Biomedical subjects

D B Carter

Publications and source records attributed to D B Carter.

At least 37 records · Page 2Linked to original sources

PubMed1996

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

Animals↗

PubMed1996

Differential distribution of gamma-aminobutyric acidA receptor subunit (alpha 1, alpha 2, alpha 3, alpha 5 and beta 2 + 3) immunoreactivity in the medial prefrontal cortex of the rat.

A detailed mapping of the gamma-aminobutyric acid (GABA)A receptor subunits (alpha 1, alpha 2, alpha 3, alpha 5 and beta 2 + 3) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The alpha 1 and beta 2 + 3 subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the alpha 1-positive cell bodies corresponded to that of small interneurons (range, 20-55 microns2; mean +/- SEM, 37 +/- 5.5 microns2) as well as pyramidal cells or large interneurons (range, 87-135 microns2; mean +/- SEM, 103.4 +/- 9.7 microns2). However, beta 2 + 3 antibody immunostained only small cell bodies. Immunoreactivity for alpha 2 was restricted to layers I and II, whereas alpha 3 and alpha 5 subunit expression was seen only in layer VI. The antibody to the alpha 2 subunit immunostained small cell bodies (range, 29-63 microns2; mean +/- SEM, 32 +/- 4.5 microns2) in layer II, resembling interneurons. Conversely, both alpha 3 and alpha 5 antibodies immunostained large cell bodies (range, 94-151 microns2; mean +/- SEM, 115.7 +/- 13.4 microns2), consistent with pyramidal cell labelling in layer VI.

Animals↗

PubMed1996

Characterization of U-101017 as a GABA(A) receptor ligand of dual functionality.

Drugs acting on the benzodiazepine site of GABA(A) receptors are much safer than barbiturates, but are still liable to abuse. Recently, we have reported that a benzodiazepine site agonist, U-97775 (a dihydroimidazoquinoxaline analog), may have minimal abuse liability because of its interaction with a second, low-affinity site on GABA(A) receptors, the occupancy of which, at high drug concentrations, leads to a reversal of its agonistic activity on the benzodiazepine site and inhibition of GABA-induced Cl- currents [Br. J. Pharmacol. 115 (1995)19-24]. Here we report that U-101017 (7-chloro-5[(cis-3,5-dimethylpiperazine)carbonyl]imidazo[1,5a]quinoline- 3-carboxylate) is another similar benzodiazepine site agonist possessing the ability to reverse its agonistic activity at higher concentrations, but its ability to inhibit GABA currents is considerably milder than that of U-97775. In the alpha 6 beta 2 gamma 2 subtype where these drugs have no agonistic activity, for instance, U-101017 at concentrations up to 80 mu M, showed no appreciable effect on GABA currents, whereas U-97775 inhibited the currents with an IC(50) value of 10 mu M as measured with the whole cell patch clamp techniques in human embryonic kidney cells expressing recombinant receptors. Similar, milder inhibition of GABA currents by U-101017 was observed in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes. Furthermore, U-101017 was of higher efficacy in the alpha 1 beta 2 gamma 2 than alpha 3 beta 2 gamma 2 subtypes as compared to diazepam, although its binding affinity was not appreciably different in the two subtypes. We conclude that U-101017 is a partial benzodiazepine agonist, somewhat selective to the alpha 1 beta 2 gamma 2 subtype, and with the ability to limit its own agonistic activity over a wide range of doses through its interaction with the low affinity site, but without potential convulsant activity, inherent to agents which block GABA currents.

Animals↗

PubMed1996

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

Animals↗

PubMed1996

Induction of apolipoprotein E mRNA in the hippocampus of the gerbil after transient global ischemia.

B/A4 is the major component of brain amyloid plaque, one of the hallmarks of Alzheimer's disease (AD). B/A4 is a product of proteolytic processing of its precursor, the Alzheimer amyloid precursor protein (APP). Recently, apolipoprotein E (APO-E) has also been shown to be associated with Alzheimer's disease pathology because it is localized to plaques and tangles, and the gene encoding one of the isoforms of APO-E (E4) is associated with late-onset familial and sporadic AD. In addition, APO-E exhibits high affinity for binding to the B-peptide (B/A4). In this study, we have investigated changes in the steady state levels of APP, APO-E, and the astrocyte-specific marker, glial fibrillary acidic protein (GFAP) mRNA in the gerbil hippocampal CA1 region after a 10-min period of bilateral carotid occlusion-induced forebrain ischemia. Following this insult, we observed a loss of 90% of the CA1 neurons by 72 h post-ischemia. The mRNA levels on day 1 through day 7 post-ischemia were quantitated using an image analyzer. There was an increase in the transcription of APO-E and GFAP mRNAs, with the levels of APO-E mRNA being the highest (3-fold increase on day 7 post-ischemia) (P < 0.005). However, we did not see an increase in APP mRNA. In a parallel study [Hall, E.D. et al., Exp. Neurol., 135(1995) 17-27], we have also seen an increase in levels of APO-E and GFAP protein measured by immunocytochemistry. However, in contrast to the lack of an increase in APP mRNA, immunocytochemical measurement of APP did show an increase, perhaps due to delayed translation of previously formed mRNA. We suggest that neuronal injury or insult results in the induction of certain genes (and, therefore, protein synthesis) in the surrounding reactive astrocytes, and these proteins may contribute to post-injury amyloidogenesis.

