Organophosphorus poisoning manifests as a cholinergic syndrome due to an inhibition of acetylcholinesterase. It is treated symptomatically by anticholinergics and oxime reactivators are used as causal antidotes. Reactivators possess a complex mechanism of action and interact at various levels of the cholinergic transmission. The aim of this study was to investigate the effect of standard oxime reactivators (HI-6, obidoxime, trimedoxime, methoxime and pralidoxime) on the hemicholinium-3 sensitive carriers, which are involved in the high-affinity choline uptake (HACU) transport, a key regulatory step in the synthesis of acetylcholine. The activity of the carriers was estimated in vitro on hippocampal synaptosomes using the substrate (3H)-choline and the competitive inhibitor (3H)-hemicholinium-3. Furthermore, the effect of the reactivators on the fluidity of hippocampal membranes was assessed. All tested compounds, except methoxime, showed an acute inhibitory effect on the carriers, however, only at μM concentrations. Trimedoxime showed the highest potency to inhibit HACU among all tested compounds (I(max) 62%, IC(50)=3 μM). All compounds, except HI-6, influenced also a membrane fluidity in the region of the hydrophilic heads of phospholipid bilayer, nevertheless, only methoxime was able to penetrate more deeply into the hydrocarbon core. We suggest that the direct interaction of oxime reactivators with the carrier protein (HI-6 and trimedoxime) and/or the changes in carrier conformation mediated by alterations in membrane fluidity (trimedoxime, obidoxime and pralidoxime) could occur here. The influence of reactivators on the carriers could be unfavorable in the case of their prolonged administration in vivo. From this point of view, the application of methoxime appears to be the best.

• MeSH
• acetylcholinesterasa metabolismus   MeSH
• anizotropie MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• cholin metabolismus   MeSH
• fluidita membrány účinky léků   fyziologie   MeSH
• hemicholinium 3 metabolismus   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• membránové transportní proteiny účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• reaktivátory cholinesterasy metabolismus   farmakologie   MeSH
• synaptozomy účinky léků   metabolismus   MeSH
• tritium MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• cholin MeSH
• choline transporter MeSH Prohlížeč
• hemicholinium 3 MeSH
• membránové transportní proteiny MeSH
• reaktivátory cholinesterasy MeSH
• tritium MeSH

Brains of Alzheimer disease patients in early stages of dementia contain an increased 24(S)-hydroxycholesterol (cerebrosterol)/cholesterol ratio when compared to controls. In this study, effects of amyloid beta peptides and of racemic 24-hydroxycholesterol were evaluated in vitro on undepleted or cholesterol-depleted hippocampal synaptosomes of young and old rats via a high-affinity choline transport and membrane anisotropy measurements. Depletion of membrane cholesterol decreased the transport of [3H]choline, increased the specific binding of [3H]hemicholinium-3 and decreased membrane anisotropy. However, less alterations were found in old when compared to young brains. 500 nM nonaggregated peptides were ineffective but aggregated fragment 1-42 evoked marked drops in the transport and anisotropy values on depleted synaptosomes. 50 microM 24-hydroxycholesterol inhibited choline transport on depleted synaptosomes but it did not influence membrane anisotropy. Peptides eliminated the actions of oxysterol on choline carriers in young but not in old rats. On the other hand, oxysterol eliminated the effects of peptides on membrane anisotropy. Our study suggests a possible role of membrane cholesterol in the regulation of choline carriers and supports data reporting a protective role of membrane cholesterol against toxic effects of amyloid beta peptides. Moreover, via Raman spectroscopy we demonstrate for the first time that peptides form a complex with 24-hydroxycholesterol.

• MeSH
• amyloidní beta-protein chemie   toxicita   MeSH
• anizotropie MeSH
• cholinergní látky farmakologie   MeSH
• difenylhexatrien MeSH
• fluidita membrány účinky léků   MeSH
• fluorescenční barviva MeSH
• hemicholinium 3 farmakologie   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• hydroxycholesteroly chemie   toxicita   MeSH
• interpretace statistických dat MeSH
• krysa rodu Rattus MeSH
• membránové transportní proteiny metabolismus   MeSH
• membrány účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• proteiny přenášející neurotransmitery metabolismus   MeSH
• Ramanova spektroskopie MeSH
• stárnutí fyziologie   MeSH
• synaptozomy účinky léků   metabolismus   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 24-hydroxycholesterol MeSH Prohlížeč
• amyloidní beta-protein MeSH
• choline transporter MeSH Prohlížeč
• cholinergní látky MeSH
• difenylhexatrien MeSH
• fluorescenční barviva MeSH
• hemicholinium 3 MeSH
• hydroxycholesteroly MeSH
• membránové transportní proteiny MeSH
• proteiny přenášející neurotransmitery MeSH

