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PubMed2003

Phosphorylation influences neurosteroid modulation of synaptic GABAA receptors in rat CA1 and dentate gyrus neurones.

The neurosteroid 5beta-pregnan-3alpha-ol-20-one (5beta3alpha) is a potent, endogenous, positive allosteric modulator of the GABA(A) receptor. Relatively low concentrations of 5beta3alpha (10-100 nM), thought to occur physiologically, caused a concentration-dependent slowing of the decay of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from hippocampal CA1 pyramidal neurones. However, much greater concentrations of this neurosteroid (> or =300 nM) were required to similarly influence dentate granule cell mIPSCs. By contrast, the allosteric modulators pentobarbitone and flunitrazepam were equi-effective in prolonging mIPSCs in both neuronal types. Hence, the neurosteroid selectively differentiates between the synaptic GABA(A) receptors of these hippocampal neurones. Inhibition of either protein kinase A, or C, greatly reduced the sensitivity of CA1 synaptic GABA(A) receptors to 5beta3alpha, but not pentobarbitone, whereas stimulation of PKC had no effect on steroid sensitivity. However, in dentate gyrus granule cells, activation of PKC made mIPSCs sensitive to a previously ineffective concentration of 5beta3alpha. Collectively, these results suggest that the GABA-modulatory effects of physiological levels of the neurosteroid will not be uniformly experienced throughout the central nervous system, or even within the same brain region such as the hippocampus, but will be neurone-specific and will be dependent on the phosphorylation status of the GABA(A) receptor, or associated proteins.

Animals↗
PubMed1999

Subtype-selective inhibition of [methyl-3H]-N-methylscopolamine binding to muscarinic receptors by alpha-truxillic acid esters.

Seven esters of alpha-truxillic acid have been synthesized: bis-3-piperidylpropyl ester and its quaternary bis-N-ethyl derivative, bis-N-diethylaminopropyl ester and its quaternary bis-N-methyl derivative, and bis-4-piperidylbutyl ester and its quaternary bis-N-methyl and bis-N-ethyl derivatives. All esters inhibited the specific binding of muscarinic receptor antagonist [methyl-3H]-N-methylscopolamine ([3H]-NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype. In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium). All investigated esters slowed down the dissociation of [3H]-NMS from the M2 muscarinic receptor subtype. [3H]-NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis-4-piperidylbutyl aminoester and also found to be decelerated. It is concluded that the esters of alpha-truxillic acid act as M2-selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.

Allosteric Regulation↗
PubMed1997

Tolerance to diazepam and changes in GABA(A) receptor subunit expression in rat neocortical areas.

Long-term treatment with diazepam, a full allosteric modulator of the GABA(A) receptor, results in tolerance to its anticonvulsant effects, whereas an equipotent treatment with the partial allosteric modulator imidazenil does not produce tolerance. Use of subunit-specific antibodies linked to gold particles allowed an immunocytochemical estimation of the expression density of the alpha1, alpha2, alpha3, alpha5, gamma(2L&S) and beta(2/3) subunits of the GABA(A) receptor in the frontoparietal motor and frontoparietal somatosensory cortices of rats that received long-term treatment with vehicle, diazepam (three times daily for 14 days, doses increasing from 17.6 to 70.4 micromol/kg), or imidazenil (three times daily for 14 days, doses increasing from 2.5 to 10.0 micromol/kg). In this study, tolerance to diazepam was associated with a selective decrease (37%) in the expression of the alpha1 subunit in layers III-IV of the frontoparietal motor cortex, and a concomitant increase in the expression of the alpha5 (150%), gamma(2L&S) and beta(2/3) subunits (48%); an increase in alpha5 subunits was measured in all cortical layers. In the frontoparietal somatosensory cortex, diazepam-tolerant rats had a 221% increase in the expression of alpha5 subunits in all cortical layers, as well as a 35% increase in the expression of alpha3 subunits restricted to layers V-VI. Western blot analysis substantiated that these diazepam-induced changes reflected the expression of full subunit molecules. Rats that received equipotent treatment with imidazenil did not become tolerant to its anticonvulsant properties, and did not show significant changes in the expression of any of the GABA(A) receptor subunits studied, with the exception of a small decrease in alpha2 subunits in cortical layers V-VI of the frontoparietal somatosensory cortex. The results of this study suggest that tolerance to benzodiazepines may be associated with select changes in subunit abundance, leading to the expression of different GABA(A) receptor subtypes in specific brain areas. These changes might be mediated by a unique homeostatic mechanism regulating the expression of GABA(A) receptor subtypes that maintain specific functional features of GABAergic function in cortical cell layers.

