Studies on the release of exogenous and endogenous GABA and glutamate from rat brain synaptosomes

The aim of the present study was to determine whether exogenous radioactive GABA and glutamate previously taken up by rat brain synaptosomes are released preferentially with respect to the endogenous unlabeled amino acids. Preferential release was monitored by comparing the specific radioactivity of the amino acids released to that present in synaptosomes at the beginning and at the end of the release period. The GABA released spontaneously or by depolarizing the synaptosomes with high K⁺ in the presence of Ca²⁺ had the same specific radio-activity as that present in synaptosomes before or after superfusion. Depolarization with veratridine or superfusion with OH-GABA caused a moderate increase (15–20%) in the specific radioactivity of the GABA released and a corresponding slight decrease in that of superfused synaptosomes. In conditions causing a supraadditive release of exogenous and endogenous GABA (see ref. 13), the specific radioactivity of the GABA released was increased 20–30%. The GABA with higher-than-average specific radioactivity is probably representative of the cytoplasmic pool of this amino acid. The glutamate released spontaneously had a specific radioactivity lower than that present in synaptosomes at the start of superfusion, and also the specific radioactivity in superfused synaptosomes was lower than at the start of superfusion. The glutamate released by aspartate (by heteroexchange), by veratridine, or by high K⁺ had a specific radioactivity higher than that of the amino acid released spontaneously, similar to that present in synaptosomes at the start of superfusion, and higher than that found in superfused synaptosomes. These findings suggest that exogenous radioactive glutamate is released preferentially with respect to the endogenous amino acid and to the glutamate synthesized from glucose during the superfusion period.     

Effects of 2-substituted-4-phenylquinolines on uptake of serotonin and norepinephrine by isolated brain synaptosomes

In this present communication, the in vitro inhibition of the uptake of [3H]-L-norepinephrine ([3H] NE) and [3H]-Serotonin ([3H] 5-HT) by eleven synthesized 2-substituted-4-phenyl quinolines were studied using rat brain synaptosomal preparations. Compounds with an open side chain were relatively weak inhibitors of the synaptosomal uptake of [3H] NE and [3H] 5HT. Compounds having a distance of three atoms between the terminal basic nitrogen of the side chain and the quinoline ring were better inhibitors of serotonin uptake than those compounds having a four-atom distance. The replacement of the side chain with a piperazine ring produced compounds which were more potent and selective inhibitors of the uptake of either [3H] 5-HT or [3H] NE. Further structure-activity relationships are also discussed.     

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5–30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that comparment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.     

The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain

Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.     

The gamma-aminobutyric acid receptor in catfish brain

When the binding of [3H]gamma-aminobutyric acid (GABA) to its receptor in catfish synaptic membranes was studied, a high affinity (Kd = 8.4 nM) and a low affinity (Kd = 65 nM) binding component was observed. Muscimol, thiomuscimol, tetrahydroisoxazole-5,4-c-pyridin-3-ol, imidazole acetic acid and bicuculline each competitively inhibited both high affinity and low affinity [3H]GABA binding. The potency of these inhibitors was similar to that reported for the GABA receptor from mammalian brain. It is concluded that the GABA receptor from catfish brain has very similar properties to the receptor from mammalian central nervous system and consequently has not undergone any obvious evolutionary changes.     

Calciumm-dependent release of endogenous serotonin,dopamine and norepinephrine from nerve endings

The release of endogenous serotonin, dopamine, norepinephrine and 5-hydroxyindoleacetic acid was studied in static incubations of synaptosome (P₂) preparations from the telencephalon of the rat. Elevated potassium medium specifically stimulated the release of the biogenic monoamines while the deaminated metabolite of serotonin was not effected. The release of the monoamines was also sensitive in part to the presence of calcium in the incubation medium.     

Neuropharmacology of supraoptic nucleus neurons: norepinephrine and gamma-aminobutyric acid receptors

