5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes

The interaction of 5-aminolevulinic acid (ALA) with GABA_A receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [³H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1–10 mM) significantly inhibited the binding of [³H]muscimol (12 nM), with a similar potency in rat and human membranes (IC₅₀ = 199 vs. 228 M, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [³H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.     

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.     

3H-diazepam binding to brain synaptic membranes during the development of generalized epileptic activity

The development of bemegride-induced generalized epileptic activity in rats was shown to reduce the constant (CB). of specific 3H-diazepam binding with synaptic membranes from 0.23 nM-1 to 0.15 nM-1 and to increase the maximum number of membrane binding sites (Bmax) from 410 fmol/mg protein to 550 fmol/mg protein. It is assumed that the changes of benzodiazepine receptor properties are due to alteration in physico-chemical characteristics of synaptic membrane lipids resulting from the activation of lipid peroxidation.     

A novel specific binding site for anxiolytic homophthalazines in the rat brain.

Radioligand binding studies were performed in order to elucidate the mechanism of action of anxiolytic-neuroleptic homophthalazines. Rat striatal membrane preparations were found to bind 3H-EGIS 6775 [3H-GYKI-52 322, 3H-(1-(4-aminophenyl)-4-methyl-7,8-dimethoxy-5H-homophthalazine)] in a specific and displaceable manner. Several other brain regions tested were devoid of similar binding activity. Saturation analysis revealed that binding affinity was in the 10(-8)-10(-7) M range. Binding was enhanced by Mg2+ ions and, to a smaller extent by Ca2+ ions. The binding principle was sensitive to heat or trypsin treatment. This specific binding site appears, according to competition studies, different from the receptors whose presence in the rat striatum has been reported earlier.     

Control of rat gastric somatostatin release by gamma-aminobutyric acid (GABA)

The influence of gamma-aminobutyric acid (GABA) on gastric somatostatin and gastrin release was studied using an isolated perfused rat stomach preparation. GABA dose-dependently inhibited somatostatin release (maximal inhibition of 44% at 10(-5)M GABA), whereas gastrin secretion was not affected. The GABA agonist muscimol led to a decrease in somatostatin release of similar magnitude. The GABA-induced changes were partially reversed by 10(-5)M atropine. Gastrin secretion was not influenced by either protocol. It is concluded that GABA as a putative neurotransmitter in the enteric nervous system is inhibitory to rat gastric somatostatin release in vitro via cholinergic pathways.     

Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system

In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.     

Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

Summary The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.This work was supported by the grants from the Ministry of Education, Science, and Culture, Japan     

Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.     

Piperidinyl-3-phosphinic acids as novel uptake inhibitors of the neurotransmitter gamma-aminobutyric acid (GABA)

Piperidinyl-3-phosphinic acid 2, piperidinyl-3-methylphosphinic acid 3 and N-(4,4-diphenyl-3-butenyl)piperidinyl-3-phosphinic acid 4 have been synthesized as bioisosteres of the corresponding amino carboxylic acids, which are potent and specific GABA-uptake inhibitors. The novel amino phosphinic acids were tested for their GABA-uptake inhibitory activity and 2 and 4 were identified as the first phosphinic acid based GABA-uptake inhibitors. The methylphosphinic acid 3 was found to be inactive.     

Effects of anticonvulsants and gamma-aminobutyric acid (GABA)-mimetic drugs on immunoreactive somatostatin and GABA contents in the rat brain

Immunoreactive somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) contents in the rat brain were investigated to study chronic effects of the treatment with anticonvulsants, carbamazepine (CBZ), valproic acid (VPA) and phenytoin (PHT). Decreased IR-SRIF levels were found in several brain regions after chronic treatment with VPA and CBZ. GABA concentrations were found to be increased significantly in chronic CBZ and VPA treatment in the rat brain, especially in limbic structures. PHT had no effect on both IR-SRIF and GABA contents in the rat brain. Effects of several GABA-mimetic drugs also were studied on IR-SRIF contents in the rat brain. Aminooxyacetic acid an inhibitor of GABA transaminase, induced a decrease in IR-SRIF concentration in the pyriform and entorhinal cortex, whereas ethanolamine-o-sulfate, another GABA-transaminase inhibitor and muscimol, a GABA receptor agonist had no effect on brain IR-SRIF after acute administration. The present results suggest that endogenous somatostatin has an important role for anticonvulsant properties of CBZ and VPA, but not of PHT. The relationship between the changes in IR-SRIF and the GABA transmitter system in the anticonvulsant action of CBZ and VPA remains to be clarified.     

