Inhibition of Na(+),K(+)-ATPase in the hypothalamus, pons and cerebellum of the offspring rat due to experimentally-induced maternal hypothyroidism.

Neurodevelopment is known to be particularly susceptible to thyroid hormone insufficiency and can result in extensive structural and functional deficits within the central nervous system (CNS), subsequently leading to the establishment of cognitive impairment and neuropsychiatric symptomatology. The current study evaluated the effects of gestational and/or lactational maternal exposure to propylthiouracil (PTU)-induced hypothyroidism (as a suggestive multilevel experimental approach to the study of hypothyroidism-induced changes that has been developed and characterized by the authors) on crucial brain enzyme activities of 21-day-old Wistar rat offspring in a CNS region-specific manner. The activities of acetylcholinesterase (AChE), Na(+),K(+)-ATPase and Mg(2+)-ATPase in the offspring hypothalamus, cerebellum and pons were assessed. The study demonstrated that maternal exposure to PTU (0.05% w/v in the drinking water) during the critical periods of neurodevelopment can result in an inhibition of hypothalamic, pontine and cerebellar Na(+),K(+)-ATPase; a major marker of neuronal excitability and metabolic energy production as well as an important regulator of important systems of neurotransmission. On the other hand, no significant changes in the activities of the herein offspring CNS regions' AChE and Mg(2+)-ATPase were recorded. The observed Na(+),K(+)-ATPase inhibition: (i) is region-specific (and non-detectable in whole brain homogenetes), (ii) could constitute a central event in the pathophysiology of clinically-relevant hypothyroidism-associated developmental neurotoxicity, (iii) occurs under all examined experimental schemes, and (iv) certainly deserves further clarification at a molecular and histopathological level. As these findings are analyzed and compared to the available literature, they also underline the need for the adoption and further study of Na(+),K(+)-ATPase activity as a consistent neurochemical marker within the context of a systematic comparative study of existing (and novel) simulation approaches to congenital and early age hypothyroidism.

The effects of rivastigmine plus selegiline on brain acetylcholinesterase, (Na, K)-, Mg-ATPase activities, antioxidant status, and learning performance of aged rats.

UNLABELLED: We investigated the effects of rivastigmine (a cholinesterase inhibitor) and selegiline ((-)deprenyl, an irreversible inhibitor of monoamineoxidase-B), alone and in combination, on brain acetylcholinesterase (AChE), (Na(+), K(+))-, Mg(2+)-ATPase activities, total antioxidant status (TAS), and learning performance, after long-term drug administration in aged male rats. The possible relationship between the biochemical and behavioral parameters was evaluated. METHODS: Aged rats were treated (for 36 days) with rivastigmine (0.3 mg/kg rat/day ip), selegiline (0.25 mg/kg rat/day im), rivastigmine plus selegiline in the same doses and way of administration as separately. Aged and adult control groups received NaCl 0.9% 0.5 ml ip. RESULTS: TAS was lower in aged than in adult rats, rivastigmine alone does not affect TAS, decreases AChE activity, increases (Na(+), K(+))-ATPase and Mg(2+)-ATPase activity of aged rat brain and improves cognitive performance. Selegiline alone decreases free radical production and increases AChE activity and (Na(+), K(+))-ATPase activity, improving cognitive performance as well. In the combination: rivastigmine seems to cancel selegiline action on TAS and AChE activity, while it has additive effect on (Na(+), K(+))-ATPase activity. In the case of Mg(2+)-ATPase selegiline appears to attenuate rivastigmine activity. No statistically significant difference was observed in the cognitive performance. CONCLUSION: Reduced TAS, AChE activity and learning performance was observed in old rats. Both rivastigmine and selesiline alone improved performance, although they influenced the biochemical parameters in a different way. The combination of the two drugs did not affect learning performance.

Equilibrated diet restores the effects of early age choline-deficient feeding on rat brain antioxidant status and enzyme activities: the role of homocysteine, L-phenylalanine and L-alanine.

