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Identification using versatile sampling and analytical methods of volatile compounds from Streptomyces albidoflavus grown on four humid building materials and one synthetic medium.

UNLABELLED: The Streptomyces spp. form a common group of bacteria found in the indoor air of water-damaged buildings. They are known for their capability to produce compounds, like geosmin, with low odor thresholds. In this study, two strains of Streptomyces albidoflavus were cultivated on pinewood, gypsum board, particle-board, sand and tryptone glucose extract agar (TGEA). Air samples from the cultures were collected on six different adsorbents and chemosorbents to sample a wide range of compounds such as VOCs, aldehydes, amines and lightweight organic acids. The samples were analyzed with gas chromatography, high-pressure liquid chromatography and ion chromatography. Mass spectrometry was used for identification of the compounds. Metabolites were found and identified in air samples from cultures on all materials except sand. Alcohols and ketones were the dominating compound groups produced by cultures grown on pinewood and gypsum board. Few metabolites were produced on particle-board. The culture growing on TGEA produced mainly sulfur compounds and sesquiterpenes. Ammonia, methylamine, diethylamine, ethylamine and one unidentifiable amine were also found from cultivation on TGEA. The growth medium was of crucial importance to the production of potentially irritating metabolites. PRACTICAL IMPLICATIONS: Microbial growth and the production of volatile metabolites is one possible explanation for building-related health problems. Streptomyces spp. are frequently found in water-damaged buildings. This study shows that Streptomyces spp. are able to produce not only odorous compounds like geosmin, but also potentially irritating compounds. This finding should be of interest in indoor air investigations.

Agar↗

Tetrahydro-beta-carbolines and corresponding tryptamines: In vitro inhibition of serotonin and dopamine uptake by human blood platelets.

Tetrahydro-beta-carbolines (THBCs), 6-methoxyharmalan and norharman and the corresponding open chain tryptamines studied inhibited t-hydroxytrypere better inhibitors of DA than 5-HT uptake but THBCs generally were far more potent inhibitors of 5-HT uptake than of DA uptake. 6-methoxy-1,2,3,4-tetrahydro-beta-carboline was as potent as 5-HT itself 3H-5-HT uptake inhibition in platelets and the inhibition was competitive. All the beta-carbolines studied were more potent inhibitors of 3h-da uptake than DA itself. Contrary to results in rat brain synaptosomes, THBCs were more potent in platelets than the corresponding tryptamines with the freely rotating ethylamine side chain. Unsaturated beta-carbolines were weaker inhibitors than THBCs. The clear difference in the rank order of potencies of these compounds in human platelets and rat brain synaptosomes demonstrates that these different model systems for amine uptake studies do not always give comparable results. The results also suggest that there are differences in the uptake systems for 5-HT and DA in human platelets.

Blood Platelets↗

Possible regulatory role of histamine in human platelet function examined by thrombin-induced serotonin release.

The influence of histamine on human platelet function was studied by thrombin-induced serotonin release. The thrombin-induced 3H-serotonin release was confirmed to be a rapid process which does not require external calcium. Histamine was found to reduce the release of serotonin and the inhibition was abolished when H1-plus H2-antagonists were added together with histamine. H1- and H2-receptor stimulation was examined in two ways, by a combination of histamine with cimetidine or diphenhydramine and by the selective agonists 2-(2-pyridyl)-ethylamine and impromidine. In both instances H1- and H2-stimulation was found to reduce the platelet serotonin release. These results suggest a regulatory role of histamine in the platelet function by stimulation of platelet H1- and H2-receptors.

Blood Platelets↗

Analysis of amines and other bacterial products by head-space gas chromatography.

A gas chromatographic (GC) head-space technique is presented, which is suitable for the analysis of volatile products in bacterial broth cultures. This is exemplified by studies on Clostridium septicum, Klebsiella pneumoniae and Proteus mirabilis. The media were acidified or made alkaline and after heating, samples of the gas phase above the media were directly injected into the gas chromatograph. A gas chromatograph equipped with dual columns and flame ionization detectors was used, employing Porapak Q and Chromosorb 103 as stationary phases. Analysis of acidified media, using Porapak Q, gave chromatograms representing acidic and neutral volatile products, while when analysing samples made alkaline, using Chromosorb 103, alkaline and neutral compounds could be detected. Interest was particularly concentrated on the analysis of bacterial amines. P. mirabilis was found to produce isobutylamine and isopentylamine, which were identified by mass spectrometry and GC retention times C. septicum produced ethylamine. The GC head-space technique described constitutes a means for rapid identification of microorganisms. It is adaptable for use on a routine basis in the clinical microbiological laboratory.