The neurosecretory neurons in the mammalian hypothalamic supraoptic nucleus receive prominent GABAergic and noradrenergic projections arising from local interneurons and the A1 cells in the ventrolateral medulla, respectively. Intracellular recordings in in vitro perfused hypothalamic explants reveal an abundance of spontaneous inhibitory postsynaptic potentials (IPSPs) and a compound IPSP after electrical stimulation in the diagonal band of Broca area. The sensitivity of both spontaneous and evoked IPSPs to intracellular chloride injection, bicuculline, and pentobarbital is consistent with a GABA-activated, chloride-mediated inhibitory synaptic input. Parallel changes in membrane voltage and conductance are present during applications of GABA and muscimol, with similar sensitivity to ionic manipulation, bicuculline, and pentobarbital. These observations contrast with the consistently excitatory effects that follow either the stimulation of A1 cells or the application of norepinephrine and alpha 1-adrenergic agonists. Norepinephrine induces membrane depolarizations and bursting activity patterns that are blocked by the selective alpha 1 antagonist prazosin. Membrane response to norepinephrine is voltage dependent and is associated with little change in conductance. GABA and norepinephrine are proposed as transmitters in the final central pathways that mediate information to supraoptic vasopressinergic neurons from peripheral baroreceptors and chemoreceptors, respectively.     

Compartmentation and release of exogenous GABA in sheep brain synaptosomes

Exogenous tritiated -aminobutiric acid ([³H]GABA) is retained in two compartments in sheep cortex synaptosomes, corresponding to cytoplasmic and vesicular spaces, assuming that freeze-thawing the synaptosomes loaded with [³H]GABA releases the cytoplasmic [³H]GABA (81±3.9%), and that subsequent solubilization of the synaptosomes with 1% sodium cholate releases the vesicular [³H]GABA (19±3.9%). Depolarization of synaptosomes with 40 mM K⁺ in a Na⁺-medium, in the absence of Ca²⁺, releases 20.3±2.7% of the [³H]GABA retained in the synaptosomes. The [³H]GABA released under these conditions comes predominantly from the cytoplasm. The presence of 1 mM Ca²⁺ during depolarization releases and additional 13% (a total of about 33.5±9.9%) of the releasable [³H]GABA, and the [³H]GABA release which is Ca²⁺-dependent also comes mostly from the cytoplasmic compartment. When choline replaces external Na⁺, the [³H]GABA release is absolutely Ca²⁺-dependent, and the [³H]GABA released also comes mostly from the cytoplasmic pool. Therefore, it appears that [³H]GABA taken up by synaptosomes is accumulated mostly in the cytoplasmic compartment from which it is released upon depolarization. The technique described permits distinguishing the effect of different factors on the two pools of accumulated [³H]GABA.     

Action of neurotropic substances on gamma-aminobutyric acid release from brain synaptosomes during K+ depolarization

The effects of aminazine (0.25 mM), phthoracyzine (0.5 mM), trifluperidole (0.5 mM) and imipramine (0.5 mM) on GABA release from rat brain synaptosomes depolarized with K+ (50 mM) were investigated. Incubation of synaptosomes with aminazine led to a 2-fold and that with phthoracyzine, trifluperidole and imipramine to a 1.5-fold increase in GABA release from synaptosomes as compared with its basic level. The raising of K+ in the incubation medium to 50 mM brought about a 2-fold augmentation of GABA release. Exposure of synaptosomes to drugs and a higher K+ concentration at a time did not change GABA release as compared to its basic level. Introduction into the incubation medium of the Ga-ionophore A23187 together with 50 mM K+ and trifluperidole or with K+ and imipramine led to the same increase in GABA release from synaptosomes as that produced by the psychotropic drugs as regards native synaptosomes. It is assumed that the lack of the influence of the psychotropic drugs under study of GABA release from synaptosomes depolarized with K+ is caused by blockade of synaptic membrane conductibility for Ca2+.     

Alpha-melanotropin in the frog brain: regional distribution, quantification and subcellular distribution

The distribution of alpha-MSH containing neurons was studied by immunofluorescence in the brain of the frog Rana ridibunda. Most immunoreactive cell bodies were found in the ventral hypothalamic area. A rich network of fluorescent fibers was observed in the ventral infundibular region, coursing towards the preoptic area and the ventral telencephalon. Some fibers, directed backwards, project into median eminence. By means of a specific radioimmunoassay, the concentrations of alpha-MSH immunoreactive material has been determined in 10 different regions of the brain. The highest concentrations were observed in the infundibular and the preoptic regions. Using the immunogold technique, electron microscopy showed that immunostaining was restricted to 70-100 nm dense core vesicles in positive cell bodies and fibers. These results suggest that, in addition to well known hormonal (melanotropic) activity, alpha-MSH could play the role of a neurotransmitter in the frog brain.     

Efflux of gamma-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca2+, or when Ca2+ was added to synaptosomes pretreated with Ca2+ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of gamma-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [14C]arachidonic acid were depolarized in the presence of Ca2+, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of gamma-aminobutyric acid in the absence of Ca2+. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the gamma-aminobutyric acid efflux.