Animals↗
PubMed2005

The acute anticonvulsant effects of deoxycorticosterone in developing rats: role of metabolites and mineralocorticoid-receptor responses.

PURPOSE: The mechanisms that mediate the acute anticonvulsant effects of deoxycorticosterone (DOC) were investigated in young rats. METHODS: Fifteen-day-old rats were pretreated with a variety of compounds, including (a) agonists of the receptors that bind DOC (mineralocorticoid receptors); (b) the DOC 5alpha- and 5alpha-3alpha-reduced metabolites, plus agonists that bind the receptors of the 5alpha-reduced metabolite of DOC (progesterone receptors); and (c) DOC itself in the presence and absence of metabolism and receptor blockers. Fifteen minutes later, pentylenetetrazol (PTZ) was administered, and maximal pentylenetetrazol (MMT) seizure responses were scored. RESULTS: Agonists of mineralocorticoid receptors increased the latency to forelimb flexion in PTZ seizures and sometimes suppressed the seizures completely. At low, nonconvulsant doses, spironolactone (a mineralocorticoid-receptor antagonist) blocked the anticonvulsant effects of a nonsedating, but not a sedating, dose of DOC. These data suggest the possible direct involvement of mineralocorticoid receptors in the anticonvulsant effects of DOC. At low, nonconvulsant doses, finasteride (which blocks the metabolism of DOC) partially blocked the protective effects of DOC, suggesting the contribution of metabolites to the anticonvulsant actions of DOC. Dihydrodeoxycorticosterone (DHDOC)-the first metabolite of DOC, an agonist at progesterone receptors, and an allosteric modulator of the gamma-aminobutyric acid (GABA)(A) receptor-and tetrahydrodeoxycorticosterone, a secondary metabolite of DOC and an allosteric modulator of the GABA(A) receptor, both blocked MMT seizures. CONCLUSIONS: These findings suggest that both DOC and its metabolites may contribute to the anticonvulsant effects seen in young rats, perhaps acting via interactions with several different receptors.

Animals↗
PubMed1996

Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

Animals↗
PubMedDate not supplied

Neurosteroid coadministration prevents development of tolerance and augments recovery from benzodiazepine withdrawal anxiety and hyperactivity in mice.