We investigated the influence of the amyloid-beta-peptide(1-42) on hemicholinum-3-sensitive high-affinity choline uptake in NG108-15 cells. RT-PCR analysis revealed the presence of mRNA for a choline transporter-like protein but not for cholinergic high-affinity choline transporter. Differentiation of cells increased both hemicholinum-3-sensitive choline uptake and high-affinity hemicholinium-3 binding. This transport was not influenced by tenfold excess of carnitine. Continuous presence of submicromolar concentrations of amyloid-beta-peptide(1-42) during differentiation resulted in a decrease of both choline uptake and hemicholinium-3 binding. These effects were not present when amyloid-beta-peptide(1-42) was added 5 min prior to measurements. Neither differentiation nor amyloid-beta-peptide(1-42) treatment changed levels of choline transporter-like protein mRNA. Protein kinase C inhibition by staurosporine or its inactivation by continuous presence of tetradecanoyl phorbol acetate prevented the inhibitory effect of amyloid-beta-peptide(1-42) treatment on choline uptake. Activation of protein kinase C by tetradecanoyl phorbol acetate during measurement had inhibitory effect on choline uptake in control but not amyloid-beta-peptide(1-42)-treated cells. The concentration of amyloid-beta-peptide(1-42) maximally effective on hemicholinium-3-sensitive choline uptake had no effect on cell growth, oxidative activity, membrane integrity, number of surface muscarinic receptors, caspase-3 and -8 activities, or uptake of deoxyglucose. Results demonstrate that long-term treatment with non-toxic concentrations of amyloid-beta-peptide(1-42) downregulates choline uptake presumably mediated by a choline transporter-like protein through activation of protein kinase C signaling. The decrease of choline uptake may have relevance to the pathogenesis of Alzheimer's disease.

• MeSH
• amyloidní beta-protein aplikace a dávkování   fyziologie   MeSH
• buněčná diferenciace fyziologie   MeSH
• buněčné linie MeSH
• cholinergní látky farmakologie   MeSH
• hemicholinium 3 farmakologie   MeSH
• krysa rodu Rattus MeSH
• membránové transportní proteiny účinky léků   genetika   metabolismus   MeSH
• messenger RNA analýza   MeSH
• myši MeSH
• neurony účinky léků   enzymologie   metabolismus   MeSH
• peptidové fragmenty aplikace a dávkování   fyziologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteinkinasa C metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloid beta-protein (1-42) MeSH Prohlížeč
• amyloidní beta-protein MeSH
• choline transporter MeSH Prohlížeč
• cholinergní látky MeSH
• hemicholinium 3 MeSH
• membránové transportní proteiny MeSH
• messenger RNA MeSH
• peptidové fragmenty MeSH
• proteinkinasa C MeSH

Vulnerability of hippocampal hemicholinium-3 (HC-3)-sensitive carriers to ethanol was evaluated in vitro during rat postnatal development. The high-affinity uptake of [3H]choline (HACU) and the specific binding of [3H]HC-3 were measured on synaptosomes from 7-, 14-, and 60-day-and 3-month-old male and female Wistar rats. Marked increases of basal (between 7 and 60 days of age) and of stimulated HACU levels via K(+)-depolarization (between 14 days and 3 months) but only a mild elevation in [3H]HC-3 binding (between 7 days and 3 months) associated with alterations in the binding site number were found. On the mature tissue, ethanol at high concentrations (5%) moderately inhibited the choline transport under basal conditions but totally eliminated depolarization effects. However, both age- and sex-dependent alterations in basal HACU mediated by high or low pharmacologically relevant alcohol concentrations (50-100 mM) were observed in the immature tissue. Namely, the dose- and incubation time-dependent inhibition of HACU associated with changes in the transport velocity was found in postnatal male but not female tissue. [3H]HC-3 binding site was not markedly sensitive to ethanol actions. Anisotropy measurements in the region of the hydrophilic heads of phospholipid bilayers and in the membrane hydrocarbon core indicated penetration of 100 mM ethanol to immature female but not male tissue. Our results suggest the noncompetitive binding of alcohol to choline carriers from immature male tissue and correspond with data reporting significant sexual dimorphism of postnatal hippocampal neurons. The direct effects of ethanol on male choline carriers can contribute to the inhibition of acetylcholine synthesis and to sex-dependent neurotoxic effects of alcohol applied in vivo during early and late postnatal period.

