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T T Gibbs

Publications and source records attributed to T T Gibbs.

31 records · Page 2Linked to original sources
PubMed1990

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.

Affinity Labels↗
PubMed1990

gamma-Aminobutyric acidA receptor regulation in culture: altered allosteric interactions following prolonged exposure to benzodiazepines, barbiturates, and methylxanthines.

In previous reports, we have described the use of primary neuronal cultures derived from chick brain to study the regulation of the gamma-aminobutyric acidA (GABAA) receptor complex. Chronic exposure of cultures to GABA, benzodiazepines, or methylxanthines results in decreased enhancement of [3H]flunitrazepam binding by GABA, consistent with an allosteric uncoupling of GABA and benzodiazepine recognition sites of the GABAA receptor. In the present communication, we extend our studies of the pharmacology of benzodiazepine- and methylxanthine-induced uncoupling of GABA/benzodiazepine recognition site interactions and present evidence to show that certain barbiturates (barbital and pentobarbital) also induce uncoupling. Chronic exposure to flurazepam (a high efficacy benzodiazepine) elicits no change in the number of benzodiazepine binding sites or the affinity of benzodiazepine binding in the absence of GABA. Whereas flurazepam and theophylline decrease coupling, Ro15-1788 (a low efficacy benzodiazepine) inhibits flurazepam-induced but not theophylline-induced uncoupling, suggesting that theophylline and flurazepam act through separate receptors. Flurazepam-induced uncoupling is not prevented by SR-95531 or picrotoxin (specific inhibitors of GABA action) and, therefore, is not an indirect effect mediated by endogenous GABA. The onset of flurazepam-induced uncoupling (EC50 approximately 1 microM) exhibits a t 1/2 of about 18 hr, in general agreement with the half-time for receptor turnover. Uncoupling is reversible following washout and recovery at 37 degrees. These results are discussed in terms of mechanisms of GABAA receptor regulation in response to chronic exposure to functionally homologous or heterologous ligands.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Inverse modulation of gamma-aminobutyric acid- and glycine-induced currents by progesterone.

The ability of certain synthetic and endogenous steroids to modulate neuronal responses to gamma-aminobutyric acid (GABA) is well documented, but little is known of the effect of steroids on glycine responses. We show here that in voltage-clamped neurons progesterone (10-100 microM) itself enhances GABA-induced chloride currents but, surprisingly, antagonizes those induced by glycine. Some, but not all, progesterone metabolites also display these effects. The effects of progesterone on GABA and glycine responses are dose dependent, with EC50 values of 26 and 16 microM and maxima of +156 and -60%, respectively. Progesterone and its reduced metabolite 5 alpha-pregnan-3 alpha-ol-20-one potentiate GABA responses by acting through a common site. The site through which progesterone acts to inhibit glycine responses is distinct from the strychnine and glycine binding sites. These results not only provide an important distinction between chloride-mediated GABA and glycine responses but also suggest that endogenous progesterone or its metabolites may differentially modulate the inhibitory actions of these two neurotransmitters.

Animals↗
PubMed1989

Transmembrane topology of the gamma-aminobutyric acidA/benzodiazepine receptor: subcellular distribution and allosteric coupling determined in situ.

The subcellular distribution, allosteric coupling, and topology of the gamma-aminobutyric acidA (GABA)/benzodiazepine receptor were investigated in situ. We have shown previously that a transmembrane fragment (Mr = 24,000) of the benzodiazepine receptor remains in the plasma membrane after trypsin treatment of embryonic brain neurons maintained in primary monolayer cell culture. Here we report a study of the effects of exhaustive trypsinization on the binding of ligands to the GABA recognition site. Approximately 60% of the binding sites for [3H]muscimol in intact cells are inactivated by extracellular trypsin and, therefore, are associated with cell surface GABA receptors, whereas 40% of the sites are trypsin resistant. GABA potentiates [3H]flunitrazepam binding to intact cells and trypsin-treated intact cells. GABA-induced enhancement of [3H]flunitrazepam binding to intact cells is eliminated when cell-surface benzodiazepine receptors are blocked with Ro7-0213 (a charged benzodiazepine), but some enhancement remains after extracellular trypsinization. This provides indirect evidence for the existence of a population of trypsin-resistant GABA recognition sites associated with cell-surface receptors and indicates that trypsin-resistant cell-surface GABA recognition sites are allosterically linked to trypsin-resistant cell-surface benzodiazepine recognition sites, which we have shown previously to be associated with the trypsin-generated Mr 24,000 transmembrane fragment. The results are discussed in terms of a model of the transmembrane topology of the GABA/benzodiazepine receptor.

