The interaction of standard oxime reactivators with hemicholinium-3 sensitive choline carriers.

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.

Novel acetylcholinesterase reactivator K112 and its cholinergic properties.

The oxime reactivator K112 is a member of the new group of xylene linker-containing AChE reactivators. Its cholinergic properties could be of importance at OP poisoning and are not related to the AChE reactivation that has been studied. It has been found that, despite of reactivating potency, this compound has additional effects. These cholinergic effects include a weak inhibition of AChE (IC(50)=43.8 ± 4.88 µM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 µM) and finally, the inhibition of HACU (68.4 ± 9.9%), a key regulatory step in the synthesis of ACh. The inhibition of the binding of (3H)-HC-3 (64.7 ± 4.7%) and the influence on the membrane fluidity have also been observed. Blocking properties of K112 on the muscarinic receptors have been revealed in the in vitro experiment (rat urinary bladder) and in the in vivo experiment (rat heart BPM) as well. All these cholinergic properties could significantly contribute to the antidotal effect of K112 at the poisoning by the organophosphates.

Sex-dependent actions of amyloid beta peptides on hippocampal choline carriers of postnatal rats.

It is suggested that amyloid beta peptides (Abeta) play a role in the pathogenesis of Alzheimer disease but their physiological function is still unknown. However, low pM-nM concentrations mediate a hypofunction of a basal forebrain cholinergic system without marked signs of neurotoxicity. In this study, we compared in vitro effects of soluble nonaggregated human Abeta 1-40 and 1-42 either on synaptosomal hemicholinium-3 sensitive choline carriers or on membrane fluidity in hippocampi of male and female Wistar rats aged 7 and 14 days or 2-3 months. The results indicate age- and sex-dependent effects mediated by peptides at nM concentrations but no significant differences between both fragments. Namely, opposite actions were observed in 14-day (the increase in the choline uptake and membrane fluidity) when compared to 7-day old and adult males (the mild drops). Lineweaver-Burk plot analysis revealed that the enhancement of the high-affinity choline transport in 14-day old males occurs via alterations in K (M )and the change was accompanied by a mild increase in the specific binding of [3H]hemicholinium-3. On the other hand, no age-dependent differences were found in females. Rat Abeta 1-40 mediated similar effects on 14-day old rats as the corresponding human fragment. Moreover, higher levels of soluble peptides were detected in immature when compared to mature male brains by means of competitive ELISA. Our study indicates that Abeta could play a role in postnatal sexual differentiation of hippocampal cholinergic system.

Exposure of postnatal rats to a static magnetic field of 0.14 T influences functional laterality of the hippocampal high-affinity choline uptake system in adulthood; in vitro test with magnetic nanoparticles.

Our previous experiments indicated an age- and sex-dependent functional lateralization of a high-affinity choline uptake system in hippocampi of Wistar rats. The system is connected with acetylcholine synthesis and also plays a role in spatial navigation. The current study demonstrates that a single in vivo exposure of 7- or 14-day-old males to a static magnetic field of 0.14 T for 60-120 min evokes asymmetric alterations in the activity of carriers in adulthood. Namely, the negative field (antiparallel orientation with a vertical component of the geomagnetic field) mediated a more marked decrease in the right hippocampus. The positive field (parallel orientation) was ineffective. Moreover, differences between the carriers from the right and the left hippocampi were observed on synaptosomes pretreated with superparamagnetic nanoparticles and exposed for 30 min in vitro. The positive field enhanced more markedly the activity of carriers from the right hippocampus, the negative that from the left hippocampus, on the contrary. Our results demonstrate functionally teratogenic risks of the alterations in the orientation of the strong static magnetic field for postnatal brain development and suggest functional specialization of both hippocampi in rats. Choline carriers could be involved as secondary receptors in magnetoreception through direct effects of geomagnetic field on intracellular magnetite crystals and nanoparticles applied in vivo should be a useful tool to evaluate magnetoreception in future research.

Brain maldevelopment and neurobehavioural deviations in adult rats treated neonatally with indomethacin.

