Biomedical subjects
Publications and source records attributed to L Ossola.
Although their body weights were decreased by about 77% and their brain weights by about 30%, high-affinity [3H] muscimol binding to a cerebral membrane fraction was not altered in hereditary pituitary dwarf mice. Marked changes in the level of pituitary growth-associated hormones do not appear to be associated with a change in cerebral GABA-receptors.
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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.
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"Specific" binding of [3H]muscimol (11.5 nM) related to GABA receptors, occurred in subcellular particles prepared from 4-, 8-, and 12-day cultures of embryonic (14- to 15-day-old) mouse cerebrum, but not to particles prepared from 20- to 45-day cultures. Phase-contrast microscopy revealed that the presence of neuronal elements (neuroblasts, neurites) paralleled the binding of [3H]muscimol. Further evidence is thus provided for using high-affinity [3H]muscimol binding as a "neuronal marker."
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Although these studies have provided some further insight into GABA binding processes associated with transport and receptor-activation, such attempts remain limited due to the lack of specificity of the ligands used to displace these components. Thus, BMI appeared to interact with both transport and synaptic receptor sites for GABA. Perhaps certain GABA-agonists (e.g., muscimol, isoguvacine) will be shown to interact more specifically with synaptic GABA-receptors? So-called "specific" binding of GABA and its analogues, or antagonists might not be the issue to be kept in focus in this type of experiment, especially since it cannot be separated into presynaptic, postsynaptic and non-synaptic components. The specificity of an interaction of ligand with membrane site may be determined by other requirements of the particular system concerned. In any case, it seems essential to examine the binding sites, themselves, in order to define more clearly which of these are involved in receptor-activation and in uptake. An approach involving the isolation and purification of GABA-receptors should provide evidence for specific binding of GABA to its postsynaptic receptors.
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The binding of [3H]GABA and retention of [14C]sucrose have been studied in freshly prepared "synaptosomal-mitochondrial" (P2) fractions of rat cerebral cortex and liver using bicarbonate-buffered medium (containing 147 mEq/liter of Na+), and in frozen/thawed crude membrane fractions of rat whole brain and liver using Na+-free Tris HCl medium. GABA-sensitive sites (GSS) and bicuculline-methiodide- (BMI-) sensitive sites (BMI-SS) were defined as those amounts of [3H]GABA that were sensitive to the displacement by 10(-3) M unlabeled GABA or BMI. In the presence of added Na+, two high-affinity GABA-binding processes were detected in the P2 fraction of cerebral cortex. The lower-affinity process (likely related mainly to uptake sites) had KB approximately equal to 10(-5) M, Bmax for GSS approximately equal to 3 nmol/mg protein, and Bmax for BMI-SS approximately equal to 0.5 nmol/mg protein, whereas the higher-affinity process (likely related to synaptic GABA receptors) had KB approximately equal to 10(-7) M, BMAX for GSS approximately equal to 43 pmol/mg protein, and BMAX for BMI-SS approximately equal to 2 pmol/mg proteins. Only the higher-affinity process was detected in the liver P2 fraction, and it had KB approximately equal to 3.7 x 10(-8) M, BMAX for GSS approximately equal to 0.48 pmol/mg protein, and BMAX for BMI-SS approximately equal to 0.1 pmol/mg protein (i.e., about 1/100 and 1/20 the receptive BMAX values of cerebral cortex). This binding process of the liver P2 fraction could represent sites involved in mitochondrial GABA transport. In Na+-free Tris HCl medium, high-affinity [3H]GABA binding appeared to exist in frozen/thawed membrane preparations of both brain and liver when data were expressed on a protein basis. However, this binding to liver membranes was not displaceable by 10(-5) M unlabeled GABA, and when these data were expressed on a weight basis and corrected for [3H]GABA present in trapped supernatant fluid of the pellets, no [3H]GABA binding was detected in the liver preparation.
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.
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