Panagoula Angelogianni

Protective effect of L-phenylalanine on rat brain acetylcholinesterase inhibition induced by free radicals.

OBJECTIVE To investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe) could reverse the free radical effects on brain acetylcholinesterase (AChE) activity, since it has been reported that Phe binds hydroxyl radicals ((*)OH). DESIGN AND METHODS Two well established systems were used for production of free radicals: (a) FeSO(4) (84 microM) plus ascorbic acid (400 microM), and (b) FeSO(4), ascorbic acid and H(2)O(2) (1 mM) at 37 degrees C in homogenates of adult rat whole brain. Changes in brain AChE activity were studied in the presence of each system separately. RESULTS AChE was inhibited (18-28%) by both systems of free radicals. This inhibition was reversed when the brain homogenate was preincubated with Phe 1.8 mM. CONCLUSIONS In accordance with our previous reports, Phe could protect against the direct action of (*)OH radicals on brain AChE and in this way it might be useful in the prevention of certain cholinergic neural dysfunctions.

Protective effect of L-cysteine and glutathione on rat brain Na+,K+-ATPase inhibition induced by free radicals.

Abstract The aim of this study was to investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe), L-cysteine (Cys) or reduced glutathione (G SH) could reverse the free radical effects on Na+,K+-ATPase activity. Two well established systems were used for the production of free radicals: 1) FeSO4 (84 μᴍ ) plus ascorbic acid (400 μᴍ ) and 2) FeSO4, ascorbic acid and H2O2 (1 mᴍ) for 10 min at 37 °C in homogenates of adult rat whole brain. Changes in brain Na+,K+-ATPase activity and total antioxidant status (TAS) were studied in the presence of each system separately, with or without Phe, Cys or GSH. TAS value reflects the amount of free radicals and the capacity of the antioxidant enzymes to limit the free radicals in the homogenate. Na+,K+-ATPase was inhibited by 35-50% and TAS value was decreased by 50-60% by both systems of free radical production. The enzymatic inhibition was completely reversed and TAS value increased by 150-180% when brain homogenates were preincubated with 0.83 mᴍ Cys or GSH. However, this Na+,K+-ATPase inhibition was not affected by 1.80 mᴍ Phe, which produced a 45-50% increase in TAS value. It is suggested that the antioxidant action of Cys and GSH may be due to the binding of free radicals to sulfhydryl groups of the molecule, so that free radicals cannot induce Na+,K+-ATPase inhibition. Moreover, Cys and GSH could regulate towards normal values the neural excitability and metabolic energy production, which may be disturbed by free radical action on Na+,K+-ATPase.

Ontogenesis of Proopiomelanocortin and Its Processing to β-Endorphin by the Fetal and Neonatal Rat Brain

A number of reports suggest that β-endorphin (β-END) may play an important role in the regulation of cell proliferation and neuronal differentiation. Proopiomelanocortin (POMC), the common precursor ofadrenocorticotropic hormone and β-END, is detected very early in embryonic life in hypothalamic neurons of the developing rat. However, very little is known about the degree to which POMC is processed to β-END during fetal and early postnatal life. Thus, it was the objective of the present study to estimate the hypothalamic content of POMC mRNA, as well as the biosynthesis and posttranslational processing of POMC by hypothalamic neurons on fetal day 20 and on days 1, 8 and 22 of postnatal life. Hypothalamic POMC mRNA, as determined by Northern blot analysis, was higher on fetal day 20 than on postnatal days 1, 8 and 22. A higher rate of incorporation of [3H]phenylalanine into β-END immunoreactive peptides was observed on fetal day 20 than on postnatal day 1. However, the rate of incorporation was significantly increased by day 8 of postnatal life and was similar to that on day 22. POMC was processed to β-lipotropin (β-LPH) and β-END at all ages examined, but the relative proportions of POMC:β-LPH:β-END changed during development. Thus, β-END accounted only for 34.89 ± 6.14% of the total [3H]phenylalanine-labeled β-END immunoreactive peptides on fetal day 20, while it accounted for 57.37 ± 5.20, 62.81 ± 1.38 and 79.25 ± 6.57% on days 1, 8 and 22 of postnatal life, respectively. Thus, POMC is processed to a considerable extent into β-END-sized peptides by the fetal hypothalamus and may influence brain development. Furthermore, the rate of processing of hypothalamic POMC into β-END increases with development, probably due to the increased activity of the enzymes specific for POMC processing.

Effect of CDP-choline on hippocampal acetylcholinesterase and Na+,K(+)-ATPase in adult and aged rats.