The risk of neurodevelopmental toxicity was studied in indomethacin (INDO), an inhibitor of prostaglandin synthesis, which is used in at-risk neonates to prevent the consequences of brain intraventricular haemorrhage or to accelerate the closure of patent ductus arteriosus. Model experiments were carried out in rats of the Wistar strain and Konárovice breed. The drug dose (2 mg/kg, s.c.) was applied to rat pups either once or twice in the following way: (1) on postnatal day 4 (PD:4) or postnatal days 4 and 5 (PD:4-5), i.e. model of brain ontogenic developmental stage in human fetus/preterm neonate of 7-month-gestational age; (2) on postnatal day 9 (PD:9) or postnatal days 9-10 (PD:9-10), i.e. model of brain ontogenic stage in full-term human newborn. The rats were followed up during development (body weight, maturation) until adulthood (age 3-9 months) using tests of behaviour (open field, social memory), nociception (tail flick, plantar test), reproduction and brain neurobiological analysis. The results were evaluated by comparison of litter-mates: treated vs control. No differences between INDO and controls were found in developmental landmarks, adult social memory or reproduction. The pattern of behavioural and neuroendocrine deviations in adult animals was dependent on the ontogenic stage exposed to drug insult. INDO rats of the groups PD:4 and PD:4-5 revealed depression of open field motor activity and emotional reactivity, and higher pituitary weight with lower TSH content. On the other hand, deviations in adult INDO groups PD:9 and PD:9-10 were characterized by pain hypersensitivity, lower pituitary weight with unchanged TSH content and deficit of monoamine transmission in the hypothalamus.

Amyloid beta peptide 1-40 and the function of rat hippocampal hemicholinium-3 sensitive choline carriers: effects of a proteolytic degradation in vitro.

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.

Neuro-immuno-teratogenicity of drugs used in neonatal pharmacotherapy in relation to the ontogenic stage at the time of their administration.

The risk of functional teratogenicity of two drugs used in neonatal pharmacotherapy was studied: indomethacin (INDO) and dexamethasone (DEX). Model experiments were carried out in Wistar strain rats, breed Konárovice, which received single subcutaneous drug injection (INDO 2 mg/kg, DEX 1 mg/kg) on postnatal day 4 (PD:4; model of human fetus/preterm newborn of 6-7-month-gestational age) or on postnatal day 9 (PD:9; model of full-term human neonate). The rats were followed up during development (body weight, maturation) till late adulthood (age 6-8 months) using tests of cognition, immune reactivity and biochemical brain analysis. The results evaluated by comparing treated and control litter-mates indicated that the functional teratogenic risk was significantly higher in DEX than in INDO. DEX-rats revealed disorganization of developmental processes: retardation of body growth, but acceleration of sensory development (pinna and eye opening), retarded male sexual maturation. Adult DEX-rats (age 6 months) of both series (PD:4, PD:9) had deficit of short-term memory (social recognition test). Disturbances of immune reactivity (decrease of humoral and rise of cell-mediated immune response) appeared both in adult INDO and DEX-rats (age 7 months), but only in the PD:9 series i.e. when the drugs were administered at a higher stage of the ontogenic development simulating neonatal period in humans. This finding may be warning from the clinical point of view for the neonatological practice.

Effects of K+-depolarization, arachidonic acid, ethanol, and aging on the high-affinity choline transport in rat hippocampus.

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.

Effects of aging and dementia on the levels of thiobarbituric-acid-reactive products stimulated by L-glutamic acid in human autopsy and biopsy brain tissue.

Basal and stimulated (by L-glutamic acid, GA) levels of thiobarbituric-acid-reactive products were estimated in the brain tissue (hippocampus, cortex and cerebellum) from autopsy samples of people with Alzheimer disease (AD), multi-infarct dementia (MID) and from nondemented control patients. The experiment was also performed on biopsy brain tissue (cortex) of nondemented controls. The biopsy brain tissue influenced by normal aging in vivo showed a limited susceptibility to undergo lipid peroxidation stimulated by GA in vitro in comparison with the younger tissue. A significant decrease in the ratio of stimulated to basal levels was found in the cerebellum of MID patients in comparison with nondemented controls and AD patients.

In vitro effect of Ginkgo biloba extract (EGb 761) on the activity of presynaptic cholinergic nerve terminals in rat hippocampus.