Neurosteroids are potent and specific modulators of the GABAA receptors which regulate the neuronal activity through diverse neurotransmitter mechanisms. In the present study we investigated the effects of concomitant treatment with various neurosteroids on the development of tolerance and recovery from withdrawal anxiety and hyperactivity to chronic benzodiazepines. Long-term treatment of mice with full allosteric modulator (triazolam 0.25 mg/kg/day for 8 days) or selective allosteric modulator (diazepam 20 mg/kg/day for 21 days) of GABAA receptor induced tolerance to behavioral sedation on actimeter and anxiolytic effects on plus-maze, and produced a marked withdrawal anxiety and hyperactivity syndrome upon abrupt cessation of treatment, respectively. Concomitant progesterone (10 mg/kg, s.c.), a neurosteroid precursor, of 4'-chlordiazepam (0.25 mg/kg, i.p.), a mitochondrial diazepam binding inhibitor (DBI) receptor (MDR) ligand, prevented the development of tolerance and significantly augmented the recovery from withdrawal-induced anxiety and hyperlocomotion to diazepam. When administered alone for 21 days, neither progesterone nor 4'-chlordiazepam produced any per se effects on actimeter or plus-maze when tested on post-withdrawal days. Coadministration of neurosteroid allopregnanolone (AP) (0.25 and 0.5 mg/kg), or pregnenolone sulfate (PS) (2 mg/kg), but not dehydroepiandrosterone sulfate (2 mg/kg), abolished the development of tolerance and attenuated withdrawal-induced anxiety and hyperlocomotion due to triazolam, without producing any per se behavioral effects when tested at 1 and 2 days after the last injection. Coadministration of flumazenil (5 mg/kg), progesterone (10 mg/kg), 4'-chlordiazepam (0.25 mg/kg), hydrocortisone (100 mg/kg) or nifedipine (2 mg/kg) also prevented the development of tolerance and suppressed the triazolam withdrawal syndrome. However, pretreatment with PK11195 (2 mg/kg), a MDR partial antagonist, reversed the effects of 4'-chlordiazepam on triazolam tolerance and recovery from chronic triazolam. When injected simultaneously, nifedipine, a Ca2+ channel antagonist, potentiated the progesterone- and 4'-chlordiazepam-induced attenuation of triazolam tolerance and withdrawal behavior. These findings suggest that coadministration of neurosteroids allopregnanolone, pregnenolone sulfate and progesterone, and MDR ligand 4'-chlordiazepam prevents the development of tolerance to benzodiazepines and augments the recovery from chronic benzodiazepines. These results indicate that coadministration of neurosteroids may facilitate discontinuation of benzodiazepines in long-term therapy.

Animals↗
PubMedDate not supplied

A new approach to insect-pest control--combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity.

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing anti-insect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion alpha-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.

Amino Acid Sequence↗
PubMed1995

Imidazenil, a new anxiolytic and anticonvulsant drug, attenuates a benzodiazepine-induced cognition deficit in monkeys.

Imidazenil is a highly potent partial allosteric modulator of gamma-aminobutyric acid action at a great variety of gamma-aminobutyric acid(A) receptors, whereas alprazolam is a full allosteric modulator at these receptors. Data in rats indicate that imidazenil, like alprazolam, has pharmacological effects in animals indicative of anxiolytic, antipanic and anticonvulsant activity in humans, but unlike alprazolam, in animals imidazenil does not produce ataxia, sedation, tolerance or dependence nor does it potentiate the effects of ethanol. To extend the study of the imidazenil pharmacological profile, we administered this drug and alprazolam, alone and in combination, in monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance), the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.01-0.32 mg/kg, p.o.) was administered alone, the overall response rate in both learning and performance decreased and the percent errors in both components increased with increasing doses. Learning, however, was more sensitive than performance; i.e., error-increasing effects were seen in learning at doses that had no effect on performance accuracy.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprazolam↗
PubMed2006

The allosteric potentiation of nicotinic acetylcholine receptors by galantamine ameliorates the cognitive dysfunction in beta amyloid25-35 i.c.v.-injected mice: involvement of dopaminergic systems.

Galantamine, a drug for Alzheimer's disease, is a novel cholinergic agent with a dual mode of action, which inhibits acetylcholinesterase and allosterically modulates nicotinic acetylcholine receptors (nAChRs), as a result stimulates catecholamine neurotransmission. In the present study, we investigated whether galantamine exerts cognitive improving effects through the allosteric modulation of nAChR in the intracerebroventricular beta amyloid (Abeta)(25-35)-injected animal model of Alzheimer's disease. Galantamine (3 mg/kg p.o.) significantly increased the extracellular dopamine release in the hippocampus of saline- and Abeta(25-35)-injected mice. The effects of nicotine on the extracellular dopamine release were potentiated by galantamine, but antagonized by mecamylamine, a nAChR antagonist. Abeta(25-35)-injected mice, compared with saline-injected mice, could not discriminate between new and familiar objects in the novel object recognition test and exhibited less freezing response in the fear-conditioning tasks, suggesting Abeta(25-35) induced cognitive impairment. Galantamine improved the Abeta(25-35)-induced cognitive impairment in the novel object recognition and fear-conditioning tasks. These improving effects of galantamine were blocked by the treatment with mecamylamine, SCH-23390, a dopamine-D1 receptor antagonist, and sulpiride, a dopamine-D2 receptor antagonist, but not by scopolamine, a muscarinic acetylcholine receptor antagonist. This study provides the first in vivo evidence that galantamine augments dopaminergic neurotransmission within the hippocampus through the allosteric potentiation of nAChRs. The improving-effects of galantamine on the Abeta(25-35)-induced cognitive impairment may be mediated through the activation of, at least in part, dopaminergic systems, and the enhancement of dopamine release may be one of multiple mechanisms underlying the therapeutic benefit of galantamine.