Acids↗

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily.

We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.

Chromatography, Gel↗

5-Hydroxytryptamine is a substrate for both species of monoamine oxidase in beef heart mitochondria.

The activity of beef heart mitochondrial monoamine oxidase towards 5-hydroxytryptamine (5-HT) is inhibited by the selective inhibitors clorgyline, PCO [5-phenyl-3-(N-cyclopropyl)-ethylamine-1,2,4-oxidiazole] and Deprenyl with a biphasic dependence on the inhibitor concentration. The activities towards tyramine, dopamine and tryptamine were also inhibited in a biphasic manner, but the apparent proportions of the two enzyme species active on dopamine and tryptamine depended on the inhibitor used. Phenethylamine oxidation was inhibited in a monophasic manner suggesting that only a single enzyme species was responsible for the oxidation of this substrate. The biphasic response of 5-HT oxidation to inhibition by clorgyline persisted when functionally competent mitochondria were used and was unaffected by the soluble amine oxidase inhibitors semicarbazine and aminoguanidine. These results indicate that the behaviour of the beef heart enzyme towards selective inhibitors is considerably different from that of any preparations previously studied and suggest that the classification of monoamine oxidase activites into A and B types may be only of limited usefulness.

Amines↗

Fluorescence spectroscopic analysis of the hydrogen bonding properties of catecholamines, resorcinolamines, and related compounds with phosphate and other anionic species in aqueous solution.

Fluorescence spectroscopic analysis based upon the difference in fluorescent behaviour of several phenol, catechol and resorcinol derivatives in 0.2 M phosphate and water, at different pH values ranging from 7.4 to 1.0, produced evidence for the existence of complexes between phosphate anions and these compounds. This complex formation was also found with acetate, citrate and glutamate but not with sulphate and borate. A comparison of different ligand structures revealed that the complex formation observed with unsubstituted phenol, catechol or resorcinol was not influenced by ethyl substitution; however, ethylamine or ethanolamine substitution favoured the formation of the complex. The findings indicate the formation of 1:1 intermolecular complexes between mono- and dihydroxyphenethylamines and several anionic species with participation of two hydrogen bonds. The predictions of a theoretical model describing this interaction are confirmed by the experimental data. Association constants of isoprenaline with phosphate and glutamate were found to be 5.1 and 26.6 litres mol-1 respectively; for the complexes of m-tyramine and terbutaline with phosphate, association constants of 15.3 and 6.9 litres mol-1 respectively, were obtained.

Catecholamines↗

Deamination of aliphatic amines of different chain lengths by rat liver monoamine oxidase A and B.

Monoamines with from 1 to 18 straight chain carbon atoms have been analysed as rat liver monoamine oxidase substrates. Methylamine and ethylamine are clearly not substrates of monoamine oxidase (MAO). n-Propylamine, n-butylamine, n-dodecylamine and n-octadecylamine are relatively poor substrates, i.e. with high Km and low Vmax values for the enzyme. n-Pentylamine, n-hexylamine, n-heptylamine, n-octylamine, n-nonylamine and n-decylamine are all very good MAO substrates. All these aliphatic amines are found to be typical type B substrates according to the sensitivities of the enzyme towards the selective MAO-B inhibitor selegiline and the MAO-A inhibitor, clorgyline. The sensitivity towards selegiline with respect to these amines is even higher, i.e. Ki = 1 x 10(-9) M for butylamine, than that of the typical type B substrate beta-phenylethylamine (Ki = 1 x 10(-8) M). The sensitivity towards selegiline decreases slightly with increasing chain length of these aliphatic amines.

Amines↗

Correlation between structures of organic cations and their binding behaviours to brush border membrane isolated from rat small intestine.

