Behavioral effects of GABA agonists in relation to anxiety and benzodiazepine action

A considerable body of biochemical and neurophysiological evidence implicates GABA in anxiety and in benzodiazepine action. The present article surveys the behavioral effects of GABA agonists and their interactions with drugs acting at the benzodiazepine receptor in animal anxiety paradigms. Certain GABA agonists, notably valproate, simulate many behavioral actions of benzodiazepines. Moreover, several behavioral studies of the interaction of GABA agonists with benzodiazepines support the hypothesis of a benzodiazepine receptor complex with one or more GABA, benzodiazepine and probably other binding sites. However, there are also a number of anomalous findings of GABA agonist action alone and in combination with benzodiazepines. It is argued that these paradoxical results can better be accounted for in terms of the receptor complex and the distribution of the drugs, rather than by suggesting that the anxiolytic actions of benzodiazepines are not mediated by GABA systems. The potential clinical usefulness of GABA agonists in anxiety is commented upon.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine-binding site of GABA_A receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K_i values of around 0.20 nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis in an overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat ??₁??₃??₂, ??₂??₃??₂, ??₃??₃??₂, and ??₅??₃??₂ subtypes, and displayed selectivity for the ??₁??₃??₂ isoform.     

GABA spillover activates postsynaptic GABA(B) receptors to control rhythmic hippocampal activity

In the hippocampus, interneurons provide synaptic inhibition via the transmitter GABA, which can activate GABA(A) and GABA(B) receptors (GABA(A)Rs and GABA(B)Rs). Generally, however, GABA released by a single interneuron activates only GABA(A)Rs on its targets, despite the abundance of GABA(B)RS. Here, I show that during hippocampal rhythmic activity, simultaneous release of GABA from several interneurons activates postsynaptic GABA(B)Rs and that block of GABA(B)Rs increases oscillation frequency. Furthermore, if GABA uptake is inhibited, even GABA released by a single interneuron is enough to activate GABA(B)Rs. This occurs also on cells not directly contacted by that interneuron, indicating that GABA has to overcome uptake and exit the synaptic cleft to reach GABA(B)RS. Thus, activation of extrasynaptic GABA(B)Rs by pooling of GABA is an important mechanism regulating hippocampal network activity.     

Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABA(A) receptors

The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha- and a gamma-subunit in the GABA(A) receptor, selected compounds were tested on the alpha(1)beta(2)gamma(2s), alpha(2)beta(2)gamma(2s) and alpha(3)beta(2)gamma(2s) GABA(A) receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for alpha(1)- versus alpha(2)- and alpha(3)-containing receptors, and high-affinity ligands essentially selective for alpha(1) over alpha(3) were developed.     

Conformational changes at benzodiazepine binding sites of GABA(A) receptors detected with a novel technique

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.     

Pyritinol inhibition of the GABA and benzodiazepine receptor

In 100 and 200 mumol/l concentration, pyritinol inhibited GABA binding to the GABA receptors of brain synaptosomal membranes. GABA receptors from the cerebral cortex, diencephalon and striatum were inhibited to approximately the same degree; those from the cerebellum and spinal cord were inhibited more. Both high and low affinity receptors were inhibited. Pyritinol did not greatly affect the number of binding sites (Bmax), but reduced the affinity (raised the dissociation constant KD) of both receptors. The benzodiazepine receptor, which is connected with the postsynaptic GABAA receptor, was also inhibited by pyritinol. The character of inhibition was the same as for GABA receptors, i.e. there was no change in the number of binding sites, but there was a decrease in their affinity. It is assumed that the similarity of the effect on GABA and benzodiazepine receptors is associated with their occurrence on one, or on two relatively firmly interconnected, protein molecules. Depression of the affinities of GABA and the associated benzodiazepine receptor, together with inhibition of GABA synthesis, in the presence of pyritinol indicate that diminished activity of the GABA system in the brain might be related to the activating effect of pyritinol.     

