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Biomedical subjects

T T Gibbs

Publications and source records attributed to T T Gibbs.

At least 19 recordsLinked to original sources
PubMed1997

17beta-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors.

Several lines of evidence suggest that 17beta-estradiol (betaE2) has neuroprotective properties. The risk and severity of dementia are decreased in women who have received estrogen therapy, and betaE2 protects neurons in vitro against death from a variety of stressors. Neuroprotection by betaE2 has been suggested to be due to free radical scavenging. We demonstrate an additional neuroprotective mechanism whereby betaE2 protects against NMDA-induced neuronal death by directly inhibiting the NMDA receptor.

Animals↗
PubMed1997

Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids.

Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.

Animals↗
PubMed1996

gamma-aminobutyric acidA receptor regulation: heterologous uncoupling of modulatory site interactions induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment in culture.

Prolonged administration of anxiolytic, sedative, and anticonvulsant drugs that act through the GABAA receptor (GABAAR) can evoke tolerance and dependence, suggesting the existence of an endogenous mechanism(s) for altering the ability of such agents to interact with the GABAAR. Uncoupling appears to be one such mechanism. This is a decrease in the allosteric interactions between the benzodiazepine (BZD) recognition site and other agonist or modulator sites on the GABAAR, as measured by potentiation of [3H]flunitrazepam ([3H]FNZ) binding. To investigate the mechanism(s) of uncoupling, neuronal cultures were treated chronically with 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone), pentobarbital, flurazepam, or GABA, then tested for enhancement of [3H]FNZ binding by these substances. The results indicate that BZDs, barbiturates, and steroids, as well as GABA itself, are capable of inducing both heterologous and homologous uncoupling. Surprisingly, different chronic drug treatments produce different patterns of homologous and heterologous uncoupling. Chronic exposure to pregnanolone, GABA, flurazepam or pentobarbital induces complete uncoupling of barbiturate-BZD site interactions, partial uncoupling of GABA-BZD site interactions, but different amounts of uncoupling of steroid-BZD site interactions. In addition, the EC50 for pregnanolone-induced homologous uncoupling (1.7 microM) is over an order of magnitude greater than that for heterologous uncoupling of GABA and BZD sites (82 nM). Moreover, heterologous uncoupling by pregnanolone is inhibited by the GABA site antagonist SR-95531, whereas homologous uncoupling by pregnanolone is resistant to SR-95531. Therefore, there are at least two distinct ways in which GABAAR modulatory site interactions can be regulated by chronic drug treatment.

Animals↗
PubMed1994

Phosphorylation factors control neurotransmitter and neuromodulator actions at the gamma-aminobutyric acid type A receptor.

Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50. Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations. Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However, use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization. Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such as steroids, benzodiazepines, and barbiturates.

Adenosine Triphosphate↗
PubMed1993

Dual activation of GABAA and glycine receptors by beta-alanine: inverse modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one.

The differential sensitivity of the glycine and GABAA receptors to modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha) was used to determine whether beta-alanine acts through its own receptor, or through the glycine and/or GABAA receptor(s). The response to beta-alanine resembles the glycine response as it is inhibited by strychnine (a competitive glycine antagonist) or progesterone (a negative modulator of the glycine response). Significantly, the response to beta-alanine also resembles the GABA response in that it is inhibited by 2-(carboxy-3'-propyl)-3-amino-6-paramethoxy-phenylpyridazinium+ ++ bromide (SR-95531; a competitive GABA antagonist) and potentiated by 5 alpha 3 alpha (a positive modulator of the GABA response). The efficacy of beta-alanine at the GABAA receptor is comparable to that of GABA. Similarly, the efficacy of beta-alanine at the glycine receptor is comparable to that of glycine. The greater potency of beta-alanine at the glycine receptor indicates that, if beta-alanine is a neurotransmitter, its effects are more likely to be mediated by glycine receptors than by GABAA receptors. However, activation of the GABAA receptor by beta-alanine may become important in the presence of steroid modulators such as progesterone or 5 alpha 3 alpha.

Animals↗
PubMed1993

Gamma-aminobutyric acidA receptor regulation: chronic treatment with pregnanolone uncouples allosteric interactions between steroid and benzodiazepine recognition sites.

Pregnanolone [5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha)] and allopregnanolone [5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha)] are neuroactive steroids that are reduced metabolites of progesterone. Both 5 beta 3 alpha and 5 alpha 3 alpha are potent positive modulators of the gamma-aminobutyric acid response that enhance the binding of [3H]flunitrazepam ([3H] FNZ) to the gamma-aminobutyric acid type A receptor. Chronic (48 hr) exposure of brain neurons in culture to 5 beta 3 alpha or 5 alpha 3 alpha abolishes potentiation of [3H]FNZ binding by these steroids. This uncoupling, or loss of allosteric interactions between steroid and benzodiazepine recognition sites, is dose dependent, stereospecific, and reversible. The number and affinity of [3H]FNZ binding sites are unaffected. In contrast, the steroids 5 beta-pregnan-3 beta-ol-20-one, beta-estradiol, testosterone, progesterone, deoxycorticosterone, and dexamethasone, which show little capacity to potentiate [3H]FNZ binding, are also much less effective in inducing uncoupling of steroid and benzodiazepine recognition sites. These results suggest a mechanism whereby neurons could become refractory to long term modulation by neuroactive steroids. The results are discussed in terms of their possible relevance to premenstrual anxiety and enhanced frequency of seizures in certain women.

Allosteric Site↗
PubMed1991

Pregnenolone sulfate: a positive allosteric modulator at the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) receptor is believed to play a major role in learning and in excitotoxic neuronal damage associated with stroke and epilepsy. Pregnenolone sulfate, a neurosteroid, specifically enhances NMDA-gated currents in spinal cord neurons, while inhibiting receptors for the inhibitory amino acids glycine and gamma-aminobutyric acid, as well as non-NMDA glutamate receptors. This observation is consistent with the hypothesis that neurosteroids such as pregnenolone sulfate are involved in regulating the balance between excitation and inhibition in the central nervous system.

Allosteric Regulation↗
PubMed1991

Negative modulation of the gamma-aminobutyric acid response by extracellular zinc.

We have studied the effects of divalent cations on the gamma-aminobutyric acid (GABA) response of voltage-clamped spinal cord neurons, using the whole-cell recording configuration. Zn, Cd, Ni, and Mn (but not Ba, Ca, or Mg) inhibit GABA-induced whole-cell currents when applied extracellularly. Although Zn is an effective inhibitor when applied extracellularly, it is ineffective when applied intracellularly. Inhibition by these cations is mediated by a common saturable recognition site that is distinct from the recognition sites for GABA, benzodiazepines, barbiturates, picrotoxin, or steroids. The maximal inhibition, or efficacy of inhibition, of GABA-induced currents is greater for Zn than for Cd, Ni, or Mn. The order of potency is Cd greater than Zn much greater than Ni much greater than Mn. Inhibition by Zn is partially surmountable by GABA, consistent with a decrease in both the maximum response and the affinity for GABA. The dose-response curve for inhibition of the GABA response by Zn is shifted to the right at a high GABA concentration but is unaffected by the presence of chlordiazepoxide, pentobarbital, or 5 beta-pregnan-3 alpha-ol-20-one. The results are consistent with a model in which a Zn-sensitive modulatory site exerts negative allosteric control over GABA receptor function.

Allosteric Regulation↗
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↗