spectra and mass ion abundance ratios established the identity of N tau - and N pi - methylimidazoleacetic acids in the biological extracts and of imidazoleacetic acid in urine, but not in cerebrospinal fluid, plasma, and brain. The methylimidazoleacetic acids as n-butyl esters were quantified by electron impact selected ion monitoring of m/e 95 esters at different retention times. 3-Pyridylacetic acid was used as an internal standard and monitored at m/e 93. The levels of N tau-methylimidazoleacetic acid and N pi-methylimidazoleacetic acid were, respectively (picomoles/g or picomoles/ml +/- S.E.), for brain, 373, 19 +/- 13.08 and 110.33 +/- 12.44; for cerebrospinal fluid, 22.77 +/- 2.15 and 80.76 +/- 18.92; and for plasma, 84.57 +/- 13.64 and 73.64 +/- 14.50. In urine, the respective levels were 20.75 +/- 1.30 and 73.02 +/- 38.22 nmol/mg of creatinine. The origin of N pi-methylimidazoleacetic acid is not certain.

Superoxide Production by Eosinophils: Activation by Histamine

Both guinea pig peritoneal exudate and human peripheral blood eosinophils produce large amounts of superoxide anion when stimulated by preopsonized zymosan or phorbol myristate acetate (PMA). Superoxide production is also activated by histamine but not the histamine metabolite, imidazole acetic acid. Supernatants from degranulated rat mast cells stimulate superoxide production. In studies of both human and guinea pig eosinophils, the H1-antagonist, chlorpheniramine (10-3 M and 10-4 M), preopsonized zymosan histamine) production of superoxide anion but the H2-antagonist, cimetidine, only modestly inhibited superoxide anion production (zymosan, PMA), These studies provide direct evidence for the influence of histamine on the oxidative metabolism of eosinophils. These results are consistent with the hypothesis that histamine interacts with eosinophils predominantly via an H1 receptor site. Furthermore, they suggest that eosinophils may participate in immediate hypersensitivity reactions by the release of superoxide anion in response to stimulation by histamine.

Diamine oxidase activity and imidazoleacetic acid formation in the foetal and maternal guinea pig liver

Diamine oxidase activity and imidazoleacetic acid formation in the foetal and maternal guinea pig liver during gestation were examined. DAO activity and IMAA formation in the foetal liver increased continuously, while maternal enzyme activity and ImAA formation in the second half of pregnancy simultaneously decreased. The roles of GABA and ImAA are discussed.

Pharmacological investigation of gamma-aminobutyric acid (gaba) and fully-developed generalized seizures in the amygdala-kindled rat

The effects of GABA-modulating drugs were assessed in a pharmacological study of amygdala-kindled seizures in the rat. Fully-kindled subjects were tested with a randomized dose regimen, including drug vehicle, for each of seven drugs. Afterdischarge duration, motor seizure latency, motor seizure duration and motor seizure stage were scored. The GABA synthesis inhibitor, 3-mercaptopro-pionic acid, the GABA antagonist, bicuculline, and the chloride ionophore blocker, picrotoxin, all decreased motor seizure latency, but did not otherwise alter the kindled seizure duration or seizure stage. The inhibitor of GABA metabolism, gamma-vinyl-GABA, and pentobarbital, which competes for the picrotoxin binding site, both antagonized kindled seizures. Gamma-vinyl-GABA, however, did not appear to antagonize kindled seizures by a specific effect on GABA neurotransmission. The GABA agonists, imidazole acetic acid and ¦alpha-(chloro-4'phenyl)fluoro-5-hydroxy-2-benzylidene-amino¦-4-butyramide (SL 76-002), did not alter the kindled seizures. The results of these experiments are not consistent with the hypothesis that kindled seizures result from a loss of GABA-mediated inhibition: however, GABA may have a role in the modulation of kindled seizure activity.

Ontogenesis of GABA receptor sites in chick embryo cerebellum

The time-course of the development of GABA receptor sites in chick embryo cerebellum was correlated with the appearance of synaptic junctions in the cerebellar cortex. At 13 days of incubation, the earliest stage examined, specific [ 3H]GABA binding was only 19% of that found in cerebella of adult chicks. Between 15 days of incubation and hatching, specific [ 3H]GABA binding increased 3-fold, already reaching at birth adult values. During this period the number of synaptic junctions also increased. Scatchard analysis of the binding data obtained at birth revealed two binding sites of K d s 40 and 174 nM and a maximal number of binding sites (n) of about 1.6 and 4.0 pmol/mg protein, respectively. The high-affinity binding site for [ 3H]GABA was inhibited by muscimol, GABA, imidazoleacetic acid and bicuculline (IC 50: 0.007, 0.020, 0.1 and 10 μM, respectively). These values correspond to the potencies shown by those compounds in the binding to the synaptic GABA receptor. Treatment of the synaptic membranes with Triton X-100 enhanced [ 3H]GABA binding depending on the developmental stage studied, suggesting that GABAmodulin that inhibits the binding also appears during that period.