The effects of Ginkgo biloba extract (EGb) applied in vitro to hippocampal synaptosomes from young Wistar rats on the specific binding of [3H]hemicholinium-3 ([3H]HCh-3), high-affinity choline uptake (HACU) and activity of Na+,K(+)-ATPase were examined. EGb at a concentration of 100 micrograms/ml markedly elevated the specific binding of [3H]HCh-3 (to 306%) and moderately elevated HACU values (to 115%). Scatchard analysis revealed an increase in the Bmax for [3H]HCh-3 binding. Lineweaver-Burk analysis an increase in the Vmax for choline uptake. No marked changes in the activity of the sodium pump were discovered. EGb was not able to influence the specific 'second messenger' effect of arachidonic acid.

In vitro effects of arachidonic and L-glutamic acids on the high-affinity choline transport in rat hippocampus.

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.

Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue.

Studies of age-related changes based on the thiobarbituric acid (TBA) test appear to be inconsistent and contradictory. In our work, real basal (hypothetical, corrected to the zero concentration of atmospheric oxygen), basal (atmospheric oxygen-stimulated) and stimulated (L-glutamic and hydrochloric acids) levels of TBA-reactive products (TBARP) were estimated in the brain (hippocampus--HPC, cortex--COR, cerebellum--CRB) from young (3 months) and old (28 months) male and female Wistar rats. The values of basal levels of TBARP were different in young (HPC > COR > CRB) as well as in old animals (COR > CRB > HPC). Thus, the process of aging caused a significant decrease in the HPC (to 72%) and no change in the COR and CRB. Levels stimulated by L-glutamic acid were significantly decreased (COR, CRB) and unchanged (HPC) in old compared to young animals. The real basal levels estimated by regression analysis seem to be higher in the brain tissue damaged by aging. We discuss the usefulness of the TBA test applied to aged tissue, the effect of acidosis, the effectivity of L-glutamic acid to generate free radicals and the differences between individual brain areas.

[Perinatal pharmacotherapy and the risk of functional teratogenic defects]. / Perinatální farmakoterapie a riziko funkcne teratogenních defektu.

Perinatal period, which is characterized by intensive histogenesis and cytodifferentiation of the already shaped organs, is a highly vulnerable phase for fetal/neonatal brain and immune system-organs with high similarity in receptor equipment. Even fine deviations in the programmed developmental processes induced by drugs initiate disorders in the formation of neural network, cytoarchitectonics and receptor-transmitter communication systems. This pathology is not evident at birth, but forms the basis for various functional defects of neuro-psycho-immunocompetence which become apparent gradually during further maturation or even in adulthood. Clinical recognition of such functional teratogenic action of drugs is hampered by the long time interval (upto decades) between the drug administration and its consequences, making the identification of causal relations very difficult. Consequently, experimental research is necessary under the precondition of adequate animal models with sufficient validity for the extrapolation on human level. The authors suggest the principles of such approach using drug application in neonatal rats with lifelong follow-up of behaviour, immune reactivity and brain biochemical analysis. The evaluation of functional teratogenic risk in three drugs used in the treatment of risk pregnancies and risk neonates (dexamethazone, fenoterol, diazepam) is presented.

(3H)hemicholinium-3 binding sites in postmortem brains of human patients with Alzheimer's disease and multi-infarct dementia.

(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.

Early postnatal diazepam treatment of rats and neuroimmunocompetence in adulthood and senescence.

Functional teratogenic risk of perinatal diazepam (D) treatment was studied in animal model experiments using early postnatal D administration in rats (single dose of 10 mg/kg sc in 7-day-old pups) and long-term follow-up till the age of 18 months with monitoring of behavior, reproductive functions, brain biochemical variables, and immune system reactivity. Behavioral tests carried out at the age of 6, 12, and 18 months indicated higher emotionality and deviations of novelty reaction in D rats in comparison with controls, and these differences decreased with aging. However, no deficits were found in memory testing. D rats revealed some transitional alterations of monoamine neurotransmission in the hypothalamus (5-HT) and striatum (DA) and minor defects in reproductive functions (irregular estrous cycles in females). Significant depression of immune response in D rats persisting for the whole life may be considered as a serious risk of neonatal D treatment.