Amyloid beta-Protein Precursor↗

PubMed1996

Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2.

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.

Animals↗

PubMed1995

Increased amyloid protein precursor and apolipoprotein E immunoreactivity in the selectively vulnerable hippocampus following transient forebrain ischemia in gerbils.

The postischemic time course of amyloid protein precursor (APP), beta-amyloid protein (beta-AP), and apolipoprotein E (APO-E) immunoreactivity were examined in comparison to neuronal necrosis in the selectively vulnerable hippocampal CA1 region of gerbils subjected to 10 min of bilateral carotid occlusion-induced forebrain ischemia. Loss of 90% of the CA neurons occurred between 24 and 72 h after ischemia, after which no further neuronal necrosis was observed. At 24 h postischemia, there was a decrease in APP and beta-AP immunostaining in the CA1 region. However, beginning at 2 days, there was a dramatic increase in the staining for both proteins. This coincided with a progressive increase in the expression of APO-E and glial fibrillary acidic protein (GFAP) staining between Days 2 and 6, indicative of an activation of astrocytic protein synthesis. Each of the immunocytochemical markers also increased in the less vulnerable CA3 region. However, the peak increase in that region was much less than that in CA1 and, by 7 days, only the GFAP staining remained significantly above the sham level. It has been shown that the E4 isoform of APO-E, when oxidized, avidly binds to beta-AP and thus increases the likelihood of co-beta-AP/APO-E deposition. Therefore, it is postulated that the increased levels of amyloid proteins coincident with an increased production of APO-E in response to ischemic neuronal necrosis may provide conditions that are favorable for the postischemic formation of amyloid deposits.

Amyloid beta-Protein Precursor↗

PubMed1995

GABAA, GABAC, and NMDA receptor subunit expression in the suprachiasmatic nucleus and other brain regions.

Identification of the neurotransmitter receptor subtypes within the suprachiasmatic nuclei (SCN) will further understanding of the mechanism of the biological clock and may provide targets to manipulate circadian rhythms pharmacologically. We have focused on the ionotropic GABA and glutamate receptors because these appear to account for the majority of synaptic communication in the SCN. Of the 15 genes known to code for GABA receptor subunits in mammals we have examined the expression of 12 in the SCN, neglecting only the alpha 6, gamma 3, and rho 2 subunits. Among glutamate receptors, we have focused on the five known genes coding for the NMDA receptor subunits, and two subunits which help comprise the kainate-selective receptors. Expression was characterized by Northern analysis with RNA purified from a large number of mouse SCN and compared to expression in the remaining hypothalamus, cortex and cerebellum. This approach provided a uniform source of RNA to generate many replicate blots, each of which was probed repeatedly. The most abundant GABA receptor subunit mRNAs in the SCN were alpha 2, alpha 5, beta 1, beta 3, gamma 1 and gamma 2. The rho 1 (rho 1) subunit, which produces GABAC pharmacology, was expressed primarily in the retina in three different species and was not detectable in the mouse SCN despite a common embryological origin with the retina. For several GABA subunits we detected additional mRNA species not previously described. High expression of both genes coding for glutamic acid decarboxylase (GAD65 and GAD67) was also found in the SCN. Among the NMDA receptor subunits, NR1 was most highly expressed in the SCN followed in order of abundance by NR2B, NR2A, NR2C and NR2D. In addition, both GluR5 and GluR6 show clear expression in the SCN, with GluR5 being the most SCN specific. This approach provides a simple measure of receptor subtype expression, complements in situ hybridization studies, and may suggest novel isoforms of known subunits.

Animals↗

PubMed1995

Cloning, sequence analysis and expression of two forms of mRNA coding for the human beta 2 subunit of the GABAA receptor.