Choline is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions, that affect the developing brain. The aim of this study was to: (a)examine the effects of early age choline deficient diet (CDD) administration on the total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na(+),K(+))-ATPase and Mg(2+)-ATPase in the rat brain, (b)investigate the effect of feeding restoration into an equilibrated diet on the above parameters, and (c)study the role of homocysteine (Hcy), L: -phenylalanine (Phe) and L: -alanine (Ala) in certain of the above effects. Male and female Wistar rats were continuously kept off choline (Ch) during their gestational period of life, as well as during the first 6 weeks of their post-gestational life. The animals were sacrificed by decapitation and their whole brains were rapidly removed and homogenated. Their enzyme activities were measured spectrophotometrically. Moreover, in vitro experiments were conducted in order to estimate the effects of Hcy (0.3 mM), Phe (1.2 mM) and/or Ala (1.2 mM) on the above parameters. The administration of CDD led to a statistically significant decrease of the rat brain TAS (-29%, p < 0.001) and to a significant increase of both AChE (+20%, p < 0.001) and (Na(+),K(+))-ATPase (+35%, p < 0.001) activities. Mg(2+)-ATPase activity was found unaltered. Equilibrated diet, administered to early age CDD-treated rats of both sexes for an additional period of 18 weeks, restored the above parameters to control levels. Moreover, the in vitro experiments showed that Hcy could simulate these changes (at least under the examined in vitro conditions), while both Phe and Ala act protectively against the CDD-induced effects on the examined rat brain enzyme activities. The effects of early age CDD-feeding on the examined parameters are proved to be reversible through restoration to equilibrated diet, while our data suggest a role for Hcy (as a causative parameter for the CDD-induced effects) and a possible protective role for Phe and Ala (in reversing the observed CDD-induced effects).

Headspace solid phase micro extraction gas chromatographic determination of fenthion in human serum.

A simple and effective analytical procedure was developed for the determination of fenthion residues in human serum samples. The sample treatment was performed using the headspace solid-phase micro extraction with polyacrylate fiber, which has the advantage to require low amount of serum (1 mL) without tedious pre-treatment. The quantification of fenthion was carried out by gas chromatography-mass spectrometry and the recoveries ranged from 79 to 104% at two spiking levels for 6 replicates. Detection and quantification limits were calculated as 1.51 and 4.54 ng/mL of serum respectively. Two fenthion metabolites fenoxon and fenthion-sulfoxide were also identified.

Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

Effects of acute and chronic cadmium administration on the vascular reactivity of rat aorta.

The effect of acute and chronic cadmium (Cd) administration on the vascular function of the rat aorta was studied. The rats were randomly divided into four main groups (A: saline controls under chronic administration, B: Cd-treated rats under chronic administration, C: saline controls under acute administration, D: Cd-treated rats under acute administration). After their sacrifice, the aortic rings were divided into rings with endothelium (E+) and without (E-), and suspended in an isolated organ bath with Krebs - Henseleit buffer. Maximal tension (T max, in g) was measured in response to potassium chloride (KCl) and phenylephrine (PE) in all aortic rings. Relaxation response to acetylcholine (ACh) administration was expressed as percent of maximal tension induced by PE. Chronic administration: A statistically significant increase of the contraction was observed between groups B (i.m. Cd 0.5 mg/kg for 120 days) and A (i.m. 0.9% NaCl for 120 days) in response to KCl (20-60 mM) and the T max as well (in both the E+ and the E- subgroups). No statistically significant difference was observed in response to PE and ACh exposure. Acute administration: A statistically significant increase was observed between group D(E+) (i.m. Cd 2 mg/kg, 8 h before sacrifice) and group C(E+) (i.m. 0.9% NaCl, 8 h before sacrifice) in response to 10-30 mM of KCl, and a significant decrease between D(E-) and C(E-) in response to 10(-7)-10(-6) M of PE, though T (max) was increased between D(E-) and C(E-) with PE exposure. The contractile response levels of the E+ aortic rings to PE and ACh showed no statistically significant difference.

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na(+), K (+))- and Mg ( 2+ )-ATPase activities of adult rat hypothalamus and cerebellum.

UNLABELLED: Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na(+), K(+))- and Mg(2+)-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (-23%, p < 0.001) and the Na(+), K(+)-ATPase activities (-26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (-17%, p < 0.001) and Na(+), K(+)-ATPase (-27%, p < 0.001). Mg(2+)-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. IN CONCLUSION: neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na(+), K(+)-ATPase activities. The decreased (by the THs) Na(+), K(+)-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.

Cadmium effects on brain acetylcholinesterase activity and antioxidant status of adult rats: modulation by zinc, calcium and L-cysteine co-administration.