We have investigated the correlation between the molecular structures of various organic cations and their binding to rat small intestinal brush border membrane. The binding of small quaternary ammonium compounds such as tetramethylammonium and choline to brush border membrane was not sufficient to inhibit methylchlorpromazine binding. However, lauryltrimethylammonium and cetyltrimethylammonium, both quaternary amines with a long carbon chain, inhibited binding significantly. The inhibition was competitive. When the unbranched hydrocarbon chain of the quaternary amines was extended in steps from C1 (methyl) to C16 (cetyl), the inhibitory effect increased sharply with length from C7 (heptyl) to C16. These results suggest that the size of the hydrophobic part of the molecule is an important factor in binding of quaternary ammonium compounds to the brush border membrane. The structure of the hydrophilic part was another factor. In imipramine-related compounds, the order of binding was N-didesmethylimipramine (primary amine) greater than desipramine (secondary) greater than imipramine (tertiary) greater than methylimipramine (quaternary). However, with the small molecular ethylamine-related compounds, binding properties did not reflect differences in the hydrophilic component. Therefore, the effect of the hydrophilic part may be secondary and may depend on the size of the hydrophobic part. We suggest that organic cations which are amphiphilic can bind to a common binding site on brush border membrane through hydrophobic and/or hydrophilic interactions.

Animals↗

Inhibition of [3H]dopamine uptake by platelets by the dopamine-D2 receptor agonist RU 24926.

We have examined the effect of the dopamine-D2 receptor agonist RU 24926 (N-n-propyl-di-beta(3-hydroxy-phenyl)-ethylamine HCl) on [3H]dopamine uptake by human platelets. RU 24926 reduced the uptake of [3H]dopamine by platelet-rich plasma and this affect was not reversed by the dopamine-D2 receptor antagonist haloperidol, or the dopamine-D1 receptor antagonist SCH 23390 (8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol). These data suggest that RU 24926 reduces [3H]dopamine uptake by platelets by competing for the dopamine uptake mechanism on the platelet and not by activation of the dopamine-D2 receptor.

Benzazepines↗

Brain pharmacokinetics of a nonpeptidic corticotropin-releasing factor receptor antagonist.

Corticotropin-releasing factor (CRF) is known to play an important role in the body response to stress. Butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-ethylamine (CP-154,526) is a CRF(1) antagonist showing anxiolytic activities in rats in behavioral models, suggesting that CP-154,526 crosses the blood-brain barrier. However, there is no direct evidence for this. This study determined the pharmacokinetic profile of CP-154,526 in rats after i.v. and p.o. application. After i.v. bolus, the concentration declined in a biphasic manner, the first half-life being 0.9 h and the terminal one being 51 h. Systemic clearance was 36 ml/min/kg, and the volume of distribution was 105 l/kg. Oral bioavailability reached 27%. To study brain pharmacokinetics, rats were given a single dose of CP-154,526 p.o. or i.v. and sacrificed after different post-treatment times. Plasma, cortex, striatum, hypothalamus, hippocampus, and cerebellum concentrations were measured. After i.v. bolus, maximal brain concentration was reached after 20 min. The hypothalamus displayed significantly lower concentrations compared with the other brain tissues. In the p.o. study, the maximal plasma concentration was reached after 30 min, whereas the maximal brain concentration was observed after 1 h and remained stable until 2 h post-treatment, without significant differences between the brain tissues. The unidirectional brain extraction ratio was 27 and 9% at vascular concentrations of 0.08 and 16 nmol/ml, respectively. These results demonstrate a large brain penetration of CP-154,526 after i.v. and p.o. applications and a comparable distribution among the brain regions, except for the hypothalamus, which displayed lower concentrations after i.v. bolus.

Animals↗

Ocular hypotensive action of topical flunarizine in the rabbit: role of sigma 1 recognition sites.