Allosteric Regulation↗
PubMed1988

Ethanol potentiates GABA- and glycine-induced chloride currents in chick spinal cord neurons.

The effects of acute ethanol exposure of chick spinal cord neurons were studied in tissue culture, using whole-cell voltage-clamp techniques. Results indicate that ethanol produces a persistent increase in the sensitivity of spinal neurons to GABA and glycine, with no change in input resistance or resting membrane potential. Glutamate responses, in contrast, are unaffected by ethanol.

Animals↗
PubMed1987

Identification, characterization, and developmental regulation of embryonic benzodiazepine binding sites.

We report the identification and characterization of 2 classes of benzodiazepine binding sites in the embryonic chick CNS. Binding was examined by competition and saturation binding experiments, using as radioligands 3H-flunitrazepam, a classical benzodiazepine anxiolytic, and 3H-Ro5-4864, a convulsant benzodiazepine. The results demonstrate that high-affinity (KD = 2.3 nM) 3H-flunitrazepam binding sites (site-A) are present by embryonic day 5 (Hamburger and Hamilton stage 27) and increase throughout development (Bmax = 0.3 and 1.3 pmol/mg protein in 7 and 20 d brain membranes, respectively). When 7 or 20 d brain membranes are photoaffinity-labeled with 3H-flunitrazepam and ultraviolet light, the radioactivity migrates as 2 bands on SDS-PAGE, consistent with Mrs of 48,000 and 51,000. GABA potentiates 3H-flunitrazepam binding at both 7 and 20 d of development, indicating that site-A is coupled to receptors for GABA early in development. Importantly, we have also identified a novel site (site-B) that binds classical benzodiazepine agonists with low affinity (micromolar) but displays high affinity for Ro5-4864 (KD = 41 nM). Site-B displays characteristics expected for a functional receptor, including stereospecificity and sensitivity to inactivation by heat and protease treatment. Saturation binding studies employing 3H-Ro5-4864 indicate that the levels of site-B are similar in 7 and 20 d brain (ca. 2.5 pmol/mg protein). The function of site-B is not known, but its preponderance in 7 d brain, relative to site-A, suggests that it might be important during early embryonic development.

Affinity Labels↗
PubMed1985

Benzodiazepine receptor photoaffinity labeling: correlation of function with binding.

Exhaustive photoaffinity coupling of flunitrazepam to living spinal cord neurons reduced the capacity of benzodiazepines to potentiate the electrophysiologically measured GABA response. In qualitative agreement with reversible binding data the dose-response curve for enhancement of the GABA response by benzodiazepines was shifted to the right, indicating that the remaining reversible benzodiazepine binding sites have lower affinity for benzodiazepines. Photoaffinity labeling did not reduce inhibition of the GABA response by beta-carbolines and there was only a small decrease in beta-carboline binding. In both control and photoaffinity-labeled cultures, the inhibitory effect of beta-carbolines on the GABA response was reversed in the presence of excess benzodiazepine. The results indicate that the effects of photoaffinity labeling are confined to the BZD recognition site, and that coupling between benzodiazepine receptors and GABA receptors remains intact.

Affinity Labels↗
PubMed1983

Multiple embryonic benzodiazepine binding sites: evidence for functionality.

We have found high-affinity binding (site-A) and low-affinity binding (site-B) of benzodiazepines to membrane homogenates of embryonic chick brain and spinal cord. A new technique was developed to permit the determination of complete electrophysiological dose-response curves on single neurons in cell culture, eliminating cell-to-cell variability as a problem that complicates the interpretation of pooled data. The electrophysiological potencies and binding affinities of a series of benzodiazepines correlate well for site-A but not for site-B or the micromolar site reported in adult rat brain. Site-A and the electrophysiological response are sensitive to photo-affinity blockade with flunitrazepam (FNZM) by about 75% while site-B is resistant to blockade. The FNZM-photolinked benzodiazepine receptor/GABA receptor complex is not chronically potentiated and thus exists in an 'unpotentiated' state. These experiments suggest that site-A in embryonic CNS membranes corresponds to a functional benzodiazepine receptor/GABA receptor complex in spinal cord cell cultures.

Animals↗