Degradation of gamma-aminobutyric acid (GABA) by marine microorganisms

Abstract By degrading the settlement inducer gamma-aminobutyric acid (GABA), bacteria may affect the larval settlement of sedentary marine invertebrates. Nearly one third of bacterial isolates from surfaces suitable for abalone ( Haliotis ) settlement were able to grow on GABA as sole carbon source. Compared with similar compounds, GABA was a good source of carbon, nitrogen and energy, and it was utilized concomitantly with glucose. GABA-metabolizing enzymes were constitutive in Pseudomonas fluorescens and inducible in Aeromonas hydrophila . High-affinity ( K _m: 20–50 μ M) and low-affinity ( K _m: 7–9 mM) uptake systems were produced in response to low and high GABA concentrations, respectively, in the growth medium. Within the ecologically significant temperature range (12–24°C), specific GABA degradation rates varied 2.5-fold in young cells of P. fluorescens . This organism los its ability to degrade GABA during the stationary phase. The results suggest that marine bacteria have the potential to affect invertebrate larval settlement by removing GABA from the settlement habitat.     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Measurement of gamma-aminobutyric acid (GABA) in blood.

Blood GABA levels can be readily determined using a radioreceptor assay or gas chromatography-mass spectrometry. After withdrawal of blood, GABA levels remain stable with 25–50% of the GABA in whole blood found in the plasma fraction. Whole blood GABA concentrations range from 500 pmoles/ml to 1200 pmoles/ml in 8 mammalian species with human values being about 900 pmoles/ml. $\text{in vivo}$ administration of aminooxyacetic acid increases both blood and brain GABA levels to a similar extent.     

Effects of GABA antagonist-induced seizures on 3H-muscimol and 3H-diazepam binding in the rat striatum]

The binding of 3H-muscimol and 3H-diazepam to rat striatum membranes after picrotoxin- and bicuculline-induced seizures was characterized. No alteration in the maximal binding capacity (Bmax) of 3H-muscimol was observed. However, bicuculline produced a 27% decrease in Kd. Both picrotoxin and bicuculline increased the binding capacity of 3H-diazepam. Bicuculline produced a 86% increase in Kd. These results suggest that the GABA antagonists-induced seizures may modulate 3H-muscimol and 3H-diazepam binding in rat striatum.     

Regional distribution of gamma-aminobutyric acid (GABA) in brain of the rhesus monkey

—The concentrations of γ-aminobutyric acid (GABA) in twenty different regions of Rhesus monkey brain were studied. The highest levels were found in the substantia nigra, globus pallidus, and hypothalamus. Regions of the cerebral cortex and thalamus contained low amounts and white matter the lowest. Indirect evidence supporting an inhibitory transmitter role for GABA is discussed.     

The effect of ionizing radiation on the binding of muscimol by synaptic membranes in the rat brain

A study was made of the effect of gamma-radiation on binding of muscimol, a GABA agonist, by synaptic membranes of rat brain cortex. Exposure to 2 Gy radiation was shown to reduce [3H]-muscimol binding to membranes.     

Autoradiographic studies of the gamma-aminobutyric acid (GABA) system in the rat pancreas

The beta-cells of the pancreatic islets have been shown to contain gamma-aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in beta-cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

Kinetics of [3H]muscimol binding to the GABAA receptor in bovine brain membranes

The binding of the GABA receptor agonist [3H]muscimol to membrane preparations from bovine cerebral cortex has been investigated in equilibrium and kinetic experiments. Equilibrium binding curves are biphasic and suggest that [3H]muscimol binds to both high-affinity (Kd approximately 10 nM) and low-affinity (Kd approximately 0.5 microM) sites. Binding to each class of sites is inhibited by GABA and by the specific GABAA receptor antagonist bicuculline. The kinetics of [3H]muscimol binding have been measured by using both manual filtration assays and an automated rapid filtration technique which permits the measurement of ligand dissociation on subsecond time scales. Association and dissociation curves are biphasic at all concentrations of [3H]muscimol studied, even under conditions of low receptor saturation when no significant occupancy of the low-affinity sites would be expected. These results cannot be simply explained by the presence of two populations of binding sites in the membrane preparations but suggest the existence of two forms of the monoliganded receptor. Dissociation constants for these two forms have been estimated to be 16 and 82 nM at 23 degrees C. At higher ligand concentrations, kinetic measurements have suggested that the binding of [3H]muscimol to low-affinity sites is accompanied by a slow conformational change of the receptor-ligand complex.