Amyloid beta-Peptides↗
PubMed2006

Heparin modulates the 99-loop of factor IXa: effects on reactivity with isolated Kunitz-type inhibitor domains.

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.

Alanine↗
PubMed2005

Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy.

Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.

Allosteric Regulation↗
PubMed2004

Anticonvulsant efficacy of the low-affinity partial benzodiazepine receptor agonist ELB 138 in a dog seizure model and in epileptic dogs with spontaneously recurrent seizures.

PURPOSE: Ataxia, sedation, amnesia, ethanol and barbiturate potentiation, loss of efficacy (tolerance), development of dependence, and the potential for drug abuse limit the clinical use of benzodiazepines (BZDs) for long-term treatment of epilepsy or anxiety. BZD ligands that are in current use act as full allosteric modulators of gamma-aminobutyric acid (GABA)-gated chloride channels and, on long-term administration, trigger a functional uncoupling between the GABAA and BZD recognition sites. Partial allosteric modulators, which have a low intrinsic activity at the BZD recognition site of the GABAA receptor, might eventually overcome the limitations of full agonists such as diazepam (DZP). METHODS: In the present study, the new low-affinity partial BZD-receptor agonist ELB 138 [former name AWD 131-138; 1-(4-chlorophenyl)-4-morpholino-imidazolin-2-one] was evaluated in a dog seizure model and in epileptic dogs with spontaneously recurrent seizures. RESULTS: ELB 138 was shown to increase potently the pentylenetetrazole (PTZ) seizure threshold in dogs. Prolonged oral administration with twice-daily dosing of ELB 138 with either 5 or 40 mg/kg over a 5-week period was not associated with loss of anticonvulsant efficacy in the PTZ dog model. To study whether physical dependence developed during long-term treatment, the BZD antagonist flumazenil was injected after 5 weeks of treatment with ELB 138. Compared with prolonged treatment with DZP, only relatively mild abstinence symptoms were precipitated in dogs treated with ELB 138, particularly at the lower dosage (5 mg/kg, b.i.d.). In a prospective trial in dogs with newly diagnosed epilepsy, ELB 138 markedly reduced seizure frequency and severity without significant difference to standard treatments (phenobarbital or primidone) but was much better tolerated than the standard drugs. In dogs with chronic epilepsy, most dogs exhibited a reduction in seizure frequency and severity during add-on treatment with ELB 138. CONCLUSIONS: The data demonstrate that the partial BZD receptor agonist ELB 138 exerts significant anticonvulsant efficacy without tolerance in a dog seizure model as well as in epileptic dogs with spontaneously recurrent seizures. These data thus substantiate that partial agonism at the BZD site of GABAA receptors offers advantages versus full agonism and constitutes a valuable approach for treatment of seizures.

Animals↗
PubMed1996

Low level hyperbaric antagonism of diazepam's locomotor depressant and anticonvulsant properties in mice.

