Effect of hypermethioninemia on some parameters of oxidative stress and on Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in hippocampus of rats

In the present study we investigated the effect of chronic administration of methionine, a metabolite accumulated in many inherited pathological conditions such as methionine adenosyltransferase deficiency and homocystinuria, on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), catalase activity and total thiol content, as well as on Na⁺,K⁺-ATPase activity in rat hippocampus. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that chronic hypermethioninemia significantly increased TBARS, decreased Na⁺,K⁺-ATPase activity but did not alter catalase and total thiol content. Since chronic hypermethioninemia altered TBARS and Na⁺,K⁺-ATPase activity at 12 h after methionine administration, we also investigated the effect of acute administration of this amino acid on the same parameters studied after chronic methionine administration. For acute treatment,29-day-old rats received one single injection of methionine (2.68 μmol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that rats subjected to acute hypermethioninemia presented a reduction of Na⁺,K⁺-ATPase activity and an increase in TBARS when the animals were killed at 3 and 12 h, but not at 1 h, after methionine administration. These data indicate that hypermethioninemia increases lipid peroxidation which may, at least partially, explain the effect of methionine on the reduction in Na⁺,K⁺-ATPase activity. If confirmed in human beings, our findings could suggest that the induction of oxidative stress and the inhibition of Na⁺,K⁺-ATPase activity caused by methionine might contribute to the neurophysiopathology observed in patients with severe hypermethioninemia.     

Intrastriatal Hypoxanthine Reduces Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity and Induces Oxidative Stress in the Rats

The main objective of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, a metabolite accumulated in Lesch Nyhan disease and possibly involved in its neuropathology, on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress, namely chemiluminescence (an index of lipid peroxidation), total radical-trapping antioxidant parameter—TRAP (an index of total antioxidant capacity of the tissue) and total thiol protein membrane content, in striatum, cerebral cortex and hippocampus of rats. Results show that hypoxanthine significantly decreased Na⁺,K⁺-ATPase activity and TRAP while increased chemiluminescence in all ipsislateral structures tested. However, no effect on total thiol protein membrane content was detected. We suggest that hypoxanthine induces oxidative stress in all cerebral structures studied (striatum, hippocampus and cerebral cortex) and that the reduction of Na⁺,K⁺-ATPase activity was probably mediated by reactive oxygen species.     

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)- and Mg <Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-ATPase activities of adult rat hypothalamus and cerebellum

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 Mg²⁺-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25μg/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). Mg²⁺-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.     

Suckling Rat Brain Regional Distribution of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the In Vitro Galactosaemia: The Effect of L-Cysteine and Glutathione

Inhibition of Na⁺,K⁺-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-1-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na⁺,K⁺-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na⁺,K⁺-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20–30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50–60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na⁺,K⁺-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.     

The Protective Effect of L-Cysteine and Glutathione on the Adult and Aged Rat Brain (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase and Mg<Superscript>2+</Superscript>-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 37C 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.     

Changes in Antioxidant Status,Protein Concentration,Acetylcholinesterase, (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-, and Mg<Superscript>2+</Superscript>-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 g/100 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.     

Homocysteine alters glutamate uptake and Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and oxidative status in rats hippocampus: protection by vitamin C

In the present study we investigate the effect of homocysteine on glutamate uptake, Na⁺,K⁺-ATPase, enzymatic antioxidant defenses, as well as reactive species levels in hippocampus of rats. The influence of vitamin C, a classic antioxidant, on the effects elicited by homocysteine was also tested. Results showed that chronic hyperhomocysteinemia decreased glutamate uptake and the activities of Na⁺,K⁺-ATPase, catalase and superoxide dismutase in hippocampus of rats. Reactive species levels were increased by chronic homocysteine administration. Concomitant administration of vitamin C significantly prevented these alterations caused by homocysteine. According to our results, it seems possible to suggest that the reduction in glutamate uptake and Na⁺,K⁺-ATPase activity may be mediated by oxidative stress, since vitamin C prevented these effects. We suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diet in homocystinuria.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Level of CD14<Superscript>+</Superscript>-endothelial progenitor cells is not associated with coronary artery disease or cardiovascular risk factors

There is evidence for two subpopulations among circulating endothelial progenitor cells (EPCs), i.e., CD34⁺-EPCs and CD14⁺-EPCs. Prior studies on the relationship between the level of EPCs and coronary artery disease (CAD), either did not distinguish between the two types of EPCs or studied only CD34⁺-EPCs. We therefore investigated whether the number of circulating CD14⁺-EPCs correlates with either CAD and/or cardiovascular risk factors. Circulating CD14⁺-EPCs—as defined by the surface markers CD14⁺KDR⁺—were analyzed by flow cytometry in 100 individuals [34 control subjects, 41 patients with stable CAD and 25 patients with acute coronary syndromes (ACS)]. The level of circulating CD14⁺-EPCs was not significantly different in patients with normal coronary arteries compared to those with stable CAD or ACS. Neither was there any association between the severity of CAD or risk factors and the number of circulating CD14⁺-EPCs. Thus, the number of circulating CD14⁺-EPCs was not significantly correlated either with the severity of coronary disease or with cardiovascular risk factors.     

Reduced Na++K+-ATPase activity in peripheral nerve of streptozotocin-diabetic rats: a role for protein kinase C?

