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Biomedical subjects

D H Farb

Publications and source records attributed to D H Farb.

At least 37 records · Page 2Linked to original sources
PubMed1995

From ion currents to genomic analysis: recent advances in GABAA receptor research.

The gamma-aminobutyric acid type A (GABAA) receptor represents an elementary switching mechanism integral to the functioning of the central nervous system and a locus for the action of many mood- and emotion-altering agents such as benzodiazepines, barbiturates, steroids, and alcohol. Anxiety, sleep disorders, and convulsive disorders have been effectively treated with therapeutic agents that enhance the action of GABA at the GABAA receptor or increase the concentration of GABA in nervous tissue. The GABAA receptor is a multimeric membrane-spanning ligand-gated ion channel that admits chloride upon binding of the neurotransmitter GABA and is modulated by many endogenous and therapeutically important agents. Since GABA is the major inhibitory neurotransmitter in the CNS, modulation of its response has profound implications for brain functioning. The GABAA receptor is virtually the only site of action for the centrally acting benzodiazepines, the most widely prescribed of the anti-anxiety medications. Increasing evidence points to an important role for GABA in epilepsy and various neuropsychiatric disorders. Recent advances in molecular biology and complementary information derived from pharmacology, biochemistry, electrophysiology, anatomy and cell biology, and behavior have led to a phenomenal growth in our understanding of the structure, function, regulation, and evolution of the GABAA receptor. Benzodiazepines, barbiturates, steroids, polyvalent cations, and ethanol act as positive or negative modulators of receptor function. The description of a receptor gene superfamily comprising the subunits of the GABAA, nicotinic acetylcholine, and glycine receptors has led to a new way of thinking about gene expression and receptor assembly in the nervous system. Seventeen genetically distinct subunit subtypes (alpha 1-alpha 6, beta 1-beta 4, gamma 1-gamma 4, delta, p1-p2) and alternatively spliced variants contribute to the molecular architecture of the GABAA receptor. Mysteriously, certain preferred combinations of subunits, most notably the alpha 1 beta 2 gamma 2 arrangement, are widely codistributed, while the expression of other subunits, such as beta 1 or alpha 6, is severely restricted to specific neurons in the hippocampal formation or cerebellar cortex. Nervous tissue has the capacity to exert control over receptor number, allosteric uncoupling, subunit mRNA levels, and posttranslational modifications through cellular signal transduction mechanisms under active investigation. The genomic organization of the GABAA receptor genes suggests that the present abundance of subtypes arose during evolution through the duplication and translocations of a primordial alpha-beta-gamma gene cluster. This review describes these varied aspects of GABAA receptor research with special emphasis on contemporary cellular and molecular discoveries.

Animals↗
PubMed1994

Mapping of the beta 2 subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABAA receptor isoform.

The gamma-aminobutyric acid receptor (GABAAR) is a multisubunit Cl- channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABAAR subunits, including alpha 1-6, beta 1-4, gamma 1-4, delta, and rho 1-2, suggesting that an enormous number of combinations of subunits are possible. Here we report that the beta 2 gene is located on chromosome 5q34-q35, defining a cluster comprising alpha 1, beta 2, and gamma 2 genes that together code for the most abundant GABAAR isoform. The fact that intron position is conserved in the beta 1-3 genes, taken together with the observation that chromosomes 4 and 15 also contain distinct alpha-beta-gamma gene clusters, strongly suggests that an ancestral alpha-beta-gamma cluster was duplicated and translocated to at least two different chromosomes. This organization of GABAAR gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression.

Amino Acid Sequence↗
PubMed1994

3 alpha-Hydroxy-5 beta-pregnan-20-one sulfate: a negative modulator of the NMDA-induced current in cultured neurons.

