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PubMed1996

Quantitative determination of imidazenil, a novel imidazobenzodiazpine carboxamide derivative, by normal phase high-performance liquid chromatography.

Imidazenil, an imidazobenzodiazepine carboxamide derivative, is a novel anxiolytic and anti-convulsant agent recently characterized as a partial allosteric modulator of GABAA receptors. Owing to the pharmacological and pharmacokinetic importance of plasma-level determination, a HPLC method has been developed. Imidazenil was extracted from a plasma sample after a partition with diethyl ether, using alprazolam as internal standard. The analysis was performed by a normal-phase HPLC method with UV detection at 255 nm. The limit of quantitation was 6 ng corresponding to 30 ng/ml of plasma concentration. This procedure has been successfully applied to the quantitation of imidazenil plasma levels in primarily pharmacokinetic studies after a single i.v. and an oral administration of the compound to the rat.

Administration, Oral↗
PubMed1995

Pentobarbital modulatory effect on GABA binding sites in developing chick optic lobe.

Barbiturates are allosteric modulators of the CNS GABAA receptor, increasing [3H]-GABA binding to its receptor sites. In the present work we have studied the modulatory effect of the barbiturate pentobarbital on low-affinity GABA binding sites during ontogenetic development of the chick optic lobe. Our results indicate that [3H]-GABA binding enhancement by pentobarbital shows a differential profile during development, following a two-component enhancement model at early stages of development and a single-component enhancement model in the adult stage. Kinetic analysis performed at different stages of development showed that barbiturate enhancement was invariably due to an increase in [3H]-GABA binding affinity, while maximal binding capacity remained unchanged. Using GABA antagonists, picrotoxinin and bicuculline, convulsant sensitivity of high-affinity barbiturate modulatory sites was found at early stages. These data suggest that barbiturate action displays receptor heterogeneity during development, with high- and low-affinity modulatory sites only at early stages, while the high-affinity sites disappear between hatching and adulthood. Kinetic data indicate that both barbiturate modulatory sites are coupled to the GABAA receptor at early stages. The presence of high-affinity modulatory sites at early stages and at hatching suggests a major role during visual pathway maturation.

Animals↗
PubMed1990

Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor.

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.

Animals↗
PubMed1992

Ornithine decarboxylase induction and polyamine synthesis in the kindling of seizures: the effect of alpha-difluoromethylornithine.

It has been suggested that the kindling of seizures may depend on the induction of genes encoding enzymes involved in neurotransmission. Experimental seizures are followed by an especially rapid and massive induction of brain ornithine decarboxylase (ODC), an enzyme which catalyses the rate-limiting step in the synthesis of polyamines. The latter compounds have been shown to act as positive allosteric modulators of the NMDA receptor, and also to play an important role in cell growth and differentiation. The induction of ODC by seizures has accordingly been suggested to play a pivotal role in the changes in synaptic structure and function that underlie kindling. In the present study we examined the progress of kindling during treatment with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We found that progressive increase in the duration and severity of kindled seizures and in the duration of local afterdischarges was unaffected by daily injections of DFMO in doses previously shown to cause substantial depression of brain ODC activity. Treatment with DFMO also failed to produce significant anticonvulsant or proconvulsant effects. Progressive increase in seizure activity during kindling is therefore unlikely to depend to any appreciable extent on enhanced synthesis of polyamines by ODC.

Animals↗
PubMed1989

Allosteric interactions of three muscarine antagonists at bovine tracheal smooth muscle and cardiac M2 receptors.

The kinetics of [3H]dexetimide dissociation from muscarine receptors in bovine cardiac left ventricular and tracheal smooth muscle membranes were studied in the absence and presence of three muscarine antagonists. It was found that [3H]dexetimide dissociation from cardiac muscarine receptors was monophasic and very fast (half life less than 1 min) and was slowed by the cardioselective muscarine antagonists, gallamine, methoctramine and AF-DX 116, concentration dependently. [3H]Dexetimide dissociation from tracheal muscarine receptors was biphasic, with a fast phase (half-life less than 1 min) followed after 4-5 min by a slow phase (half-life = 38.5 min). The fast component, but not the slow component, was slowed by the muscarine antagonists with concentration dependencies very similar to those found in the heart. We conclude from these data that the major population of tracheal smooth muscle muscarine receptors resembles the cardiac M2 type not only with respect to equilibrium binding affinities but also with respect to the secondary, allosteric binding site on the muscarine receptor. The results also imply that the cardiac receptor subtype is much more sensitive to allosteric modulation than the glandular/smooth muscle receptor subtype.