Exposure to 12 atmospheres absolute (12 ATA) helium oxygen gas (heliox) (low level hyperbaric exposure) antagonizes the behavioral effects of ethanol and n-propanol, but not morphine. These and other results indicate that the mechanism of the antagonism is direct (pharmacodynamic) and selective. Our study further investigates the selectivity of low level hyperbaric antagonism by testing its effectiveness against diazepam, a high affinity binding drug that acts via allosteric modulation of GABAA receptors. C57BL/6J mice received injections i.p. of vehicle or diazepam, and were then exposed to 1 ATA air, 1 ATA heliox or 12 ATA heliox. Exposure to 12 ATA heliox antagonized the locomotor depressant effect of 4 and 6 mg/kg, but not 8 mg/kg diazepam. Hyperbaric exposure also antagonized the anticonvulsant effect of 8 and 24 mg/kg, but not 4 mg/kg, diazepam vs. 300 mg/kg isoniazid. Exposure to 12 ATA heliox did not significantly affect blood concentrations of diazepam or its metabolite n-desmethyl diazepam. The pharmacological characteristics of the antagonism (direct, surmountable, rightward shift in diazepam's dose-response curve) closely matched those seen in previous studies for hyperbaric antagonism of ethanol. The results add to the evidence that low level hyperbaric exposure is a direct, mechanistic antagonist that selectively antagonizes drugs that act via perturbation or allosteric modulation of receptor function. Moreover, the results suggest that allosteric coupling pathways, which transduce binding events on ligand-gated ion channels, may represent initial sites of action for ethanol.

Animals↗
PubMed1992

Release of endogenous benzodiazepine receptor ligands (endozepines) from cultured neurons.

Endozepines are naturally occurring small organic molecules, devoid of peptidic bonds and halogens, that act as allosteric modulators of the GABAA receptor through their actions at the benzodiazepine binding site. Endozepines are present in physiologically significant amounts in the brain and can act as potent positive allosteric modulators of the GABAA receptor. In this study, 3 endozepines present in cultured cerebellar granule cells were found to be released from neurons in a potassium-stimulated, calcium-dependent fashion. This release could also be mimicked by increasing concentrations of veratridine. Although endozepines were also found in cultured astrocytes, they could not be released in significant amounts by potassium depolarization. Differential release under depolarizing conditions and granule cell content of the various endozepines suggested a possible metabolic relationship between these two processes.

Animals↗
PubMed2006

Effects of some neurosteroids injected into some brain areas of WAG/Rij rats, an animal model of generalized absence epilepsy.

Neurosteroids are synthesized in the brain and have been demonstrated to modulate various cerebral functions. Allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), a naturally occurring neurosteroid, and ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a synthetic derivative, are two neurosteroids acting as positive allosteric modulators of the GABA(A) receptor complex acting on a specific steroid recognition site. Both agents antagonize generalized tonic-clonic seizures in various animal models of epilepsy. Pregnenolone sulphate (3beta-hydroxy-5alpha-pregnen-20-one 3-sulphate; PS) is a negative allosteric modulator of GABA(A) receptors and a positive modulator of the NMDA receptors. We have evaluated the effects of such compounds in a genetic animal model of absence epilepsy, the WAG/Rij rat. Animals were chronically implanted with five frontoparietal cortical electrodes for electrocorticogram (EEG) recordings and bilateral guide cannulae into specific brain areas of the cortico-thalamic circuit in order to evaluate the effects of these compounds on the number and duration of epileptic spike-wave discharges (SWDs). The focal and bilateral microinjection of the two GABA(A) positive modulators into some thalamic nuclei (nucleus ventralis posteromedialis, nucleus reticularis thalami, nucleus ventralis posterolateralis was usually able to significantly worsen the occurrence of SWDs in WAG/Rij rats. Whereas both compounds were able to reduce the number and duration of SWDs when microinjected into the peri-oral region of the primary somatosensory cortex. The effects of PS were more complex depending on both the dose and the site of administration, generally, at low doses in thalamic nuclei and cortex, PS induced an increase of absence activity and a reduction at higher doses. These findings suggest that neurosteroids might play a role in absence epilepsies and that it might depend on the involvement of specific neuronal areas.

Animals↗
PubMed2000

(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPA receptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rat hippocampal and frontal cortex slices.