We have previously reported that cadmium (Cd) as 3CdSO4 . 8H2O decreases rat brain total antioxidant status and L-cysteine (Cys) has a protective effect against it. The aim of the present study was to investigate the effects of subacute Cd administration, either alone or (almost simultaneously) with zinc (Zn), calcium (Ca) and Cys, on brain acetylcholinesterase (AChE) activity and brain total antioxidant status in male adult rats. All substances were administered intraperitoneally. Cadmium (1 mg/kg/day for 14 days) increased AChE activity (+46%; P<0.001) and decreased total antioxidant status (-29%; P<0.001). Zinc and Ca (both 1 mg/kg/day for 14 days) increased AChE activity (+18%; P<0.01 and +53%; P<0.001) and caused no changes on rat brain total antioxidant status. L-Cysteine (7 mg/kg/day for 14 days) impressively increased AChE activity (+185%; P<0.001) and augmented total antioxidant status (+26%; P<0.001). In the groups of Zn+Cd, Ca+Cd and Cys+Cd co-administration, AChE activity remained high (+42%; P<0.001, +41%; P<0.001 and +141%; P<0.001 respectively), while total antioxidant status returned to the saline control levels. L-Cysteine given before a toxic dose of Cd (5 mg/kg) resulted in high AChE activity (+85%; P<0.001), a total antioxidant status similar to the control values, and survival of the treated rats. In conclusion, Cd increased brain AChE activity and decreased brain total antioxidant status of adult male rats. Zinc, Ca and Cys, given just before Cd administration, modified the Cd-induced effects and restored rat brain total antioxidant status to the control levels.

Changes in antioxidant status, protein concentration, acetylcholinesterase, (Na+,K+)-, and Mg2+ -ATPase activities in the brain of hyper- and hypothyroid adult rats.

It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+ -ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 microg/l00 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg2+ -ATPase activity were found unaffected in hyperthyroidism, while AChE and Na+,K+ -ATPase activities were reduced by 25% (p < 0.01). In contrast, TAS, (Na+,K+)-ATPase and Mg2+-ATPase activities were found to be increased (approx. 23-30%, p < 0.001) in the hypothyroid brain, while AChE activity and PC were shown to be inhibited (approx. 23-30%, p < 0.001). These changes on brain enzyme activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg2+, neural excitability, as well as the uptake and release of biogenic amines.

The protective effect of L-cysteine and glutathione on the adult and aged rat brain (Na+,K+)-ATPase and Mg2+-ATPase activities in galactosemia in vitro.

The aim of this study was to evaluate whether the addition of the antioxidants L-cysteine (Cys) or the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes (Na+,K+)-ATPase, and Mg2+-ATPase in adult or aged rat brain homogenates induced by galactosemia in vitro. Mixture A [mix. A: galactose-1-phosphate (Gal-1-P, 2 mM) plus galactitol (Galtol, 2 mM) plus galactose (Gal, 4 mM) = classical galactosemia] or mixture B [mix. B: Galtol (2 mM) plus Gal (1 mM) = galactokinase deficiency galactosemia] were preincubated in the presence or absence of Cys (0.83 mM) or GSH (0.83 mM) with adult or aged brain homogenates at 37 degrees C for 1 h. TAS and the enzyme activities were determined spectrophotometrically. Mix. A or mix. B preincubation with the adult brain resulted in a significant (Na+,K+)-ATPase inhibition (-30%) and a Mg2+-ATPase stimulation (+300% and +33%, respectively), whereas lower modifications of the enzyme activities (p < 0.001) were found in the aged brain. Gal mixtures decreased TAS by 40% (p < 0.001) and by 20% (p < 0.01) in adult and aged samples, respectively. The antioxidants significantly increased TAS resulting in the reversion of (Na+,K+)-ATPase inhibition and Mg2+-ATPase stimulation by mix. B only. The inhibitory effect of Gal and its derivatives on brain (Na+,K+)-ATPase and their stimulatory effect on Mg2+-ATPase are being decreased with age, probably due to the producion of free radicals. Cys and GSH increased TAS resulting in a reversion of the inhibited (Na+,K+)-ATPase in both models of the in vitro galactosemia and the stimulated Mg2+-ATPase in galactokinase deficiency galactosemia only.

In vivo and in vitro effects of cadmium on adult rat brain total antioxidant status, acetylcholinesterase, (Na+, K+)-ATPase and Mg2+-ATPase activities: protection by L-cysteine.

