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D H Farb

Publications and source records attributed to D H Farb.

At least 55 records · Page 3Linked to original sources
PubMed1989

Identification of an intracellular pool of gamma-aminobutyric acidA/benzodiazepine receptors en route to the cell surface of brain neurons in culture.

We have previously shown that approximately 20% of the gamma-aminobutyric acid (GABA)A/benzodiazepine receptors in intact neurons are intracellular [J. Neurosci. 6:2857-2863 (1986); Mol. Pharmacol., 35: 75-84], but the nature of this pool remained unknown. In this report, we describe the synthesis, appearance in the plasma membrane, and degradation of the GABAA/benzodiazepine receptor complex in nerve cells derived from embryonic chick brain and grown in primary monolayer cell culture. Irreversible photoaffinity labeling of the benzodiazepine-sensitive modulator site on the GABAA/benzodiazepine receptor using [3H]flunitrazepam as a permeable probe was used in conjunction with exhaustive trypsinization of intact cells and competition binding using Ro7-0213 (a benzodiazepine bearing a charged quarternary ammonium moiety). Newly synthesized intracellular receptors that are transported to the surface membrane comprise at most only 4% of total receptors and up to one fifth of the steady state intracellular pool. The kinetics of receptor recovery after photoaffinity blockade are consistent with a model in which newly synthesized receptors first appear within the cell and then undergo intracellular transit before appearing on the cell surface. This suggests a "precursor-product" relationship between newly synthesized intracellular receptors and surface receptors. However, the majority of intracellular receptors are not transported to the cell surface and may represent a "nonprecursor" pool. The kinetics of degradation for intracellular receptors are consistent with the overall kinetics for total (surface plus intracellular) receptors. Intracellular and surface receptors are pharmacologically similar with regard to their sensitivity to methyl-beta-carboline-3-carboxylate binding and their affinity for flunitrazepam. Degradation of the individual receptor subunits (with apparent molecular weights of 51,000 and 48,000) was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both photoaffinity-labeled subunits degrade with indistinguishable biphasic kinetics. From these results, and the results of our previous studies, we propose a minimal model describing the dynamic cellular pathway for GABAA/benzodiazepine receptor metabolism in central nervous system neurons.

Animals↗
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↗
PubMed1988

Modulation of neurotransmitter receptor desensitization: chlordiazepoxide stimulates fading of the GABA response.

Benzodiazepines are neuromodulatory drugs that potentiate GABA-mediated conductance increases. We report the findings of an investigation into the effect of a full benzodiazepine-positive modulator (agonist), chlordiazepoxide (CDPX), on desensitization of the GABA response in chick spinal cord neurons maintained in primary monolayer cell culture. GABA application initially increases cell conductance, which then desensitizes. CDPX increases the apparent rate constant and extent of desensitization for the GABA response. The observed values for rate constant and extent of desensitization in the presence of CDPX are significantly greater than the values predicted for an equivalent peak response to GABA alone. Flunitrazepam, another full positive modulator, also stimulates the rate constant for GABA-induced desensitization. Furthermore, a weak partial benzodiazepine-positive modulator, Ro 15-1788 (an imidazodiazepine) (1) exhibits little or no potentiation of GABA-induced desensitization, and (2) antagonizes the ability of CDPX to stimulate GABA desensitization. The results demonstrate a novel form of neuromodulator action: the stimulation of receptor desensitization that cannot be explained by channel blockade. Thus, benzodiazepine modulators have the capacity to increase GABA receptor desensitization while increasing the peak response to GABA.

Animals↗
PubMed1988

Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor.

Allosteric Regulation↗
PubMed1988

Mechanism of gamma-aminobutyric acid/benzodiazepine receptor turnover in neuronal cells: evidence for nonlysosomal degradation.

In a previous report we described the use of flunitrazepam as a photoaffinity label to monitor the turnover of the gamma-aminobutyric acid/benzodiazepine receptor complex in primary brain and spinal cord cell cultures [Science (Wash. D. C.) 226:857-860 (1984)]. In the present communication we have extended our studies on the kinetics of receptor turnover and have examined the mechanism of receptor degradation. There are approximately 60,000 irreversible binding sites per neuron, and photolabeling is stereospecific. Photolabeling does not demonstrably alter the kinetics of degradation, and a complete rate equation relating the kinetic constants for degradation to receptor number is described. The rapid phase of degradation is slowed at low temperature (Q10 = 5, which corresponds to an apparent energy of activation of 25 kcal/mol) and by inhibitors of ATP production (sodium azide, 2-deoxyglucose, and 2,4-dinitrophenol). The fast phase for the degradation of photolabeled receptor is not affected by lysosomotropic agents (methylamine, ammonium chloride, and chloroquine) or by elimination of horse serum and chick embryo extract from the growth medium. In contrast, overall protein degradation is inhibited by methylamine and enhanced in serum-free medium. The results suggest that the gamma-aminobutyric acid receptor complex is degraded through an energy-dependent nonlysosomal pathway.

Affinity Labels↗
PubMed1988

"Run-down" of gamma-aminobutyric acidA receptor function during whole-cell recording: a possible role for phosphorylation.