The effects of imidazole-4-acetic acid upon the energy metabolism of normoxic and hypoxic rat brain

The metabolic effects of intravenous administration of 200 mg/kg imidazole-4-acetic acid (IMA) on normoxic, hypoxemic and hypoxemic-oligemic rat brain were assessed by measurement of the cerebral contents of energy phosphates and selected glycolytic-citric acid cycle intermediates. In normoxic brain IMA was associated with unaltered adenylates, increased glucose and aspartate, and decreased pyruvate, lactate, citrate, α-ketoglutarate and malate; a pattern which is compatible with cerebral metabolic depression. In hypoxemic brain IMA was associated with a lower accumulation of lactate and a higher level of tissue glucose; again conforming to patterns documented for classical cerebral depressants. In hypoxemic-oligemic brain IMA was associated with a gross deterioration of the tissue ATP content and with a massive lactacidosis which indicated a detrimental action in this hypoxic state.

Pharmacological investigation of γ-aminobutyric acid (GABA) and the development of amygdala-kindled seizures in the rat

The effect of GABA agonists and antagonists on the rate of kindled seizure development was studied in rats. Drugs and drug doses were selected to modify GABA function by a variety of mechanisms. In the drug treatment groups, drugs were administered daily before kindling stimulation, and matched control groups received only drug vehicle before stimulation. Kindling development was (i) accelerated by the GABA-synthesis inhibitor 3-mercaptopropionic acid (20 mg/kg) and the GABA antagonist bicuculline (2.5 mg/kg); (ii) retarded by pentobarbital (5 mg/kg); and (iii) not altered by the GABA-response antagonist picrotoxin (2 mg/kg), the GABA agonist imidazole acetic acid (100 mg/kg), or the GABA metabolism inhibitor γ-vinyl GABA (100 mg/kg). These results are interpreted as evidence that endogenous GABA may play a role in opposing kindled seizure development, but that the mechanism of that development does not primarily involve failure of the GABA system.

Perturbation of benzodiazepine receptor binding by pyrazolopyridines involves picrotoxinin/barbiturate receptor sites

The two pyrazolopyridines, etazolate (SQ20009) and cartazolate (SQ65396), enhance the binding of [3H]diazepam to benzodiazepine receptor sites in rat brain. This enhancement is due to a change in affinity without a change in maximal binding. Pentobarbital also enhances [3H]diazepam binding by lowering the K(D). Pentobarbital gives a maximal enhancement of benzodiazepine binding slightly greater than that induced by etazolate, but maximal concentrations of etazolate cannot increase further the maximal enhancement by pentobarbital. By contrast, the enhancement of benzodiazepine binding by γ-aminobutyric acid (GABA) is fully additive with the effect of either etazolate or pentobarbital. The enhancement by γ-aminobutyric acid is blocked specifically by the two γ-aminobutyric acid mimetic compounds, imidazole acetic acid and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP), compounds which do not inhibit the enhancement of diazepam binding by pentobarbital or etazolate. The effects of etazolate and pentobarbital, but not those of GABA, can be blocked competitively by 1 to 10 μM picrotoxinin. Etazolate and cartazolate, like pentobarbital competitively inhibit α-[3H]dihydropicrotoxinin binding with potencies correlating well with their stimulatory properties on benzodiazepine binding. These results suggest that pyrazolopyridines, like pentobarbital, stimulate [3H]diazepam binding through a picrotoxinin-sensitive site which is distinct from the γ-aminobutyric acid receptor site. These in vitro interactions between the etazolate and benzodiazepine receptor sites can be modulated also by the GABA antagonist, bicuculline, consistent with the existence of a complex containing three receptors (GABA, benzodiazepines, and etazolate/barbiturates/picrotoxinin). Bicuculline reverses completely the effects of etazolate and GABA on benzodiazepine binding but reverses only partially the enhancement by pentobarbital. This difference between the effects of pentobarbital and etazolate, as well as differences in their maximal enhancing effects mentioned above, suggests that pentobarbital may have an action similar to etazolate on some bicuculline-sensitive benzodiazepine binding sites, but, in addition, pentobarbital can enhance some other benzodiazepine sites in a manner which is neither mimicked by etazolate nor blocked by bicuculline. (Less)

Activating effects of homotaurine and taurine on corticoreticular epilepsy.