Summary Six weeks after induction of diabetes, the rate of ouabain-sensitive ⁸⁶Rb⁺ accumulation, a parameter which reflects Na⁺+K⁺-ATPase pumping activity, was significantly reduced in endoneurial preparations of sciatic nerve from untreated diabetic rats compared with that in control rats (Trial 1, 0.19±0.09 versus 0.48±0.13 pmol/min per mg wet weight of tissue, p<0.001; Trial 2, 0.27±0.16 versus 0.47±0.18, p<0.01). This decrease in ouabain-sensitive ⁸⁶Rb⁺ uptake was not observed in nerves from diabetic rats maintained on sorbinil (an aldose reductase inhibitor) or myo-inositol diets. Protein kinase C activity was demonstrated in the soluble fraction of a sciatic nerve homogenate by assaying for lipid-activated, Ca⁺-dependent phosphorylation of calf thymus histone. No significant difference in the time course of kinase C activity was observed between cytosol fractions of nerve homogenates from control and diabetic rats (control, 6.22±0.97 pmol ³²P incorporated/mg cytosol protein in 50 min; diabetic, 5.32±0.71). Three low molecular weight neural proteins (each with M_r<29,000) were identified as substrates for protein kinase C.     

Inhibition of CO<Subscript>2</Subscript> production from glucose by arginine in brain slices of rats

In the present study we evaluated the in vivo effect of arginine on CO₂ production from glucose in a medium with physiological and high extracellular K⁺ concentrations. We also tested the influence of the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), on the effects elicited by arginine in order to investigate the possible participation of NO and/or its derivatives on the effects of arginine on CO₂ production from glucose. Sixty-day-old rats were treated with a single intraperitoneal injection of saline (control; group I), arginine (0.8 g/kg; group II), L-NAME (2.0 mg/kg; group III) or arginine (0.8 g/kg) plus L-NAME (2.0 mg/kg; group IV) and were killed 1 h later. Results showed that arginine administration inhibited CO₂ production from glucose at physiological extracellular K⁺ concentration and L-NAME prevented such effect. In contrast, arginine administration had no effect on CO₂ production from glucose at high extracellular K⁺ concentration. Based on these data, we also investigated the in vitro effect of arginine on CO₂ production from glucose in a medium with physiological extracellular K⁺ concentration in hippocampus slices. Results showed that arginine (0.1–1.5 mM) when added to the incubation medium did not alter CO₂ production from glucose in hippocampus slices of untreated rats. In addition, we also demonstrated that arginine inhibits Na⁺, K⁺-ATPase activity. The data indicate that the reduction of CO₂ production by arginine was probably mediated by NO and/or its derivatives, which could act inhibiting the activity of Na⁺, K⁺-ATPase. The results suggest that arginine impairs energy metabolism in hippocampus slices of rats.     

Equilibrated diet restores the effects of early age choline-deficient feeding on rat brain antioxidant status and enzyme activities: the role of homocysteine, <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and <Emphasis Type="SmallCaps">l</Emphasis>-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).     

Effects of adult-onset streptozotocin-induced diabetes on the rat brain antioxidant status and the activities of acetylcholinesterase, (Na+,K+)- and Mg2+-ATPase: modulation by L-cysteine

Uncontrolled diabetes is known to affect the nervous system. The aim of this study was to investigate the effect of the antioxidant L-cysteine (Cys) on the changes caused by adult-onset streptozotocin (STZ)-induced diabetes on the rat brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase. Thirty-eight male Wistar rats were divided into six groups: C_A (8-week-control), C_B (8-week-control + 1-week-saline-treated), C + Cys (8-week-control + 1-week-Cys-treated), D_A (8-week-diabetic), D_B (8-week-diabetic + 1-week-saline-treated) and D + Cys (8-week-diabetic + 1-week-Cys-treated). All diabetic rats were once treated with an intraperitoneal (i.p.) STZ injection (50 mg/kg body weight) at the beginning of the experiment, while all Cys-treated groups received i.p. injections of Cys 7 mg/kg body weight (daily, for 1-week, during the 9th-week). Whole rat brain parameters were measured spectrophotometrically. In vitro incubation with 0.83 mM of Cys or 10 mM of STZ for 3 h was performed on brain homogenate samples from groups C_B and D_B, in order to study the enzymes’ activities. Diabetic rats exhibited a statistically significant reduction in brain TAS (−28%, D_A vs C_A;−30%, D_B vs C_B) that was reversed after 1-week-Cys-administration into basal levels. Diabetes caused a significant increase in AChE activity (+27%, D_A vs C_A; +15%, D_B vs C_B), that was further enhanced by Cys-administration (+57%, D + Cys vs C_B). The C + Cys group exhibited no significant difference compared to the C_B group in TAS (+2%), but showed a significantly increased AChE activity (+66%, C + Cys vs C_B). Diabetic rats exhibited a significant reduction in the activity of Na⁺,K⁺-ATPase (−36%, D_A vs C_A;−48%, D_B vs C_B) that was not reversed after 1-week Cys administration. However, in vitro incubation with Cys partially reversed the diabetes-induced Na⁺,K⁺-ATPase inhibition. Mg²⁺-ATPase activity was not affected by STZ-induced diabetes, while Cys caused a significant inhibition of the enzyme, both in vivo (−14%, C + Cys vs C_B;−17%, D + Cys vs C_B) and in vitro (−16%, D_B + in vitro Cys vs C_B). In vitro incubation with STZ had no effect on the studied enzymes. The present data revealed a protective role for Cys towards the oxidative effect of diabetes on the adult rat brain. Moreover, an increase in whole brain AChE activity due to diabetes was recorded (not repeatedly established in the literature, since contradictory findings exist), that was further increased by Cys. The inhibition of Na⁺,K⁺-ATPase reflects a possible mechanism through which untreated diabetes could affect neuronal excitability, metabolic energy production and certain systems of neurotransmission. As concerns the use of Cys as a neuroprotective agent against diabetes, our in vitro findings could be indicative of a possible protective role of Cys under different in vivo experimental conditions.