In a previous study we ascertained the presence of sigma1 and sigma2 recognition sites in the rabbit iris-ciliary body, an ocular structure involved in aqueous humor production and drainage. We characterized the sigma1 sites using the preferential ligand (+)-pentazocine, which caused a significant reduction of intraocular pressure (IOP). In the present study, flunarizine, a calcium channel blocker with a complex pharmacological profile, bound to sigma1 sites expressed in the iris-ciliary body with moderate affinity (K(i) = 68 nM). Unilateral topical flunarizine (0.01-0.1%) caused a dose-related reduction of IOP in ocular normotensive rabbits and in the alpha-chymotrypsin model of ocular hypertension, without altering the IOP of the contralateral eye. This activity was blocked by the sigma1 site antagonist NE-100 [N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine HCl] which, by itself, had no effect on IOP. Detection of flunarizine in rabbit iris-ciliary body homogenates, after topical instillation, showed that it adequately penetrates the rabbit eye. To investigate mechanisms that may contribute to ocular hypotension induced by sigma1 agonists, we carried out in vitro studies on the isolated rabbit iris-ciliary body. Flunarizine (IC50 = 5. 96 nM) and (+)-pentazocine (IC50 = 3. 81 nM) inhibited [3H]norepinephrine release. Moreover, flunarizine (IC50 = 6.34 nM) and (+)-pentazocine (IC50 = 27.26 nM) also antagonized isoproterenol-induced cAMP accumulation. The action of flunarizine and (+)-pentazocine was sensitive to NE-100 antagonism; however, this latter compound partially prevented their effect on [3H]norepinephrine and cAMP accumulation. These findings indicate that flunarizine and (+)-pentazocine interact with ocular sigma1 sites and may prove effective in the control of ocular hypertension.

Administration, Topical↗

Nerve growth factor-induced neurite sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants.

One theory concerning the action of antidepressants relates to the drugs' ability to induce an adaptive plasticity in neurons such as neurite sprouting. Certain antidepressants are known to bind to sigma-1 receptors (Sig-1R) with high affinity. Sig-1R are dynamic endoplasmic reticulum proteins that are highly concentrated at the tip of growth cones in cultured cells. We therefore tested the hypotheses that Sig-1R might participate in the neurite sprouting and that antidepressants with Sig-1R affinity may promote the neuronal sprouting via Sig-1R. The prototypic Sig-1R agonist (+)-pentazocine [(+)PTZ], as well as the Sig-1R-active antidepressants imipramine and fluvoxamine, although ineffective by themselves, were found to enhance the nerve growth factor (NGF)-induced neurite sprouting in PC12 cells in a dose-dependent manner. A Sig-1R antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE100) blocked the enhancements caused by these Sig-1R agonists. In separate experiments, we found that NGF dose and time dependently increased Sig-1R in PC12 cells. Chronic treatment of cells with (+)PTZ, imipramine, or fluvoxamine also increased Sig-1R. These latter results suggested that NGF induces the neurite sprouting by increasing Sig-1R. Indeed, the overexpression of Sig-1R per se in PC12 cells enhanced the NGF-induced neurite sprouting. Furthermore, antisense deoxyoligonucleotides directed against Sig-1R attenuated the NGF-induced neurite sprouting. Thus, when taken together, our results indicate that Sig-1R play an important role in the NGF-induced neurite sprouting and that certain antidepressants may facilitate neuronal sprouting in the brain via Sig-1R.

Animals↗

Regulation of septo-hippocampal activity by 5-hydroxytryptamine(2C) receptors.