Animals↗
PubMedDate not supplied

Some approaches to the pharmacology of multisubstrate enzyme systems.

Analytical and exploratory in vitro, in situ and in vivo, physio-pharmacotoxicology, from enzymology to population epidemiology, now embraces those approaches that correlate complex dynamic multisubstrate kinetics through conventional and more recent non-invasive quantitative methodologies. Basically, substrates may be classed as pertaining to fundamental energy turnovers (first-order cellular metabolic pathways or networks) and to iso- vs allosteric modulator systems (second-order metabolic control network). Pairs of substrates and cofactors set-up the third-order multienzyme-receptor patterns, which in intact, native in vivo structures establish and maintain the compartmentalized, dynamically superimposed overall coordination of local redox and phosphate potentials. Perturbations of the various levels of the metabolic hierarchy induced by drugs, as well their relaxations, can be readily submitted to non-invasive kinetic analysis. Both indirect and direct titrations of substrate levels, their modelling and statistical ad hoc evaluations of their interrelations can lead to the identification of the multiple sites involved in drug effects as structured at the different orders/levels of concomitant functional variations. Fractal geometries contribute towards defining the space- and time-related events.

Animals↗
PubMed2005

Galantamine-induced behavioral recovery after sublethal excitotoxic lesions to the rat medial septum.

Clinical trials show beneficial effects of acetylcholinesterase (AChE) inhibitors, including galantamine, on cognitive functions in patients with mild to moderate Alzheimer's disease. Galantamine shows a dual action profile by also acting as an allosteric modulator of nicotinic acetylcholine receptors. Nevertheless, its in vivo mechanism of action is only partly understood. Here, we first established a novel lesion model provoking significant functional impairment of the septo-hippocampal projection system without triggering massive neuronal death in the rat medial septum. Next, we studied whether galantamine, administered in doses of 1 and 3mg/kg post-lesion, promotes functional recovery of spatial navigation behaviors, and affects the output of septal cholinergic projections. Infusion of N-methyl-d-aspartate (NMDA; 30nmol/1microl) in the medial septum resulted in spatial learning deficits associated with significant shrinkage of cholinergic neurons and reduced AChE activity in the hippocampus at 7 days post-lesion. Galantamine treatment alone significantly increased the hippocampal acetylcholine concentration and attenuated the NMDA-induced spatial learning impairment. Galantamine post-treatment also affected NMDA-induced changes in AChE and choline-acetyltransferase activities. In conclusion, our data show that galantamine attenuates experimentally-induced cognitive impairments underscored by mild neuronal damage.

Acetylcholinesterase↗
PubMed2005

Age-related deficits in the retention of memories for cued fear conditioning are reversed by galantamine treatment.

Aging has diverse effects on different behaviors and underlying neural systems. This study utilized fear conditioning to determine if aged mice have deficits in the acquisition and/or retention of memories for contextual or cued fear conditioning, and to determine if galantamine, an acetylcholinesterase (AChE) inhibitor and allosteric modulator of nicotinic acetylcholinergic receptors, would alter acquisition and/or retention of fear conditioning memories in young (2-3 months) and aged (19-20 months) C57BL/6 mice. Mice were trained with two white-noise CS (85 dB, 30 s)-footshock US (0.57 mA, 2 s) presentations. In the initial study, separate groups were tested 24, 48, or 96 h post-training. All mice were retested 1 week after the initial test. Aged mice were impaired in freezing to the CS for the 48 and 96 h train-test intervals, but not the 24-h interval. When retested 1 week after the initial test, freezing to the CS was significantly lower for all train-test intervals. No age-related deficits were found in contextual fear conditioning. In the second study, 2 mg/kg galantamine was administered to young and aged mice before fear conditioning and conditioned fear was assessed 48 h later. No age-related deficits in cued fear conditioning were seen in galantamine-treated aged mice. Thus, aged C57BL/6 mice are impaired in the long-term retention of auditory cued fear conditioning, but not the acquisition of auditory cued or contextual fear conditioning. This retention deficit for cued fear conditioning is ameliorated by treatment with the AChE inhibitor galantamine.