Two forms of cDNA coding for the human GABAA beta 2 subunit have been cloned and sequenced. The two sequences differ by a 114 base pair insertion. The insert contains a phosphorylation consensus sequence for calmodulin-dependent protein kinase II. Quantitative PCR studies show that h beta 2L cDNA represents 10-15% of total h beta 2 cDNA in the 10 brain substructures tested. Analysis of human genomic southern blots suggests that the two forms might arise by differential splicing.

Blotting, Northern↗

PubMed1995

Sodium channels and GABAA receptor-channel complex as targets of environmental toxicants.

Voltage-activated sodium channels and GABAA receptor-chloride channel complex are among the most important target sites of various environmental neurotoxicants. Pyrethroids keep the sodium channels open for prolonged periods of time leading to hyperexcitation of the entire nervous system. In rat cerebellar Purkinje neurons and dorsal root ganglion neurons, only about 1% of sodium channel population needed to be modified by the pyrethroid tetramethrin to increase the depolarizing after-potential to the level of the threshold membrane potential for generation of repetitive after-discharges. This concept of toxicity amplification is applicable to other chemicals that go through a threshold phenomenon to exert their effects. The potency of pyrethroids on neuronal sodium channels increased with lowering the temperature with a Q10 value of 0.2. The selective pyrethroid toxicity between mammals and insects can be quantitatively explained on the basis of the differences in 5 factors, i.e. the intrinsic sodium channel sensitivity, the sodium channel modification due to temperature difference, the reversibility of sodium channel, the detoxication of pyrethroids, and body size. These 5 factors are multiplied to approximately 2000 which is in the same order of magnitude as that of the difference in LD50. Dieldrin had a dual action on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The initial transient potentiation of GABA-induced currents after application of dieldrin was followed by a suppression. Dieldrin-induced potentiation of current was observed only when the gamma 2 subunit was present in embryonic kidney cells (HEK-293) transfected with GABA receptor subunits. Dieldrin-induced suppression was observed in the presence and absence of the gamma 2 subunit. The dieldrin suppression of GABA-induced currents is deemed directly responsible for hyperactive symptoms of poisoning in animals.

Animals↗

PubMed1995

Interaction of beta-carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors.

1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.

Barbiturates↗

PubMed1995

Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations. Analysis of the binding data in the presence of Ro 15-1788 yielded a single low affinity site with an estimated Kd of 407 nM.5. In other alpha beta upsilon receptor subtypes, U-97775 at low concentrations enhanced Cl- currents in the alpha 3 beta 2 upsilon 2,but not in the alpha 6 beta 2 upsilon 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 upsilon 2 and alpha 1 upsilon 2 subtypes.6. Therapeutically, U-97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U-97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.

Animals↗

PubMed1995

Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Animals↗

PubMed1995

U-90042, a sedative/hypnotic compound that interacts differentially with the GABAA receptor subtypes.

U-90042 is a structurally novel compound that has comparable affinities for binding to three recombinant subtypes of the gamma-aminobutyric acidA receptor: alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2. The relatively high affinity for the alpha 6 beta 2 gamma 2 subtype is similar to the benzodiazepine (BZ) partial inverse agonist Ro 15-4513 and different from BZ sedative/hypnotics such as diazepam and zolpidem. In the present study, U-90042 (3 mg/kg i.p.) suppressed locomotor activity and impaired rotarod performance in mice. These effects were not antagonized by flumazenil. The sedative effect was further confirmed in rats (10 mg/kg i.p.) and monkeys (1 mg/kg p.o.) by an increase of behavioral sleep and a corresponding electroencephalographic frequency spectral shift. Unlike the BZ hypnotics, U-90042 (10 mg/kg i.p.) produced no amnesia in the one-trial passive avoidance response in mice but antagonized diazepam-induced amnesia. In rats trained to discriminate an injection of diazepam from saline, U-90042 produced predominantly vehicle-appropriate responses, even at depressant doses. The in vivo diazepam-antagonist effect of U-90042 is consistent with its low intrinsic activity and diazepam-antagonism at the gamma-aminobutyric acidA alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 receptor subtypes. The receptor mechanism for the sedative/hypnotic effect is not clear at this time.

Animals↗

PubMed1995

[4-Dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline] is a novel ligand to the picrotoxin site on GABAA receptors, and decreases single-channel open probability.