When using whole-cell recording methods and a minimal intracellular medium containing only inorganic ions, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, we have observed a time-dependent decrease in the responsiveness of cultured chick spinal cord neurons to gamma-aminobutyric acid (GABA). The current evoked by 30 microM GABA progressively declined to approximately 30% of its initial value after five applications at 10-min intervals. This was accompanied by an equivalent decline in the GABA-evoked membrane conductance. "Run-down" of the response was reduced when Mg2+-ATP was present in the pipet solution. Inclusion of ATP-gamma-S, an analog that donates a thiophosphate group resistant to hydrolysis, also reduced run-down. The nonhydrolyzable analog beta, gamma-imidoadenosine-5'-triphosphate was without effect. These results suggest that an ATP-dependent process, possibly phosphorylation, is involved in the maintenance of GABAA receptor function.

Adenosine Triphosphate↗
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↗
PubMed1986

Transmembrane topology and subcellular distribution of the benzodiazepine receptor.

The distribution and transmembrane topology of benzodiazepine receptors were investigated in situ using intact and saponin-treated neurons of brain cell cultures. Reversible ligand binding and photoaffinity labeling, using 3H-flunitrazepam as a probe, were employed in conjunction with trypsin-induced exhaustive proteolysis and competition binding using a novel benzodiazepine (Ro7-0213) that contains a quaternary ammonium moiety bearing a full positive charge. About 80% of the benzodiazepine receptors, with apparent subunit molecular weights of 48,000 and 51,000, are located in the surface membrane and contain one or more trypsin-sensitive sites exposed at the extracellular surface. The ligand recognition sites of the surface receptors are orientated toward the extracellular space and are sensitive to both competition by Ro7-0213 and trypsin attack. Approximately 20% of the receptors are intracellular or sequestered and are insensitive to both trypsin and competition by Ro7-0213 in intact cells. Strikingly, all of the 3H-flunitrazepam photolabeled sites inaccessible to competition by Ro7-0213 are also resistant to extracellular trypsin, but are sensitive to trypsin and Ro7-0213 in saponin-treated cells. This result provides strong evidence for protected receptors. Surface receptors are differentially sensitive to trypsin-induced inactivation such that 57% (of total) are inactivated, while 24% (of total) are cleaved but, surprisingly, retain the capacity to bind 3H-flunitrazepam. The observation that the ability of the receptor fragment to bind ligand is unaltered demonstrates that the integrity of the benzodiazepine binding site is not contingent upon the presence of about half of the intact polypeptide chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗
PubMed1985

Modulation of neurotransmitter action: control of the gamma-aminobutyric acid response through the benzodiazepine receptor.

The ability of several homologous benzodiazepine and heterologous nonbenzodiazepine ligands to alter the conductance increase induced in spinal cord neurons by gamma-aminobutyric acid (GABA) was determined. Complete dose-response curves were carried out on individual neurons, reducing error introduced by cell-to-cell variability. The efficacies of modulation differ for "classical" benzodiazepines and novel nonbenzodiazepine drugs in a manner consistent with a model of control of GABA receptor action through a common receptor. There was no apparent correlation between efficacy and potency. CL 218,872, a triazolopyridazine, gave the lowest efficacy of the tranquilizers tested. Ro 15-1788 (an imidazobenzodiazepine) potentiated g GABA with high potency and low efficacy, consistent with an action as a partial agonist and an "antagonist" of classical benzodiazepine action. In order of increasing efficacy, Ro 15-1788, CL 218,872, and flurazepam are partial agonists, whereas clonazepam, chlordiazepoxide, diazepam, and flunitrazepam are full agonists. The beta-carboline drugs methyl-beta-carboline-3-carboxylate (beta-CCM) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) are "anxiogenics" and convulsants that were found to exert through the benzodiazepine receptor-inhibitory and apparently insurmountable control of g GABA . beta-CCM and DMCM display large negative efficacies and act like effectors at a site distinct from the picrotoxin-sensitive chloride ionophore and coincident with the benzodiazepine site. The actions of these different benzodiazepine receptor ligands in vivo range from anxiolytic and anticonvulsant to anxiogenic and convulsant. Efficacy rather than potency almost certainly determines the qualitative nature of the pharmacological actions of these drugs.

Animals↗
PubMed1984

Benzodiazepine receptor synthesis and degradation by neurons in culture.

The benzodiazepine-gamma-aminobutyric acid receptor complex was used to study functional receptor synthesis and degradation in primary cultures of neurons. Fifty percent of the receptors turned over with an unusually rapid half-life (4 hours); this was followed by a second, slower phase (32 hours). These results provide the basis for elucidating the mechanism by which neurons derived from the central nervous system control neurotransmitter receptor number, an important problem in cellular neurobiology. The findings may be of significance in the study of neurological and psychiatric disorders.

Animals↗
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↗
PubMed1981

The inactivation of gamma-aminobutyric acid transaminase in dissociated neuronal cultures from spinal cord.

It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter gamma-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10(-6) M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.

4-Aminobutyrate Transaminase↗