Acoustic Stimulation↗
PubMed2006

Extracellular calcium sensing in rat aortic vascular smooth muscle cells.

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

Animals↗
PubMed2004

Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay.

In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput screening assay. In the patch-clamp assay, the alpha1 GlyR exhibited the properties expected from a strychnine-sensitive glycine-gated chloride channel. In the FMP assay exposure of the cell line to GlyR agonists elicited a concentration-dependent increase in fluorescent intensity, a signal that could be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC50 and Ki values in good agreement with previously reported values from studies of recombinant alpha1 GlyRs and native alpha1beta GlyRs. The rank orders of potencies was glycine > beta-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the alpha1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn2+ inhibited alpha1 GlyR signalling but in contrast to previous reports the metal ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited alpha1 GlyR function, the potentiation of alpha1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.

Cell Line↗
PubMed2004

Effects of anandamide on the binding and signaling properties of M1 muscarinic acetylcholine receptors.

This study investigated the effects of the endocannabinoid, anandamide, on M(1) muscarinic acetylcholine receptors (mAChRs) expressed in Chinese hamster ovary (CHO) cells. In the presence of anandamide, [(3)H]N-methylscopolamine ([(3)H]NMS) inhibition binding was characterized by Hill coefficients greater than 1 while saturation binding isotherms were characterized by a reduction in radioligand B(max). Anandamide did not affect the potency of classic agonists, antagonists or allosteric modulators to inhibit [(3)H]NMS binding, indicating that the site of anandamide action did not involve receptor regions recognized by these compounds. Although the mode of binding of anandamide was reversible, the order of ligand addition was important; the inhibitory effect was greatest when anandamide was equilibrated with the receptor prior to radioligand addition, and weakest in the converse situation. Interestingly, the inhibitory potency of anandamide was reduced on pre-equilibration with non-transfected CHO cell membranes, prior to addition of M(1) mAChR-transfected membranes. In phosphoinositide (PI) hydrolysis assays, anandamide significantly reduced the maximal response to acetylcholine, but at higher concentrations than those needed to fully inhibit radioligand binding. Studies utilizing a range of agonists with varying intrinsic activities showed that the inhibitory effects of anandamide on agonist function were most pronounced with the lowest efficacy agonists. These findings suggest that the mechanism of action of anandamide at the M(1) mAChR involves perturbation of the receptor via the membrane in a manner that is sensitive to the conformation of the receptor (occupied versus vacant).

Animals↗
PubMed2006

A role for AMPA receptors in mood disorders.

Major antidepressant agents increase synaptic levels of monoamines. Although the monoamine hypothesis of depression remains a cornerstone of our understanding of the pathophysiology of depression, emerging data has suggested that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptor may also play a pivotal role in depression. Positive allosteric modulators of AMPA receptors increase brain levels of brain-derived neurotrophic factor (BDNF) that impacts the viability and generation of neurons in key brain structures. AMPA receptor potentiators are active in rodent models predictive of antidepressant efficacy. The mechanisms by which AMPA receptor potentiators produce these biological effects, however, are uncertain. Current evidence points to an antidepressant mechanism that is independent of monoaminergic facilitation that is driven by neurogenesis, a process facilitated by increased BDNF expression. However, alternative hypotheses need to be considered given uncertainties in the relationship between BDNF increases and the effects of conventional antidepressant medications. Electrophysiological and protein conformational data indicate that structural variants of AMPA receptor potentiators can differentially modulate AMPA receptor-mediated currents, although the manner in which this impacts antidepressant efficacy is yet to be understood. Conventional antidepressants such as fluoxetine positively modulate AMPA receptors. This potentiation is engendered by specific phosphorylation pathways activated through the dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32). Other novel compounds with antidepressant-like effects in rodents may also produce their in vivo effects through potentiation of AMPA receptors. Thus, AMPA receptor potentiation might be a general mechanism through which the clinical outcome of antidepressant efficacy is achieved.