It is established that the serotonin system modulates hippocampal functions by regulating neuronal activity of both the medial septum and hippocampus. Inhibition of serotonin neurons leads to theta oscillation of septal neurons and theta wave activity in the hippocampus, indicating a tonic regulation of the septo-hippocampal system by serotonin neurons. Because the postsynaptic 5-hydroxytryptamine (5-HT) receptor subtypes mediating this tonic inhibition have not been identified, a putative role of 5-HT2C receptors has been evaluated in the present study. Extracellular single units were recorded from the medial septum/vertical limb of diagonal band (MS/DBv) and hippocampal CA1 or dentate gyrus with simultaneous hippocampal EEG recordings from anesthetized rats. Intravenous administration of 5-HT2C receptor agonists 1-(3-chlorophenyl)piperazine dihydrochloride (m-CPP) and [S]-2-(chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumarate (Ro 60-0175) dose dependently inhibited firing activity most of the recorded MS/DBv neurons and abolished theta oscillation in all tested MS/DBv and hippocampal neurons. Parallel to inhibition of theta oscillation of MS/DBv neurons, hippocampal EEG activity was desynchronized and its power spectrum was shifted to lower frequencies. The selective 5-HT2C receptor antagonist 6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-oxy)-pyrid-5-yl carbomyl] indoline (SB-242084) [but not the 5-HT2B antagonist 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropyl-pyrimidine (RS-127445) or 5-HT2A antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)-ethyl]-4-piperidinemethanol (MDL-100907)] reversed the action of 5-HT2C receptor agonists. Furthermore, in control rats 5-HT2C receptor antagonists [SB-242084 and 5-methyl-1-(3-pyridil-carbamoyl)-1,2,3,5-tetrahydropyr-rolo[2,3-f]indole hydrochloride (SB-206553)] induced or enhanced theta oscillation in MS/DBv and hippocampal neurons and theta wave activity of the hippocampus. These findings provide evidence for a tonic regulation of the activity and theta oscillation of the septo-hippocampal system via 5-HT2C receptors in the anesthetized rat, indicating that pharmacological interactions with these receptors could modulate various physiological and pathological processes associated with limbic theta activity.

Animals↗

The novel melatonin agonist agomelatine (S20098) is an antagonist at 5-hydroxytryptamine2C receptors, blockade of which enhances the activity of frontocortical dopaminergic and adrenergic pathways.

Agomelatine (S20098) displayed pKi values of 6.4 and 6.2 at native (porcine) and cloned, human (h)5-hydroxytryptamine (5-HT)2C receptors, respectively. It also interacted with h5-HT2B receptors (6.6), whereas it showed low affinity at native (rat)/cloned, human 5-HT2A (<5.0/5.3) and 5-HT1A (<5.0/5.2) receptors, and negligible (<5.0) affinity for other 5-HT receptors. In antibody capture/scintillation proximity assays, agomelatine concentration dependently and competitively abolished h5-HT2C receptor-mediated activation of Gq/11 and Gi3 (pA2 values of 6.0 and 6.1). As measured by [3H]phosphatidylinositol depletion, agomelatine abolished activation of phospholipase C by h5-HT2C (pKB value of 6.1) and h5-HT2B (pKB value of 6.6) receptors. In vivo, it dose dependently blocked induction of penile erections by the 5-HT2C agonists (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine (Ro60,0175) and 1-methyl-2-(5,8,8-trimethyl-8H-3-aza-cyclopenta[a]inden-3-yl) ethylamine (Ro60,0332). Furthermore, agomelatine dose dependently enhanced dialysis levels of dopamine in frontal cortex of freely moving rats, whereas they were unaffected in nucleus accumbens and striatum. Although the electrical activity of ventrotegmental dopaminergic neurons was unaffected agomelatine, it abolished their inhibition by Ro60,0175. Extracellular levels of noradrenaline in frontal cortex were also dose dependently enhanced by agomelatine in parallel with an acceleration in the firing rate of adrenergic cell bodies in the locus coeruleus. These increases in noradrenaline and dopamine levels were unaffected by the selective melatonin antagonist N-[2-(5-ethyl-benzo[b]thien-3-yl)ethyl] acetamide (S22153) and likely flect blockade of 5-HT2C receptors inhibitory to frontocortical dopaminergic and adrenergic pathways. Correspondingly, distinction to agomelatine, melatonin showed negligible activity 5-HT2C receptors and failed to modify the activity of adrenergic and dopaminergic pathways. In conclusion, in contrast to melatonin, agomelatine behaves as an antagonist at 5-HT2B and 5-HT2C receptors: blockade of the latter reinforces frontocortical adrenergic and dopaminergic transmission.

Acetamides↗

Protein kinase C-dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons.