This study was undertaken in order to investigate a) the short- and long-term in vivo effects of cadmium (Cd) on brain acetylcholinesterase (AChE), (Na+, K+)-ATPase activities in adult rats, b) the concentration-dependent in vitro and in vivo (acute experiment) effects of Cd on the activity of those enzymes, c) the in vivo and in vitro effects of the antioxidant L-cysteine (Cys) on the above enzyme activities, and d) the evaluation of brain total antioxidant status after in vivo Cd, L-cysteine, or L-cysteine+Cd administration in rats. In vitro, CdSO4 inhibited pure and brain AChE in concentrations higher than 0.1 mM, while it activated by approximately 70% (P<0.001) brain Na+, K+ -ATPase in concentrations up to 0.1 mM and inhibited its activity in higher concentrations. Mg2+ -ATPase was not influenced up to 0.1 mM concentration, while it was inactivated (40%, P<0.001) in higher CdSO4 concentrations. A dose-response study of brain CdSO4 (1,2 and 5 mg/kg once 8 hr before decapitation) revealed a dose-dependent decrease (-14 to -30%, P<0.001) of brain AChE activity, an increase of Na+, K+ -ATPase activity (+47 to +200%, P<0.001) and an increase of Mg2+ -ATPase only after the highest dose (5mg/kg) in the short-term treatment of rats. Long-term Cd administration (1 mg/kg rat daily for 4 months) activated brain AChE and Na+, K+ -ATPase about 50-65% (P<0.001) but not Mg2+ -ATPase. Brain total antioxidant status was decreased by Cd (30%, P<0.01), while it was increased by L-cysteine or L-cysteine+Cd (50%, P<0.001) in the short-term in vivo treatment. L-cysteine reversed the enzymatic activity changes observed with Cd alone in the high-dose short-term in vivo treatment of rats, as well as the brain AChE inhibition induced by Cd in the in vitro experiments. These results indicate that: a) Cd can influence in a different way the examined enzyme activities after short- and long-term administration, b) Cd may modulate brain cholinergic mechanism(s), neural excitability and metabolic energy production, and c) L-cysteine can have a protective antioxidant effect on the oxidative stress of the brain induced by Cd.

Selegiline long-term effects on brain acetylcholinesterase, (Na+,K+)-ATPase activities, antioxidant status and learning performance of aged rats.

The aim of this study was to investigate the effects of selegiline ((-)deprenyl), an irreversible inhibitor of monoaminoxidase-B (MAO-B): (a) on brain acetylcholinesterase (AChE), (Na(+),K(+))-ATPase and Mg(2+)-ATPase activities; (b) total antioxidant status (TAS); (c) learning performance and to evaluate possible correlation between biochemical and behavioral findings after long-term drug administration in aged male rats. Selegiline (0.25mgkg(-1) rat) was administered once every other day for 50 days. Learning parameters were tested through a two-way active avoidance procedure taking place in an Ugo-Basile automated shuttle box device. Enzyme activities and TAS were evaluated spectrophotometrically in brain homogenates of decapitated animals. TAS was significantly lower in aged compared to adult rats and this was reversed by selegiline administration. The decrease of free radical production by selegiline resulted in the stimulation of AChE and (Na(+),K(+))-ATPase. Mg(2+)-ATPase activity was not affected by the drug. Selegiline seems to improve the avoidance performance as compared to control groups. It is concluded that: (a) MAO-B inhibition and/or free radical protection on tyrosine hydroxylase by the drug may increase brain catecholamine concentrations resulting possibly in (Na(+),K(+))-ATPase stimulation; (b) AChE and (Na(+),K(+))-ATPase activation by the drug could modulate brain cholinergic, catecholaminergic and serotoninergic systems as well as the neural excitability and metabolic energy production; and (c) selegiline seems to improve the learning performance of aged rats.

Effects of (-)deprenyl (selegiline) on acetylcholinesterase and Na(+),K(+)-ATPase activities in adult rat whole brain.

(-)Deprenyl is an irreversible inhibitor of monoaminoxidase-B (MAO-B) at concentration 10(-6)M and of both MAO-A and MAO-B at concentration 10(-5)M. In this study, the effect of different concentrations (10(-7)-10(-4)M) of (-)deprenyl on the activity of acetylcholinesterase (AChE), Na(+),K(+)-ATPase and Mg(2+)-ATPase was investigated in homogenates of adult rat whole brain and in pure enzymes. Drug preincubation period with the homogenates or pure enzymes was 1 and 3h. Brain AChE activity was decreased by 30-39% (P<0.01) when exposed to 10(-4)M (-)deprenyl, by 22-25% (P<0.01) when exposed to 10(-5)M of the drug, and by 18-20% (P<0.01) after preincubation with a concentration of the drug 10(-6)M. Brain Na(+),K(+)-ATPase was stimulated by 46-162% (P<0.001) when the homogenate was preincubated with 10(-4)M (-)deprenyl and by 36-104% (P<0.001) for preincubation with drug concentration 10(-6)M. No effect was observed on the activity of brain Mg(2+)-ATPase, pure AChE or pure Na(+),K(+)-ATPase when exposed to the above concentrations of the drug. We conclude that (-)deprenyl is an indirect AChE inhibitor and Na(+),K(+)-ATPase stimulator in the rat brain (in vitro).