Comparison of the effects of aging in vivo and of oxygen free radicals in vitro on high-affinity choline uptake and hemicholinium-3 binding in the rat brain.

The effects of aging in vivo (Wistar rats aged 3-26 months) and of an oxygen free-radical generating system in vitro (Fe(2+)/ascorbic acid) on high-affinity choline uptake in the hippocampus and on (3H)hemicholinium-3 binding sites in the cortex and hippocampus are compared. The high-affinity choline transport system was found to be more damaged than the low-affinity system during aging (Na(+)-dependent part of the uptake drops to 76%: Na(+)-independent part increases to 120%). The decrease in high-affinity choline uptake values is probably more influenced by the impairment of correct function of carriers (the fall in the turnover rate of each carrier) than by a decrease in the number of transport sites (no change of the density of the carriers in the hippocampus and cortex). The causes of the defect in high-affinity choline transport during aging are discussed.

Effect of postmortem storage on the [3H]hemicholinium-3 binding site in the rat brain. Preliminary study for investigations of human patients with Alzheimer's disease.

The effect of postmortem storage at room temperature (24-26 degrees C, 0-4 h) and cold-room temperature (4 degrees C, (0-24 h) on the [(3)H]hemicholinium-3 binding sites in the brain hippocampus, cortex and cerebellum of 3-month-old Wistar rats was studied. A slow decrease in the density of the binding sites was observed at both temperatures, which was best fit by a linear model common for all three brain regions. No systematic alterations of the affinity of the binding sites for hemicholinium-3 were found. The values obtained from experiments with animals were compared with the values measured in the frontal cortex of old men. Approaches to the evaluation of data obtained from postmortem samples of human brain tissue of patients with Alzheimer's disease are proposed.

High-affinity choline uptake and muscarinic receptors in rat brain during aging.

The aim of this study was to determine the effect of aging on the high-affinity choline uptake (HACU) and the muscarinic acetylcholine receptors (mAChR) in the brain of Wistar male rats and to define more precisely the steps of the brain cholinergic degeneration in the course of the whole animal life. In 24-month-old rats, a substantial decrease in HACU values in the hippocampus (to 65-75%) and in the density of mAChR in the cortex (to 76%) was found in comparison with 3-month-old controls. The interaction of muscarinic receptor antagonist pirenzepine with [(3)H]QNB indicated a decrease in low-affinity sites (M(2)) in 24-month-old rats. The first slight changes due to aging manifested themselves by the reduction in HACU values very early (between 6 and 12 months), the decrease of the muscarinic receptor density was observed in a later stage (19-month-old animals). Regression analysis indicated considerable dependence of the HACU values on age (the correlation coefficient r = -0.689, the slope b = -0.279 pmol/4 min per mg(prot) per month, P < 0.001) while the density of muscarinic receptors does not correlate with age so markedly (r = -0.415, b = -6.316 fmol/mg(prot) per month, P = 0.018).

The assessment of the behavioural teratogenic risk of the tocolytic drug phenoterol.

Phenoterol, a tocolytic drug widely used in cases of imminent preterm labour for disruption of uterine contractions, was studied for potential harmful impact on the developing fetal brain which is just going through the vulnerable period of accelerated histogenesis and cytodifferentiation. As the developmental stage of human fetus brain in late pregnancy closely resembles the ontogenetic phase of the rat brain in the early postnatal period, the model experiments were carried out using the drug administration in the neonate rat and life-long comprehensive follow-up of sequels in behaviour, reproductive functions and brain biochemical parameters. No deviations were found when phenoterol was administered in the dose 1 mg/kg/day s.c. on the postnatal days 6-9 (clinically relevant dosage). Several minor aberrations were observed after 10 times higher dose (10 mg/kg/day s.c. on postnatal days 5-7) in preweaning period (acceleration of somatic development) and in adulthood i.e. at the age of 2-7 months (higher score of emotionality). The inferiority of phenoterol treated rats became apparent only with the onset of senescence (age 11-14 months) when lower score of memory acquisition was ascertained joined with an increase of lipid peroxidation in the brain cortex.