Intracellular calcium concentration ([Ca2+]i) plays a major role in neuronal excitability, especially that triggered by the N-methyl-d-aspartate (NMDA)-sensitive glutamatergic receptor. We have previously shown that sigma1 receptor agonists potentiate NMDA receptor-mediated neuronal activity in the hippocampus and recruit Ca2+-dependent second messenger cascades (e.g., protein kinase C; PKC) in brainstem motor structures. The present study therefore assessed whether the potentiating action of sigma1 agonists on the NMDA response observed in the hippocampus involves the regulation of [Ca2+]i and PKC. For this purpose, [Ca2+]i changes after NMDA receptor activation were monitored in primary cultures of embryonic rat hippocampal pyramidal neurons using microspectrofluorometry of the Ca2+-sensitive indicator Fura-2/acetoxymethyl ester in the presence of sigma1 agonists and PKC inhibitors. We show that successive activations of the sigma1 receptor by 1-min pulses of (+)-benzomorphans or (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784) concomitantly with glutamate time dependently potentiated before inconstantly inhibiting the NMDA receptor-mediated increase of [Ca2+]i, whereas 1,3-di-o-tolyl-guanidine, a mixed sigma1/sigma2 agonist, did not significantly modify the glutamate response. Both potentiation and inhibition were prevented by the selective sigma1 antagonist N,N-dipropyl-2-[4-methoxy-3-(211phenylethoxy) phenyl]-ethylamine monohydrochloride (NE-100). Furthermore, only (+)-benzomorphans could induce [Ca2+]i influx by themselves after a brief pulse of glutamate. A pretreatment with the conventional PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole (Gö-6976) prevented the potentiating effect of (+)-benzomorphans on the glutamate response. Our results provide further support for a general mechanism for the intracellular sigma1 receptor to regulate Ca2+-dependent signal transduction and protein phosphorylation.

Animals↗

Interaction with sigma(1) protein, but not N-methyl-D-aspartate receptor, is involved in the pharmacological activity of donepezil.

In the present study, we examined the interaction of (+/-)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-d-aspartate (NMDA) and sigma(1) receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the l-glutamate or glycine binding sites. The low potency of donepezil (IC(50) = 0.7-3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the sigma(1) receptor with high affinity (K(i) = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5-8, 1999). Thus, we sought to determine whether an interaction with the sigma(1) receptor may occur in vivo under physiologically relevant conditions by evaluating the sigma(1) receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the sigma(1) receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the sigma(1) receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective sigma(1) receptor agonist on these behavioral responses, and an interaction of the drug with the sigma(1) receptor must be considered in its pharmacological actions.

Animals↗

Antiamnesic and neuroprotective effects of donepezil against learning impairments induced in mice by exposure to carbon monoxide gas.

Donepezil is a potent acetylcholinesterase inhibitor that also interacts with the sigma1 receptor, an intracellular neuromodulatory protein. In the present study, we analyzed the antiamnesic and neuroprotective activities of donepezil in a mouse hypoxia model induced by repetitive CO exposure, comparing donepezil's pharmacological profile with other cholinesterase inhibitors tacrine, rivastigmine, and galanthamine, and the reference sigma1 agonist igmesine. CO exposure induced, after 7 days, hippocampal neurodegeneration, analyzed by Cresyl violet staining, and behavioral alterations, measured using spontaneous alternation and passive avoidance responses. When injected 20 min before the behavioral tests, i.e., 7 to 8 days after CO, all drugs showed antiamnesic properties. Preadministration of the sigma1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) blocked only the igmesine and donepezil effects. The neuroprotective activity of the drugs was tested by injection 20 min before the first CO exposure (preinsult protection) or by injection 1 h after the last CO exposure (postinsult protection). All drugs alleviated the hypoxia-induced neurodegeneration and behavioral impairments when injected before CO exposure. Preadministration of BD1047 blocked both the igmesine and donepezil effects. However, when injected after CO exposure, only igmesine and donepezil induced effective neuroprotection, and the morphological and behavioral effects were BD1047-sensitive. These results showed that donepezil is a potent antiamnesic and neuroprotective compound against the neurodegeneration induced by excitotoxic insult, and its pharmacological actions as both an acetylcholinesterase inhibitor and sigma1 receptor agonist contribute to its marked efficacy. In particular, the drug is a more potent postinsult protecting agent compared with more selective cholinesterase inhibitors.

Amnesia↗