The aim of this study was to investigate the effect of different cytidine-5′-diphosphocholine (CDP-choline) concentrations (0.1-1 mm) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities in homogenates of adult and aged rat hippocampi. Tissues were homogenised, centrifuged at 1000 × g for 10 min and in the supernatant, AChE activity and Na+,K+-ATPase and Mg2+-ATPase activities were determined according to Ellman’s method and Bowler’s and Tirri’s method, respectively. After an 1-3 h preincubation of the homogenised tissue with CDP-choline, a maximal AChE stimulation of about 25% for both adult and aged rats (p < 0.001) and a Na+,K+-ATPase activation of about 50% for adult rats (p < 0.001) and about 60% for aged rats (p < 0.001) were observed, while hippocampal Mg2+- ATPase activity was not influenced in either adult or aged animals. It is suggested that: CDP-choline can restore hippocampal AChE and Na+,K+-ATPase activities in the aged rat and thus it may play a role in improving memory performance which is impaired by aging and some neuronal disturbances.

L-Cysteine supplementation prevents exercise-induced alterations in human erythrocyte membrane acetylcholinesterase and Na+,K+-ATPase activities

Abstract Background: L-Cysteine (L-Cys) is implicated in the reduction of free radical production. The aim of this study was to investigate whether L-Cys supplementation prevents modulation of the activities of erythrocyte membrane acetylcholinesterase (AChE), Na+,K+-ATPase and Mg2+-ATPase induced by free radicals in basketball players during training. Methods: Blood was obtained from 10 basketball male players before (group A) and after a game (group B) and after 1 week of L-Cys (0.5 g/24 h orally) supplementation before (group C) and after training (group D). Lactate, pyruvate and total antioxidant status (TAS) were measured using commercial kits and the enzyme activities were determined spectrophotometrically. Results: Both lactate and pyruvate levels remarkably increased after exercise. In contrast, TAS levels significantly decreased in group B, increased in group C and then declined (group D), reaching those of group A. AChE activity was statistically increased post-exercise (3.98±0.04 × mg protein) compared with pre-training (2.90±0.05 × mg protein, p<0.01). Na+,K+-ATPase activity was also higher post-exercise (1.27±0.05 μmol Pi/h×mg protein) than that pre-exercise (0.58±0.04 μmol Pi/h×mg protein, p<0.001). When the players were supplemented with L-Cys, both AChE and Na+,K+-ATPase activities remained unaltered post-exercise. Mg2+-ATPase activities were unchanged in all groups studied. Conclusions: L-Cys supplementation may protect the enzyme activities studied against stimulation induced by free radical production during training in athletes by ameliorating their total antioxidant capacity. Clin Chem Lab Med 2007;45:67–72.

Effect of Aging on the Activities of Acetylcholinesterase, Na+, K+-ATPase and Mg2+-ATPase in Rat Pituitary and Hypothalamus

Abstract Acetylcholinesterase (AChE), Na+, K+-ATPase and Mg2+-ATPase activities were estimated in homogenised rat pituitary and hypothalamus of 4-and 22-month-old rats. AChE activity was not altered in the pituitary of aged com pared to adult rats, while it was found decreased by about 40% in the hypothalamus. Na+,K+-ATPase activity remained stable in the hypothalamus, while it was decreased by about 38% in the pituitary. Mg2+-ATPase activity remained unchanged in the hypothalamus, but was increased by about 83% in the pituitary. This pituitary Na+, K+-ATPase inactivation may result in pathological mood and decreased neural excitability and metabolic energy production in aged animals.The age-related alterations of AChE , Na+, K+-ATPase and Mg2+-ATPase activities may reflect changes in secretion and responses of some hormones of pituitary and hypothalamus.

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI

PurposeArtificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol.MethodsAged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically.ResultsOocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone.ConclusionsThis study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

The in vivo and in vitro effects of L-carnitine supplementation on the erythrocyte membrane acetylcholinesterase, Na+, K+-ATPase and Mg2+-ATPase activities in basketball players.

Abstract Background: We investigated whether the activities of erythrocyte membrane acetylcholinesterase (AChE), Na+, K+-ATPase and Mg2+-ATPase are modulated in basketball players pre- vs. post-forced training with or without L-carnitine (L-C) supplementation. Methods: Blood was obtained from 10 male players pre-game (group A) and post-game (group B) and after 1 month L-C supplementation (2 g/24 h orally) pre-training (group C) and post-training (group D). Lactate, pyruvate and total antioxidant status (TAS) were measured with commercial kits, catecholamines with HPLC and the enzyme activities spectrophotometrically. Results: Lactate, pyruvate, AChE, Na+, K+-ATPase and catecholamines were increased (p<0.001) and TAS was decreased (p<0.001) in group B. In contrast, TAS remained unaltered and the all enzyme activities were reduced (p<0.001) in group D at the same time of study. Mg2+-ATPase activity remained unchanged. In vitro incubation of the modulated AChE and Na+, K+-ATPase with L-C (25 μM) from group B and group D resulted in a non-significant reduction of the enzymes in group B and complete restoration of their activities in group D. Conclusions: The increase of AChE and Na+, K+-ATPase activities may be due to the elevation of catecholamines in group B. Carnitine utilization by the muscles during training may result in a reduction of the enzyme activities (group D). The latter is supported by the recovery of the enzyme activities after incubation of the membranes from group D with L-C. Clin Chem Lab Med 2008;46:137–42.