Homotaurine and taurine are two powerful inhibitory aminoacids with anticonvulsant properties against various experimental models of focal epilepsy. This study reports on their effects in the feline model of corticoreticular epilepsy induced by parenteral administration of large amounts of penicillin. Both aminoacids, but particularly homotaurine, remarkably potentiate epileptiform discharges in cats. Brainstem transection at the precollicular level does not modify the activation, thus ruling out the intervention of mesoromboencephalic structures in the observed effect. The opposing action of these two amino acids on focal epilepsy as compared to corticoreticular epilepsy suggests that the two types of epileptiform activity stem from very different pathophysiological mechanisms. Homotaurine is a powerful GABA agonist that exerts a central action upon parenteral administration. Other GABA analogs such as muscimol, imidazole acetic acid, and gamma-hydroxybutyrate have been reported to potentiate experimental models of spike and wave epilepsy. Thus, the activating effects of homotaurine in this epilepsy model are in keeping with the demonstrated GABAmimetic properties of the compound.

Neuropharmacological studies of phencyclidine (PCP)-induced behavioral stimulation in mice.

A variety of drugs were screened to determine which were capable of blocking the behavioral stimulation produced in mice by acute administration of phencyclidine (PCP). Chlorpromazine and clozapine blocked PCP-induced stimulation, while haloperidol, reserpine, and alpha-methyl-p-tyrosine did not. The GABA receptor agonists imidazole acetic acid and muscimol blocked PCP, but other drugs that influence GABA, such as dipropylacetic acid, baclofen, and diazepam, were ineffective. Yohimbine and methysergide also blocked PCP in high dosages, but other drugs with comparable alpha-noradrenergic and serotonergic blocking properties (phentolamine, cyproheptadine, and cinnanserin) were ineffective. Cholinergic and anticholinergic drugs, beta-noradrenergic and opiate antagonists and nonspecific sedatives and convulsants were also ineffective. These findings suggest that chlorpromazine, clozapine, yohimbine, and methysergide may share a property that is unlike their primary known modes of action on dopaminergic, alpha-noradrenergic, and serotonergic neurotransmitter systems, and that this property accounts for their ability to block PCP. However, the effectiveness of GABA agonists appears to be mediated through direct activation of GABA receptors. It is suggested that chlorpromazine and imidazole acetic acid should be considered as possible drug treatments for PCP toxicity.

Specific modulation of complement-dependent human granulocyte function by imidazole acetic acid

Because imidazole acetic acid (IAA), a product of histamine catabolism, was shown to inhibit histaminase release from human polymorphonuclear leukocytes (PMNs), the effect of this compound on other neutrophil functions was investigated. IAA at concentrations of 10 − 10 or more inhibited histaminase release induced by particle-bound C3b, the larger fragment of the activated form of the third component of complement. Release of histaminase induced by aggregated IgG, phorbal myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) and calcium ionophore was not affected by IAA. In addition IAA had no effect on release of β-glucuronidase, myeloperoxidase, and lysozyme or on phagocytosis and superoxide generation. IAA did modestly inhibit neutrophil chemotaxis. These findings suggest a highly specific modulating effect of the histamine catabolite IAA on complement-mediated PMN function.

The formation of hydrogen cyanide from histidine in the presence of amino acid oxidase and peroxidase

Conditions were sought to increase the yield of HCN from l-histidine incubated with l-amino acid oxidase ( l-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) from snake venom, and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Small amounts of histidine and high buffer concentrations favored high HCN yields, which reached a maximum of 72%. Imidazole 4-aldehyde and imidazole 4-carboxylic acid were identified among the reaction products, together with CO 2, NH 3, H 2O 2 and imidazole acetic acid. The CO 2 formed was equal to the histidine oxidized, and to the sum of NH 3 plus HCN formed. The production of HCN was associated with an increased O 2 uptake, which was established from the beginning of the reaction, with no apparent lag and ranged from 1.2 to 1.6 μmol extra O 2 taken up/μmol HCN formed. The system was inhibited by catalase, but added superoxide dismutase caused a small stimulation of both HCN production and O 2 consumption, and a larger stimulation of H 2O 2 accumulation. Added hydroxylamine was cooxidized to nitrite in an amount equimolar with the HCN formed. This nitrite formation was inhibited by superoxide dismutase. The facts could be interpreted in terms of superoxide anion formation during the HCN-producing reaction.

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Brønsted relation. With reference to this Brønsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.

Inhibition of histaminase release from human granulocytes by products of histaminase activity.

Imidazoleacetic acid, a product of the action of histaminase (E.C. 1.4.3.6) on histamine, inhibits specific release of histaminase from human peripheral blood granulocytes with an inhibition constant between 5 X 10(-9)M and 1 X 10(-8)M. Hence, modulation of enzyme release is indirectly mediated by the activity of the enzyme.