The present study describes the effect of (S)-2,3-dihydro-[3, 4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986-1), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with cognitive-enhancing effects, on (S)-AMPA-induced [3H]noradrenaline release in rat hippocampal and frontal cortex slices. (S)-AMPA significantly increased [3H]noradrenaline release in rat hippocampus and frontal cortex slices, whereas S18986-1 (3-1000 microM) alone, was inactive. However, S18986-1 between 30 and 1000 microM potently enhanced (+200%) (S)-AMPA-mediated [3H]noradrenaline release in both hippocampal and frontal cortex slices. The capacity of S18986-1 to potentiate [3H]noradrenaline release was specific for AMPA receptors as S18986-1 failed to potentiate either kainate and N-methyl-D-aspartate (NMDA)-mediated release of [3H]noradrenaline in rat hippocampal slices. Moreover, 1, 2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) and 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3, 4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-53655) but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801), inhibited (S)-AMPA and S18986-induced stimulation of (S)-AMPA-mediated [3H]noradrenaline release. In addition, S18986-1-induced stimulation of (S)-AMPA-evoked [3H]noradrenaline release was markedly attenuated in the presence of tetrodotoxin (1 microM) and in Ca(2+)-free buffer. S18986-1 enhanced (S)-AMPA-mediated [3H]noradrenaline release to a greater extent than its corresponding (R)-enantiomer S19024-1 and racemic mixture S17951-1. However, positive allosteric modulators of AMPA receptors such as aniracetam failed to potentiate AMPA-mediated noradrenaline release in hippocampal slices, whereas cyclothiazide potently enhanced (S)-AMPA-mediated [3H]noradrenaline release. These results suggest that the capacity of S18986-1 to enhance AMPA receptor-mediated release of noradrenaline in rat hippocampus and frontal cortex, could contribute to the cognition enhancing mechanisms of S18986-1.

Animals↗
PubMed2006

Identification of a putative binding site for 5-alkyl-benzothiadiazides in the AMPA receptor dimer interface.

Crystal structures of three different allosteric modulators co-crystallized with the iGluR2 ligand-binding domain are currently available. The modulators, cyclothiazide, aniracetam and CX614, bind at overlapping binding sites in the dimer interface between two iGluR2 subunits. However, pharmacological data indicate that there are one or more additional binding sites for this class of compounds. Based on differences in structure-activity relationship data we show that 5-alkyl-benzothiadiazide (5ABTD) modulators and a series of close analogs of cyclothiazide, despite having a common core structure, do not have the same binding site. In the present work, a new potential binding site for allosteric modulators has been identified in the dimer interface of the iGluR2 ligand-binding domain. By comparing different iGluR2 crystal structures including different co-crystallized agonists, this cavity is shown to be a structurally conserved part of the dimer interface. The cavity is characterized with respect to shape and potential favorable interactions with ligands and docking is used to find a reasonable binding mode for the core structure of the 5ABTDs. The extensive structure-activity data available for this series of compounds are in agreement with the proposed binding mode, supporting the conclusion that the identified cavity most likely is the binding site for the 5ABTDs.

Allosteric Site↗
PubMed2006

Determining the potency and molecular mechanism of action of insurmountable antagonists.

Insurmountable antagonism (maximal response to the agonist depressed) can result from a temporal inequilibrium involving a slow offset orthosteric antagonist or be the result of an allosteric modulation of the receptor. The former mechanism is operative when the antagonist, agonist, and receptors cannot come to proper equilibrium during the time allotted for collection of agonist response (hemi-equilibrium conditions). Allosteric effects (changes in the conformation of the receptor through binding of the allosteric modulator to a separate site) can preclude the agonist-induced production of response, leading to depression of maximal responses. In these cases, the effects on receptor affinity can be observed as well. The first premise of this article is that system-independent estimates of insurmountable antagonist potency can be made with no prior knowledge of molecular mechanism through the use of pA(2) (-log molar concentration of antagonist producing a 2-fold shift of the concentration response curve) measurements The relationship between the pA(2) and antagonist pK(B) (-log equilibrium dissociation constant of the antagonist-receptor complex) is described; the former is an extremely close approximation of the latter in most cases. The second premise is that specially designed experiments are required to differentiate orthosteric versus allosteric mechanisms; simply fitting of data to orthosteric or allosteric theoretical models can lead to ambiguous results. A strategy to determine whether the observed antagonism is orthosteric (agonist and antagonist competing for the same binding site on the receptor) or allosteric in nature is described that involves the detection of the hallmarks of allosteric response, namely saturation and probe dependence of effect.

Allosteric Regulation↗