U-93631,[4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline], represents a GABAA receptor ligand of novel chemical structure, and has been shown to induce a rapid, time-dependent decay of GABA-induced whole-cell Cl- currents in recombinant GABAA receptors (Dillon et al., 1993). In this study, we found that the drug competitively inhibited [35S]t-butylbicyclophosphorothionate binding to the picrotoxin site on a cloned GABAA receptor, alpha 1 beta 2 gamma 2, and preempted the action of picrotoxin and [35S]t-butylbicyclophosphorothionate on GABA-induced Cl- currents. We further examined the effect of U-93631 on GABA-induced single channel openings in outside-out patches. U-93631 (5 microM) showed no effect on single channel conductance, the duration of channel open states or the short closed state, but increased the duration of the long closed state and its relative contribution to total closed time. In addition, closings of very long duration (> 500 msec), albeit rare, occurred more frequently in the presence of U-93631. Thus, the probability of single channel openings decreased from 0.12 +/- 0.02 to 0.04 +/- 0.01 in the presence of U-93631 (5 microM). These properties of U-93631 are analogous to those of picrotoxin and [35S]t-butylbicyclophosphorothionate reported in earlier studies with native neurons. We conclude that U-93631 at least shares overlapping binding domains with picrotoxin and [35S]t-butylbicyclophosphorothionate, and the ability to stabilize the GABAA receptor/Cl- channel complex in an inactivated state(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

U-89843A is a novel allosteric modulator of gamma-aminobutyric acidA receptors.

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗

PubMed1994

Selective effects of dieldrin on the GABAA receptor-channel subunits expressed in human embryonic kidney cells.

We have recently demonstrated that the cyclodiene insecticide dieldrin modulates the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons in a complex manner, causing both stimulatory and inhibitory effects. We now report that the differential effects of dieldrin on the GABA-induced chloride current of human embryonic kidney cells expressing three different combinations of alpha, beta and gamma subunits. The EC50 values for GABA induction of current were estimated to be 9.8 microM for the alpha 1 beta 2 gamma 2s combination, 2.0 microM for the alpha 1 beta 2 combination and 3.0 microM for the alpha 6 beta 2 gamma 2s combination. When co-applied with GABA, dieldrin exerted a dual effect, enhancement and suppression, on the GABA-induced chloride currents in the alpha 1 beta 2 gamma 2s and alpha 6 beta 2 gamma 2s combinations. However, only suppression was observed in the alpha 1 beta 2 combination, indicating that the gamma subunit is necessary for dieldrin's enhancing effect. Dieldrin was more efficacious in enhancing the current in the alpha 6 beta 2 gamma 2s combination than in the alpha 1 beta 2 gamma 2s combination, indicating some specific role of alpha subunits in the dieldrin enhancement of current. Dieldrin suppressed the GABA-induced current in a non-competitive manner, with an EC50 value of 2.1 microM for alpha 1 beta 2 gamma 2s, 2.8 microM for alpha 1 beta 2 and 1.0 microM for alpha 6 beta 2 gamma 2s combination. These results indicated that dieldrin suppression did not require specific subunit combinations among the three tested.

Cell Line↗

PubMed1994

Antagonist, partial agonist, and full agonist imidazo[1,5-a]quinoxaline amides and carbamates acting through the GABAA/benzodiazepine receptor.

(4RS)-1-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihyd roimidazo[1,5- a]pyrrolo[2,1-c]quinoxalin-10(11H)-one (1a), 5-benzoyl-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5- dihydroimidazo[1,5-a]quinoxaline (13b), and tert-butyl (4S)-12,12a-dihydroimidazo[1,5-a]pyrrolo[2,1- c]quinoxaline-1-carboxylate (1e), as well as other imidazo[1,5-a]quinoxaline amides and carbamates, represent a new series of compounds which bind with high affinity to the GABAA/benzodiazepine receptor. These compounds exhibit a wide range of intrinsic efficacies as measured by [35S]TBPS binding ratios. The synthesis of 1a begins with the addition of DL-glutamic acid to 1-fluoro-2-nitrobenzene, followed by reduction of the nitro group and subsequent ring closure to form 3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinoxalin-2-one, followed by a second ring closure to afford (4RS)-1,5-dioxo-1,2,3,4,5,6-hexahydropyrrolo[1,2-a]quinoxali ne as the key intermediate. Appendage of a substituted imidazo ring via the anion of 5-cyclopropyl-1,2,4-oxadiazol-3-yl gives 1a. The (-)- and (+)-isomers of 1a were prepared from 1-fluoro-2-nitrobenzene and L- and D-glutamic acid, respectively. 1a and its enantiomers demonstrated affinity for the [3H]flunitrazepam binding site with Ki's of 0.87, 0.62, and 0.65 nM, respectively.

Animals↗