Wound healing activity of gamma-aminobutyric Acid (GABA) in rats

Gamma-aminobutyric acid (GABA) is a non-protein amino acid. It is well known for its role as an inhibitory neurotransmitter of developing and operating nervous systems in brains. In this study, a novel function of GABA in the healing process of cutaneous wounds was presented regarding anti-inflammation and fibroblast cell proliferation. The cell proliferation activity of GABA was verified through an MTT assay using murine fibroblast NIH3T3 cells. It was observed that GABA significantly inhibited the mRNA expression of iNOS, IL-1beta, and TNF-alpha, in LPS-stimulated RAW 264.7 cells. To evaluate in vivo activity of GABA in wound healing, excisional open wounds were made on the dorsal sides of Sprague-Dawley rats under anesthesia, and the healing of the wounds was apparently assessed. The molecular aspects of the healing process were also investigated by hematoxylineosin staining of the healed skin, displaying the degrees of reepithelialization and linear alignment of the granulation tissue, and immunostaining and RT-PCR analyses of fibroblast growth factor and platelet-derived growth factor, implying extracellular matrix synthesis and remodeling of the skin. The GABA treatment was effective to accelerate the healing process by suppressing inflammation and stimulating reepithelialization, compared with the epidermal growth factor treatment. The healing effect of GABA was remarkable at the early stage of wound healing, which resulted in significant reduction of the whole healing period.     

Antiepileptic effects of nifedipine]

In experiments on freely moving male Wistar rats it was shown that nifedipine in a dose 10 mg/kg (i.p.) suppressed the penicillin-induced focal epileptic activity in cerebral cortex. A similar suppressing effect of nifedipine was shown on acute generalized tonic-clonic pentylenetetrazol (PTZ) seizures (75 mg/kg, i.p.). Nifedipine in the same dose was not effective on chronic PTZ administration (PTZ-kindling, 30 mg/kg i.p. during 28 days): when injected 30 min before each PTZ administration it didn't delay the development of kindling induced seizure susceptibility and had no effect on the severity of seizures. The administration of nifedipine in a dose of 10 or 30 mg/kg to control kindled animals which had not been treated with nifedipine had no influence on the severity of seizures provoked by a testing dose of PTZ (30 mg/kg i.p.): its intensity was similar to that of caused by PTZ injection along.     

Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content.

The effect of various chronic dopaminergic treatments in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys on the brain gamma-aminobutyric acid type A (GABA(A)) /benzodiazepine receptor complex and GABA content was investigated in order to assess the GABAergic involvement in dopaminomimetic-induced dyskinesia. Three MPTP monkeys received for one month pulsatile administrations of the D1 dopamine (DA) receptor agonist SKF 82958 whereas three others received the same dose of SKF 82958 by continuous infusion. A long acting D2 DA receptor agonist, cabergoline, was given to another three animals. Untreated MPTP as well as naive control animals were also included. Pulsatile SKF 82958 relieved parkinsonian symptoms but was also associated with dyskinesia in two of the three animals whereas animals treated continuously with SKF 82958 remained as untreated MPTP monkeys. Chronic cabergoline administration improved motor response with no persistent dyskinesia. MPTP treatment induced a decrease of 3H-flunitrazepam binding in the medial anterior part of caudate-putamen and an increase in the internal segment of globus pallidus (GPi) which was in general unchanged by pulsatile or continuous SKF 82958 administration. Throughout the striatum, binding of 3H-flunitrazepam remained reduced in MPTP monkeys treated with cabergoline but was not significantly lower than untreated MPTP monkeys. Moreover, cabergoline treatment reversed the MPTP-induced increase in 3H-flunitrazepam binding in the GPi. GABA concentrations remained unchanged in the striatum, external segment of globus pallidus and GPi following MPTP denervation. Pulsatile but not continuous SKF 82958 administration decreased putamen GABA content whereas cabergoline treatment decreased caudate GABA. No alteration in GABA levels were observed in the GPe and GPi following the experimental treatments. These results suggest that: (1) D2-like receptor stimulation with cabergoline modulates GABA(A) receptor density in striatal subregions anatomically related to associative cortical afferent and (2) the absence of dyskinesia in dopaminomimetic-treated monkeys might be associated with the reversal of the MPTP-induced upregulation of the GABA(A)/benzodiazepine receptor complex in the Gpi.     

Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABA(A) receptors

A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed on GABA(A) receptor subtypes (alpha(1)beta(3)gamma(2s), alpha(2)beta(3)gamma(2s), alpha(4)beta(3)gamma(2s) and alpha(5)beta(3)gamma(2s)), displaying nanomolar affinities as well as selectivity for alpha1- versus alpha2- and alpha3-containing receptors by a factor of between 14 and 26.     

Synthesis and GABA(A) receptor activity of oxygen-bridged neurosteroid analogs

Three analogs of neuroactive steroids were prepared (4-6) in which 1,11- or 11,19-oxygen bridges give a constrained conformation. Their 3D structures were obtained by ab initio calculations and in the case of 3alpha-hydroxy-11,19-epoxypregn-4-ene-20-one (4), confirmed by X-ray analysis. Biological activity of the synthetic steroids was assayed in vitro using t-[(3)H]butylbicycloorthobenzoate as radiolabeled ligand for the GABA(A) receptor. The activity of compound 4 was similar to that of allopregnanolone (1). 1alpha,11alpha-Epoxypregnanolone (6) was more active than pregnanolone (2).     

3-Alkyl- and 3-amido-isothiazoloquinolin-4-ones as ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine binding site of the GABA(A) receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl- and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction between the hydrogen bond donor site H1 with the 3-carbonyl oxygen in 3-carbonylquinolin-4-ones can be replaced by an interaction between H1 and N-2 in the isothiazoloquinolin-4-ones, was confirmed. As with the 3-carbonylquinolin-4-ones, the length of the chain in position 3 is critical for an efficient interaction with the lipophilic pockets of the pharmacophore model. The most potent 3-alkyl derivative, 3-pentyl-6-methylisothiazoloquinolin-4-one, has an affinity (K(i) value) for the benzodiazepine binding site of the GABA(A) receptors of 13 nM. However, by replacing the 3-pentyl with a 3-butyramido group an even more potent compound was obtained, with a K(i) value of 2.8 nM, indicating that the amide function facilitates additional interactions with the binding site.     

Thymopentin antagonizes stress-induced changes of GABA/benzodiazepine receptor complex

Intraperitoneal administration of thymopentin, a thymopentin II-derived pentapeptide, had no stable and evident effect in the two anxiety models (elevated plus-maze and licking-conflict test) studied. However, in the elevated plus-maze test thymopentin antagonized the behavioral effects of DMCM, a beta-carboline derivative with anxiogenic properties. Further, it was demonstrated that the licking-conflict test procedure itself produced a significant elevation of plasma corticosterone levels, increased the number of [3H]flunitrazepam and decreased the number of [3H]muscimol binding sites in rat hippocampus. The forced-swimming stress similarly to the licking-conflict test also caused an increase in hippocampal [3H]flunitrazepam binding sites. Although ineffective behaviorally in the tests for anxiety, thymopentin pretreatment effectively reversed the changes in corticosterone levels caused by the licking-conflict test. Moreover, it normalized the changed number of benzodiazepine and GABA receptors after stressful stimuli. It is well known that not all anxiolytic drugs (i.e. buspirone) are equally active in behavioral tests for anxiety. According to our data we propose that thymopentin has stress-protective activity. As in vivo and in vitro thymopentin did not change [3H]-flunitrazepam and [3H]muscimol binding, the direct effect of this peptide on the GABA-benzodiazepine-Cl- ionophore receptor complex is unlikely. The action of this peptide on GABA release and/or metabolism can be suggested.     

Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.