• MeSH
• anizotropie MeSH
• biologický transport účinky léků   MeSH
• časové faktory MeSH
• cholin farmakokinetika   MeSH
• cholinergní látky metabolismus   MeSH
• ethanol aplikace a dávkování   farmakologie   MeSH
• hemicholinium 3 metabolismus   MeSH
• hipokampus metabolismus   MeSH
• kompetitivní vazba účinky léků   MeSH
• krysa rodu Rattus MeSH
• látky tlumící činnost CNS aplikace a dávkování   farmakologie   MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• membránové transportní proteiny účinky léků   metabolismus   MeSH
• novorozená zvířata růst a vývoj   metabolismus   MeSH
• pohlavní dimorfismus * MeSH
• potkani Wistar MeSH
• stárnutí metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholin MeSH
• choline transporter MeSH Prohlížeč
• cholinergní látky MeSH
• ethanol MeSH
• hemicholinium 3 MeSH
• látky tlumící činnost CNS MeSH
• lipidové dvojvrstvy MeSH
• membránové transportní proteiny MeSH

Effects of amyloid beta peptide 1-40 (Abeta) and of plant cysteine proteases bromelain and papain on the high-affinity uptake of choline (HACU) and the specific binding of [3H]hemicholinium-3 ([3H]HC-3) have been investigated on hippocampal synaptosomes from young adult male Wistar rats under basal and stimulated conditions (55 mM KCl). Depolarization increased significantly the HACU levels (the changes were predominantly in Vmax) and mildly the [3H]HC-3 binding (the changes especially in K(D)). Nonaggregated Abeta at low nM concentrations suppressed the depolarization effects but was ineffective under basal conditions during a short-term incubation. Higher microM concentrations decreased the HACU and binding under basal conditions in a time-dependent manner. The binding changes were firstly associated with alterations in K(D) and secondarily were accompanied also by a drop in Bmax. The results suggest that Abeta directly influences high-affinity carriers, inhibits their transport activity and enhances their sensitivity to proteoLytic cleavage. Stimulation increases the sensitivity of carriers to the interaction with Abeta.

• MeSH
• amyloidní beta-protein farmakologie   MeSH
• cholin metabolismus   MeSH
• hemicholinium 3 farmakologie   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• hydrolýza MeSH
• krysa rodu Rattus MeSH
• peptidové fragmenty farmakologie   MeSH
• potkani Wistar MeSH
• transportní proteiny účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amyloid beta-protein (1-40) MeSH Prohlížeč
• amyloidní beta-protein MeSH
• cholin MeSH
• hemicholinium 3 MeSH
• peptidové fragmenty MeSH
• transportní proteiny MeSH

The Na+-dependent high-affinity choline uptake (HACU) transport and the [3H]hemicholinium-3 ([3H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3-6 months) and old (22 months) Wistar rats. In vitro effects of 100-300 microM arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [3H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K+-depolarization effect on HACU and the decreased influence of AA on [3H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.

• MeSH
• biologický transport MeSH
• cholin metabolismus   MeSH
• draslík farmakologie   MeSH
• ethanol farmakologie   MeSH
• hemicholinium 3 metabolismus   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyselina arachidonová farmakologie   MeSH
• potkani Wistar MeSH
• stárnutí metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholin MeSH
• draslík MeSH
• ethanol MeSH
• hemicholinium 3 MeSH
• kyselina arachidonová MeSH