Animals↗
PubMed2005

Nutrition-/diet-induced changes in gene expression in white adipose tissue.

Nutrients regulate metabolic fluxes and homeostasis through transcriptional and translational control of enzyme concentrations and allosteric modulation of enzyme activity. Dietary omega-3 polyunsaturated fatty acids (PUFAs) have been shown to exert a variety of beneficial health effects such as reducing adiposity and increasing insulin sensitivity in rodents. It is now clear that PUFAs regulate fundamental adipose cell and liver functions through modulation of activity and abundance of key transcription factors that act as nutrient sensors, including peroxisome proliferator-activated receptors (PPARalpha/delta/gamma), sterol regulatory element binding proteins (SREBP-1/2), and liver X receptors (LXRalpha/beta). However, in the state of obesity, where adipose tissue shows elevated storage of triglycerides, many lipogenic genes that are essential for adipose cell function including PPARgamma, SREBP-1c, CCAAT-enhancer binding protein alpha and stearoyl-CoA desaturase-1 are downregulated, apparently due to desensitization of the very same crucial nutrient sensors. This chapter will summarize recent studies of PUFA- and obesity-induced changes in gene expression in white adipose tissue.

Adipose Tissue↗
PubMed2005

Valproate corrects the schizophrenia-like epigenetic behavioral modifications induced by methionine in mice.

BACKGROUND: Reelin and GAD(67) expression is downregulated in cortical interneurons of schizophrenia (SZ) patients. This downregulation is probably mediated by epigenetic hypermethylation of the respective promoters caused by the selective increase of DNA-methyltransferase 1 in GABAergic neurons. Mice receiving methionine (MET) provide an epigenetic model for neuropathologies related to SZ. We studied whether MET-induced epigenetic reelin promoter hypermethylation and the associated behavioral alterations can be reduced by valproate in doses that inhibit histone deacetylases (HDACs). METHODS: Mice treated with either methionine (MET) (5.2 mmol/kg/SC/twice daily) or valproate (1.5 mmol/kg/SC/twice daily) or MET+ valproate combination were tested for prepulse inhibition of startle (PPI) and social interaction (SI). S-adenosylmethionine, acetylated histone 3, reelin promoter methylation, and reelin mRNA were assayed in the frontal cortex. RESULTS: Valproate enhances acetylated histone 3 content, and prevents MET-induced reelin promoter hypermethylation, reelin mRNA downregulation, and PPI and SI deficits. Imidazenil, a positive allosteric modulator at GABA(A) receptors containing alpha(5) subunits but inactive at receptors including alpha(1) subunits, normalizes MET-induced behavioral changes. CONCLUSION: This MET-induced epigenetic mouse models the neurochemical and behavioral aspects of SZ that can be corrected by positively modulating the action of GABA at alpha(5)-containing GABA(A) receptors with imidazenil or by inhibiting HDACs with valproate, thus opening exciting new avenues for treatment of epigenetically modified chromatin in SZ morbidity.

Aggression↗
PubMed2005

Transmembrane residues define the action of isoflurane at the GABAA receptor alpha-3 subunit.

The gamma-aminobutyric acid type A (GABA(A)) receptor is the target of a structurally diverse group of sedative, hypnotic, and anesthetic drugs, including the volatile anesthetic isoflurane. Previous studies on the GABA(A) receptor have suggested the existence of a cavity located between transmembrane (TM) segments 2 and 3 in both alpha-1 and alpha-2 subunits, within which volatile anesthetics might bind. In this study, we have used site-directed mutagenesis to investigate the involvement of homologous residues of the GABA(A) alpha-3 subunit in allosteric modulation by isoflurane. Mutation of serine residue 294 within the TM2 to histidine or tyrosine increased the potency of GABA and decreased positive modulation by isoflurane. Mutation of alanine residue 315 within the TM3 to tryptophan increased the potency of GABA and abolished isoflurane modulation. The activity of the intravenous anesthetic propofol was unaltered from wild-type at these mutant receptors. These findings are consistent with the action of isoflurane on a critical site within the transmembrane domains of the receptor and suggest a degree of functional homology between the GABA(A) alpha-1, -2, and -3 subunits.