We have shown previously that the neurosteroid pregnenolone sulfate acts as a positive allosteric modulator at the N-methyl-D-aspartate (NMDA) receptor while inhibiting the kainate, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), the glycine, and the gamma-aminobutyric acid (GABA) responses of chick spinal cord neurons. Here, we report that 3 alpha-hydroxy-5 beta-pregnan-20-one sulfate (5 beta 3 alpha S), a sulfated form of naturally occurring 5 beta 3 alpha, inhibits both the NMDA and the non-NMDA receptor-mediated responses as measured by whole cell voltage clamp recordings. 100 microM 5 beta 3 alpha S rapidly and reversibly inhibits the response to 30 microM NMDA by 66%, 50 microM kainate by 37%, and 25 microM AMPA by 29%. Application of 50 microM nonsulfated 5 beta 3 alpha does not produce any significant effect on the NMDA response, demonstrating that the sulfate moiety is important for the effect of 5 beta 3 alpha S on the NMDA response. The effect of 5 beta 3 alpha S on the NMDA response is concentration dependent, with an EC50 of 62 microM. 5 beta 3 alpha S reduces the maximum NMDA response with little effect on the NMDA EC50, indicating that antagonism of the NMDA response by 5 beta 3 alpha S is noncompetitive. The fact that 5 beta 3 alpha S inhibition of the NMDA response is neither agonist nor voltage dependent demonstrates that 5 beta 3 alpha S does not act as an open channel blocker. Furthermore, inhibition of the NMDA response by 5 beta 3 alpha S is not reduced by the addition of a maximal concentration (10 microM) of glycine, indicating that 5 beta 3 alpha S does not act via the glycine recognition site. The inhibitory action of 5 beta 3 alpha S on the NMDA and non-NMDA receptors may provide a basis for inhibiting glutamate receptor-induced seizures and excitotoxic cell death.

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

Restriction selection cloning: a simple general method for the selection of recombinant DNA.

It is well known that the ligation of two DNA fragments which are the product of digestion from different restriction enzymes will not lead to the regeneration of either of the original blunt end or cohesive end restriction sites. This property of sequence incompatibility for the original restriction enzyme can be exploited in a general cloning procedure for both PCR products and restricted DNAs. Restriction selection is particularly useful when cloning low abundance polymerase chain reaction (PCR) products and when cloning blunt ended DNA into reporter vectors that lack a method for the selection of recombinants.

Base Sequence↗
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↗
PubMed1992

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons.

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

Animals↗
PubMed1991

gamma-Aminobutyric acid (GABA) induces a receptor-mediated reduction in GABAA receptor alpha subunit messenger RNAs in embryonic chick neurons in culture.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, is known to interact with a subclass of receptors that activate a ligand-gated chloride ion channel. Exposure of cultured embryonic chick neurons to physiological concentrations of GABA results in a time-dependent down-regulation of these GABAA receptors. To delineate the cellular mechanism(s) responsible for agonist-induced down-regulation of GABAA receptors we quantified the levels of GABAA receptor alpha subunit messenger RNAs, which encode the subunit(s) containing agonist recognition site(s), and observed a marked reduction in alpha subunit mRNAs following exposure of embryonic chick neurons to GABA. Both the down-regulation of GABAA receptors and the reduction in alpha subunit mRNAs induced by GABA were completely antagonized by the specific GABAA receptor antagonist SR-95531. These data demonstrate the presence of an agonist-induced receptor-mediated mechanism for regulating the expression of receptor subunit-encoding mRNAs that may be involved in the development of tolerance to the pharmacological actions of drugs known to act via GABAA receptors.

Animals↗
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

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

Chronic agonist exposure induces down-regulation and allosteric uncoupling of the gamma-aminobutyric acid/benzodiazepine receptor complex.

Using [3H]flunitrazepam as a probe for the benzodiazepine-sensitive modulator site located on the gamma-aminobutyric acid (GABA)A receptor complex, we have investigated the cellular regulation of the GABAA receptor in neuronal cultures derived from embryonic chick brain. Treatment of cultures with 1 mM GABA for 48 hr causes a reversible 35% decrease in the number of [3H]flunitrazepam binding sites with no change in affinity. The EC50 for chronic GABA-induced down-regulation is 94 microM and the half-time is 25 hr. The effect of GABA is blocked by SR-95531, a GABAA receptor antagonist, and mimicked by muscimol but not baclofen. Consistent with the decrease in [3H]flunitrazepam binding, chronic GABA exposure causes a 43% decrease in the binding of [35S]t-butylbicyclophosphorothionate, a ligand for the receptor-associated chloride ionophore. In addition to chronic GABA-induced down-regulation, allosteric interactions between GABA and benzodiazepine recognition sites are uncoupled by 34%. The half-time and pharmacology for chronic GABA-induced uncoupling is indistinguishable from that for GABA-induced down-regulation, consistent with the hypothesis that the action of GABA at a common site induces both down-regulation and uncoupling.

Allosteric Regulation↗
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↗