A second messenger role for arachidonic acid (AA) in the regulation of the high-affinity choline uptake (HACU) was suggested. It was reported that micromolar concentrations of AA applied in vitro decreased the HACU values and increased the specific binding of [3H]hemicholinium-3 ([3H]HCh-3). It was published that L-glutamic acid (GA) applied in vivo produced a fall in the HACU values. In addition, GA liberates free AA. In this study, an ability of GA to influence in vitro the activity of presynaptic cholinergic nerve terminals via its effect on the release of AA is investigated in hippocampal synaptosomes of young Wistar rats. Millimolar concentrations of GA decrease both the high- and low-affinity choline uptake, the specific as well as nonspecific binding of [3H]HCh-3 and the activity of Na+, K(+)-ATPase. Kinetic analysis (Lineweaver-Burk and Scatchard plots) reveals a change in Vmax and Bmax, but not in KM and KD. It appears very likely that under normal conditions GA applied in vitro is not able to change markedly the choline transport via its effect on the release of AA. Results confirm the hypothesis about an indirect inhibitory role for glutamatergic receptors on cholinergic cells.

• MeSH
• biologický transport účinky léků   MeSH
• cholin farmakokinetika   MeSH
• hemicholinium 3 metabolismus   MeSH
• hipokampus cytologie   účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyselina arachidonová metabolismus   farmakologie   MeSH
• kyselina glutamová farmakologie   MeSH
• lineární modely MeSH
• neurony účinky léků   metabolismus   MeSH
• potkani Wistar MeSH
• techniky in vitro MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholin MeSH
• hemicholinium 3 MeSH
• kyselina arachidonová MeSH
• kyselina glutamová MeSH

(3H)Hemicholinium-3 ((3H)HCh-3), a potent, selective, and competitive inhibitor of the high-affinity choline uptake process was used for the detection of high-affinity choline carriers in the hippocampus (gyrus parahippocampalis), neocortex (gyrus frontalis medius), and cerebellum (lobulus semilunaris inferior) in autopsy samples of people with Alzheimer's disease, multi-infarct dementia and from other psychiatric and nonpsychiatric patients. The effect of postmortem delay was eliminated by means of the cerebellum used as an individual standard. The density of (3H)HCh-3 binding sites was decreased in the hippocampus and neocortex from individuals with multi-infarct dementia and unchanged in the brain tissue from people with Alzheimer's disease in comparison with control patients. No changes in dissociation constants were found. In Alzheimer's disease, high-affinity choline transport appears to be reduced by a dysfunction of cholinergic neuronal membrane rather than by a significant decrease in the number of presynaptic cholinergic nerve terminals. Results provide evidence of a decrease in the number of nerve endings in people with multi-infarct dementia and suggest different vulnerability of particular brain areas to vascular disorders.

• MeSH
• Alzheimerova nemoc metabolismus   MeSH
• analýza rozptylu MeSH
• cholin metabolismus   MeSH
• hemicholinium 3 metabolismus   MeSH
• kompetitivní vazba MeSH
• lidé středního věku MeSH
• lidé MeSH
• multiinfarktová demence metabolismus   MeSH
• pitva MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• tritium MeSH
• vazebná místa MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholin MeSH
• hemicholinium 3 MeSH
• tritium MeSH

The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Dolezal and Tucek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.

• MeSH
• 4-aminopyridin farmakologie   MeSH
• acetylcholin metabolismus   MeSH
• cholin metabolismus   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosforylace MeSH
• fosforylcholin metabolismus   MeSH
• hemicholinium 3 farmakologie   MeSH
• krysa rodu Rattus MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• nervová zakončení účinky léků   MeSH
• paraoxon farmakologie   MeSH
• potkani Wistar MeSH
• takrin farmakologie   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 4-aminopyridin MeSH
• acetylcholin MeSH
• cholin MeSH
• fosfatidylcholiny MeSH
• fosforylcholin MeSH
• hemicholinium 3 MeSH
• paraoxon MeSH
• takrin MeSH

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low-potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under "resting" conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium-3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium-3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

• MeSH
• 4-aminopyridin farmakologie   MeSH
• acetylcholin metabolismus   MeSH
• cholin metabolismus   MeSH
• cholinesterasy metabolismus   MeSH
• hemicholinium 3 farmakologie   MeSH
• histologické techniky MeSH
• krysa rodu Rattus MeSH
• mozková kůra metabolismus   MeSH
• odpočinek MeSH
• takrin analogy a deriváty   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 4-aminopyridin MeSH
• 7-methoxytacrine MeSH Prohlížeč
• acetylcholin MeSH
• cholin MeSH
• cholinesterasy MeSH
• hemicholinium 3 MeSH
• takrin MeSH