Anesthetics, Inhalation↗
PubMed2005

Intracellular oxidation of allopregnanolone by human brain type 10 17beta-hydroxysteroid dehydrogenase.

Allopregnanolone is a positive allosteric modulator of GABAA receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) in astrocytes. This neuroactive steroid can be inactivated by its 3alpha-oxidation to yield 5alpha-DHP. It was found that 5alpha-DHP levels in HEK293 cells expressing type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10), but not its catalytic inactive mutant, increased significantly as allopregnanolone was added to culture media. The results demonstrate that mitochondrial 17beta-HSD10 effectively catalyzes the intracellular oxidation of allopregnanolone. Moreover, brain astrocytes contain a moderate level of 17beta-HSD10, which is elevated in activated astrocytes of brains with Alzheimer type pathology, including sporadic Alzheimer's disease (AD) and Down's syndrome with AD. The distribution of 17beta-HSD10 was found not to parallel that of 3alpha-HSD3. Cerebral cortex has the lowest level of 17beta-HSD10; whereas the hippocampus, hypothalamus, and amygdala possess relatively higher levels of this enzyme. The catalysis of 17beta-HSD10 appears to be essential for maintaining normal functions of GABAergic neurons. The elevated level of 17beta-HSD10 in activated astrocytes is a new feature found in brains of people with AD, and it may have important impact on AD pathogenesis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2006

The adult rat hippocampal slice revisited with multi-electrode arrays.

The multi-electrode arrays (MEA) technology for the recording of brain slices is available for more than 10 years. However, despite its relative simplicity, this recording technique is not widely used in academic or pharmaceutical research laboratories. We illustrate here that MEA provide multiple possibilities to investigate some network physiological properties as well as to evaluate the pharmacological effects of compounds. We first document that MEA allow to trigger and to record conventional FP which are inhibited by the block of action potential propagation (with 500 nM TTX). FP recorded with MEA are sensitive to ionic substitutions, to ionotropic glutamate receptor antagonists (CNQX or NBQX) and to energetic failure. Second, we illustrate that different "classical" protocols (paired-pulse, LTP, chemical LTD), revealing synaptic plasticity mechanisms, could be performed. Third, we document that MEA allow spatial and temporal discriminations for the effects of known pharmacological compounds such as competitive antagonist (gabazine, bicuculline) and allosteric modulators (steroids) of GABA(A) receptors. In conclusion, we illustrate that MEA recordings of adult rat hippocampal slices constitute a powerful and sensitive system to evaluate the effect of molecules on basic synaptic propagation/transmission and on synaptic plasticity processes.

Animals↗
PubMed2005

Indicators of neuroprotection with galantamine.

Alzheimer's disease is pathologically characterized by neurofibrillary tangles and beta-amyloid plaques. These observations form the basis for a large number of disease-modifying therapeutic approaches, which might ultimately lead to neuroprotection and enhanced survival of neurons. Recent data suggest a role for cholinergic stimulation, especially the alpha7 nicotinic acetylcholine receptors (nAChR), in beta-amyloid-mediated neurotoxicity. As galantamine is a modest acetylcholinesterase inhibitor in addition to being an allosteric modulator of nicotinic acetylcholine receptors, it is interesting to study the clinical effects of this compound in the light of its neurochemical properties to discern potential neuroprotective effects. The review presents the preclinical evidence in Alzheimer-related models of neuroprotection with galantamine, especially in models related to glutamate and beta-amyloid toxicity in vitro and to cholinergic stress in vivo. There is substantial evidence that these effects occur by upregulation of the protective protein bcl-2 and are mediated via alpha7 nicotinic acetylcholine receptors. The review also identifies possible clinical indicators, such as long-term studies, suggesting a neuroprotective effect for galantamine mediated by alpha7 nicotinic receptors. These clinically relevant neuroprotective properties of galantamine are worthwhile exploring further and their clinical relevance may improve the development of new disease-modifying agents.

Acetylcholine↗