L. Ossola

In vitro studies into the effect of inhibition of rat brain succinic semialdehyde dehydrogenase on GABA synthesis and degradation

The inhibitory role of GABA in certain central nervous system mechanisms is well known [ 1 ] . As GABA itself does not normally traverse the bloodbrain barrier various inhibitors of GABA transaminase have been used in order to increase its cerebral level [2]. However, most of these inhibitors which cross the blood-brain barrier act on the coenzyme, pyridoxal phosphate, and hence are very toxic and. not specific for GABA-T. Some branched chain fatty acids inhibit purified GABA-T [3-51 as well as succinic semialdehyde dehydrogenase [6-71. We describe the results of experiments designed to explore the effect of inhibition of SSADH on GABA synthesis and degradation in a rat brain 900 X g supernatant fraction.

Na+‐Independent Binding of [3H]GABA and [3H]Muscimol to Subcellular Particles of Neural Primary Cultures and Whole Brain

y-AMINOBUTYRIC acid (GABA) is bound to particulate preparations of the vertebrate CNS by highaffinity processes in the absence of added Na+, and it is believed that such processes represent interactions of GABA and its synaptic receptors (e.g. Enna and Snyder, 1975: reviews by DeFeudis, 1977 and Johnston, 1978). In order to examine this hypothesis further, the binding of [RH]GABA and of the potent GABA-agonist, [3H]muscimol, to subcellular particles of whole brain and of neurone-enriched and astroblast cultures have been compared.

High-affinity binding of [3H]muscimol to subcellular particles of a neurone-enriched culture of embryonic rat brain

High-affinity, Na+-independent binding of [3H]muscimol (KB approximately equal to 1.6 x 10(-8) M; Bmax approximately equal to 0.14 nmol/g pellet) occurred to a frozen-thawed particulate fraction of 74-h-old neurone-enriched cultures prepared from the cerebra of 12-13-day-old rat embryos. This finding provides evidence that GABA-receptors exist on cultured neurones which contain only a few synaptic connections.

Comparison of the structures of L-glutamate decarboxylases from human and rat brains

Abstract Human and rat L-glutamate decarboxylases have been purified to electrophoretic homogeneity. These two enzymes were compared using an immunochemical method, amino acid analysis and tryptic fingerprinting. Structural studies revealed several differences in the primary structure of the two enzymes, but the immunochemical method used did not distinguish between the antigenicity of the two proteins.

More muscimol binding sites than GABA binding sites in a particu1ate fraction of rat brain

Abstract Muscimol (3-hydroxy-5-aminomethylisoxazole), a conformationally-restricted analogue of γ-aminobutyric acid (GABA), exerts pronounced bicuculline-sensitive depressant actions when applied iontophoretically to neurones of the mammalian CNS (1,2). Recent in vitro studies have revealed further that muscimol is about 5 – 10 times more potent than GABA itself at competing with [ 3 H]GABA or [ 3 H]bicuculline-methiodide for membrane binding sites of rat brain (3–5) and that [ 3 H]muscimol is bound to various CNS particulate fractions by mechanisms that involve one or two populations of high-affinity sites (6–9). However, except for one preliminary report (10), no studies have been aimed at comparing the maximal binding capacities (B max ) of muscimol and GABA in a CNS preparation. Herein, we present such data.

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [3H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron‐enriched culture of embryonic rat brain were examined using Na+‐free Tris‐citrate medium and a centrifugation method. Competition for [3H]muscimol binding sites by excess(10−5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [3H]muscimol binding was potently inhibited by muscimol itself (IC50, 2.5 nM), GABA (1C50, 43 nM), isoguvacine (IC50, 61 nM), and 3‐aminopropanesulphonic acid (IC50, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC50, 800 nM). δ‐ Aminovaleric acid (IC50, 2.6 μM), the glycinelp‐alanine antagonist strychnine (IC50, 6.6 μM), and the predominantly glial GABA uptake inhibitors β‐alanine (IC50, 23 μM) and p‐proline (IC50, 66 μM) also inhibited [3H]muscimol binding. Other inhibitors of Na+‐dependent GABA uptake, (±)‐nipecotic acid, L‐ 2,4‐diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [3H]muscimol binding (IC50≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [3H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.

Comparison of the “specific” binding of [3H]GABA and [3H]muscimol to a particulate fraction of rat brain

Abstract 1. Over an identical concentration range (3.08 × 10−9−3.08 × 10−8 M), both [3H]GABA and [3H]muscimol were bound to a particulate fraction of rat brain by high-affinity processes in the absence of added Na+. 2. Components of these processes that were displaceable by excess unlabelled GABA had K B ⋍ 2 × 10 −7 M and B max ⋍ 1 pmol/mg protein for [3H]GABA and K B ⋍ 7 × 10 −8 M and B max ⋍ 1.9 pmol/mg protein for [3H]muscimol. 3. Pellet/supernatant distribution ratios and Bmax values indicated that more [3H]muscimol sites than [3H]GABA sites exist in rat brain. 4. [3H]Muscimol binding sites need not be the same as those that bind [3H]GABA to be sensitive to excess unlabelled GABA. 5. The concept of “specific” GABA binding is further qualified.

Antiserum to gangliosides inhibits [3H]GABA binding to a synaptosome-enriched fraction of rat cerebral cortex

Abstract 1. 1. The effects of various rabbit sera on the binding of [ 3 H]GABA (4.6 × 10 −9 M) and on the retention of [ 14 C]sucrose (1.04 × 10 −7 M) in a synaptosome-enriched fraction of rat cerebral cortex were examined using differential centrifugation and double-isotope methods. 2. 2. Additions of undiluted whole sera (amounting to about 1 5 th of the total volume of suspensions) decreased the binding of [ 3 H]GABA, did not affect the retention of [ 14 C]sucrose and increased pellet/supernatant [ 14 C]sucrose distribution ratios and protein contents of pellets. 3. 3. Only antiserum to total gangliosides-plus-albumin significantly decreased the binding of [ 3 H]GABA to a greater extent than did control serum; this effect was not due to albumin antibodies since antiserum to albumin did not affect [ 3 H]GABA binding and it was not due to increased protein content of pellets since serum containing hemolyzed erythrocytes (which had a higher protein content than other sera) did not affect [ 3 H]GABA binding. 4. 4. Gangliosides might be involved in modulating the interaction of GABA and its synaptic receptors and/or its transport sites.

Binding of [3H]GABA and [3H]muscimol to subcellular particles of a neurone-enriched culture of mouse brain

Binding of [3H]GABA and [3H]muscimol, indicative of GABA-receptors, has been demonstrated in a neurone-enriched culture of embryonic mouse brain using a ligand-binding technique. Evidence is provided for the existence of different populations of GABA-receptors.

Binding of muscimol and GABA in sub-fractions of a crude membrane fraction of rat brain

Abstract The binding of [ 3 H]γ-aminobutyric acid ([ 3 H]GABA) and [ 3 H]muscimol to subcellular particles of brain, which occurs in the absence of added Na + (i.e., “Na + -independent binding”), has been used to estimate synaptic GABA-receptors (1–3). This binding appears to be most enriched in crude synaptic membrane fractions of brain (4–6), but all of the binding sites do not appear to be localized to synaptic membranes (e.g., 2,7,8). Recent studies have revealed further that the highest-affinity process for [ 3 H]muscimol binding to subcellular particles of rat brain has a higher capacity than that of [ 3 H]GABA (9–12). Herein, the binding of these ligands to sub-fractions of a crude membrane fraction of rat brain are compared.