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L-Carnitine antioxidant effect on protein concentration, acetylcholinesterine, Na+,K+-ATPase and Mg2+-ATPase activities in rat brain after forced swimming Theodore Tsakiris1, Panagoula Angelogianni1, Christi Tesseromatis2, Stylianos Tsakiris1 ,* and Kleopatra H. Schulpis3 1 Department of Experimental Physiology, Medical School, Athens University, P.O. Box 65257, GR-15401 Athens, Greece 2 Department of Experimental Pharmacology, Medical School, Athens University, 75 Mikras Asias Str., Goudi, GR-11527 Athens, Greece 3 Institute of Child Health, Research Center, “Aghia Sophia” Children's Hospital, GR-11527 Athens, Greece RUNNING TITLE: L-Carnitine effects on brain enzymes in swimming rats Keywords: Carnitine, exercise, acetylcholinesterase, Na+,K+-ATPase, Mg2+-ATPase, oxidation, free radicals * Corresponding author: S. Tsakiris, PhD, Associate Professor, Tel: +302107462662 ; fax:+302107462571 e-mail adress: [email protected] (S. Tsakiris) INTRODUCTION The determinants of human exercise performance are multifactorial and complex. The performance of skeletal muscle work during exercise is dependent on the availability of biochemical energy in the form of ATP. Thus, it is not surprising that a variety of metabolic and biochemical markers are related to exercise performance and training [1,2] or that disorders of energy metabolism are frequently associated with impaired exercise performance [3]. Similarly, manipulations of bioenergetics have frequently been proposed as strategies to enhance exercise endurance or capacity. Carnitine is an endogenous molecule with well established important roles in cellular metabolism [4]. The functions of carnitine in skeletal muscle are critical to sustaining normal bioenergetics during exercise. All biochemical actions of carnitine are based on the carnitine acyltransferasemeditated reversible transfer of carboxylic acids, or “acyl” groups, between carnitine and coenzyme A in reactions of the following form: Carnitine + Acyl-CoA ↔ Acylcarnitine + Coenzyme A Coenzyme A is an important cofactor in a number of catabolic and anabolic reactions. Thus the carnitine pool, consisting of carnitine, short-chain acylcarnitines (acylcarnitines corresponding to short- and medium-chain length carboxylic acid groups), and long-chain acylcarnitines (corresponding to long-chains length carboxylic acid), will dynamically interact with multiple coenzyme A-dependent biochemical pathways. Carnitine is required for mitochondrial long-chain faty acid oxidation [4], a major source of energy during exercise [5]. Long-chain acylcarnitines transport activated long-chains fatty acids into the mitochondrial matrix for β-oxidation. Short-chain acylcarnitines are generated from short- chain acyl-CoAs. This generation of short-chain acylcarnitines may be viewed as buffering the cellular coenzyme A pool from acyl-CoA accretion, as accumulation of short-chain acyl-CoAs may adversely affect cellular metabolism [6]. The reversible transfer of short-chain acyl groups between carnitine and coenzyme A occurs dynamically in muscle during exercise. For example, acetyl- carnitine accumulation in muscle at workloads above the lactate threshold [8-10] corresponds to the accumulation of acetyl-CoA in the muscle [11]. The important biochemical functions of carnitine and the altered muscle physiology associated with clinical carnitine deficiency [7] support the critical role of carnitine in muscle bioenergetics. However, the extrapolation of these established concepts to the effects of carnitine supplementation in healthy persons or in varied pathologic conditions is less clear. It is unknown whether supraphysiologic carnitine concentrations enhance exercise performance, or which pathophysiologic conditions might be associated with decreased muscle carnitine content or increased carnitine requirements [13,14]. Humans take in carnitine primarily from diet (75%) with the remaining 25% coming from endogenous synthesis. Another major role for the carnitine is the transfer of acetyl-CoA from the mitochondria into the cytoplasma, thereby providing acetyl groups in the synthesis of the neurotransmitter acetylcholine (ACh) [15]. In keeping with polar characteristics, L-C does not bind to plasma proteins (factor unbound-1) and enters erythrocytes very slowly. The uptake of L-C into most tissues of the body, including liver, kidney, skeletal and cardial muscle, neurons and epididymal tissue, involves carrier-mediated transport systems, which maintain high tissue-to-plasma concentration ratio [4]. The transport of L-C from the plasma into the neurons is mediated by a Na+ and energy dependent process and may involve the organic cation transporter [16,17]. The most widely established function for L-C is its obligate role in the transport of long-chain fatty acids into mitochondrial matrix where they enter β-oxidation [18]. Studies in humans have also demonstrated that a decrease on muscle free carnitine concentration occurs during short- duration high intensity exercise and at the onset of exhaustive submaximal exercise and is matched by an almost equivalent increase in acetylcarnitine [18]. These findings suggest that another important role for carnitine is in buffering excess acetyl group accumulation, which occurs when the rate of acetyl-CoA formation exceeds the rate at which the tricarboxylic acid cycle can recycle CoA [19,20]. Acetylcholinesterase (AChE) (E.C. 3.1.1.7) is a biological significant component of cell membrane, contributing to its integrity and to permeability changes occuring during synaptic transmissions and conduction. It is a membrane-bound enzyme with its active side exposed in the external leaflet of the bilayer (ectoenzyme). This enzyme participates in the hydrolysis of acetylocholine (ACh) and can modulate the cholinergic function [21]. If ACh action is enhanced and due to the wide spread distribution of cholinergic fuctions, toxic effects involve the sympathetic, parasympathetic, motor and central nervous system [21,22]. Na+,K+-ATPase (E.C. 3.6.1.3) is the enzymatic base of univalent cation transport [23]. It is implicated in neural excitability [24], metabolic energy production [25], uptake and release of catecholamines [26] and serotonin [27]. Additionally, the role of Mg2+-ATPase is to control the intercellular Mg2+ concentration, changes which can modulate the activity of Mg2+-dependent enzymes and regulate the rate of protein synthesis and cell growth [28,29]. Previous in vitro studies implicated low total antioxidant status (TAS) (e.g. free radicals production) with the activities of AChE, (Na+,K+)-ATPase and Mg2+-ATPase in rat brain [30,31]/ In addition, the above membrane enzyme activities were found modulated in the presence of the free radicals [32,33]. Previous study [34] showed that free radicals production leading to a failure of mitochondria function was ameliorated by L-C administration. Similarly, pretreatment with L-X enhanced trend of AChE inhibition in all selected brain parts comparing with single galantamine administration, a competitive AChe inhibition [35]. Since L-C action and free radicals production are closely related to ACh modulation and Na+dependent energy process, we aimed to investigate AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities in rat brain homogenates pre- and post-forced swimming before and after L-C administration. In addition, in an in vitro study, we aimed to find out a direct effect of L-C on the above rat brain enzymes. MATERIALS AND METHODS Animal care and training protocol Adult male Albino Wistar rats (Saint Savvas Hospital, Athens, Greece) were used in all experiments. Body weight was 260 ± 27 g (mean ± SD). They were housed four in a cage, at a constant room temperature (22 ± 1 oC) under a 12h light:12h dark (light 08:00 – 20:00h) cycle. Foor and water were provided ad libitum. Animals were cared for in accordance with the principles of the “Guide for the Care and Use of Experimental Animals” [36]. Swimming was performed in a plastic waterpool (height 80 cm · width 50 cm and depth 55 cm), at a water temperature of 22 oC - 23 oC, between 09:00 and 14:00h. Swimming duration was 0h (animals were removed from the pool immediately after water immersion), 2h or 3h. Each of the three groups consisted of seven animals. Venditti at al. [37] found that prolonged (8h) swimming of rats induces oxidative stress, lipid peroxidation, and decreased antioxidant capacity in live homogenates, whereas swimming of moderate duration (3h) did not produce these effects. However, the aim of our study was to evaluate the total antioxidant status, the protein concentration and the enzyme activities in rat brain, which may be affected earlier than liver by exercise. So, the above parameters were investigated under short (2h) or moderate (3h) swimming of rats. Control animals (21 rats) were kept in their home cages at a constant room temperature (22 ± 1 oC) during the period of exercise and seven rats for each group (0h, 2h or 3h) were used for tissue preparation and biochemical determinations (group A). In addition, groups of seven animals either were used as controls after L-C administration (300 mg/kg) ip [38] (group C) or were treated with 2h or 3h forced swimming as above. Tissue preparation Immediately after each treatment, rats were sacrificed by decapitation. The whole brain of each animal was quickly removed and homogenized in 10 volume ice-cold (0-4 oC) medium containing 50mM tris(hydroxymethyl)-aminomethane-HCl (Tis-HCl), pH=7.4 and 30mM sucrose using an ice- chilled glass homogenizing vessel at 900 rpm (4-5 strokes). Then, the homogenate was centrifuged at 1000 x g for 10 min to remove nuclei and debris. In the resulting supernatant, the protein content was determined according to the method of Lowry et al. [39] and then the total antioxidant status (TAS) and enzymatic activities were measured. The enzyme incubation mixture was kept at 37 oC. Incubation of rat brain homogenates in the presence or absence of L-Carnitine 100μg protein of rat brain homogenate from group B (3h) or group D (3h) were incubated with 50 mM Tris-HCl, pH 7.4, 120 mM NaCl in the presence or absence of L-C (5 mM) for 1h at 37 oC. Then, AChE as well as Na+,K+-ATPase activities were evaluated in he reaction mixture [40,41]. L- C (5 mM) concentration was adapted because this value expresses the mean valua of total concentration in the vlood of healthy individuals in Greece, as previously reported (42]. Determination of brain total antioxidant status TAS was evaluated in each homogenized fresh rat brain. The total antioxidant capacity was measured spectrophotometrically by a commercial kit (Randox Laboratories Ltd., Cat. No. NX2332, UK) as previously reported [43]. 2,2'-Azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) was incubated with a peroxidase (met-myoglobin) and H2O2 in order to produce the radical cation which was measured at 600 nm. Inhibited values of TAS reflect the increase in brain free radical production, whereas stimulated TAS values show the decrease in free radical production and the increase in the antioxidant compounds. Determination of AChE activity AChE activity was determined by following the hydrolysis of acetylthiocholine according to the method of Ellman et al. [44] as described in detail previously [45]. The incubation mixture (1 ml) contained 50 mM Tris-HCl, ph 8, 240 mM sucrose, and 120mM NaCl. Pseudocholinesterase activity was not detected in the brain homogenate by using 2 · 10-2 mM quinidine sulfate in the above reaction mixture. Protein concentration of the incubation mixture was 80 – 100 μg/ml. The reaction was initiated after the addition of 0.03 ml of 5,5'-dithionitrobenzoic acid (DTNB) and 0.05 ml of acetylthiocholine iodide which was used as substrate. The final concentrations of DTNB and substrate were 0.125 and 0.5 mM, respectively. The reaction was followed spectrophotometrically by the increase of absorbance (Δ OD ) at 412 nm. Determination of Na+,K+-ATPase and Mg2+-ATPase activities Na+,K+-ATPase activity was calculated as the difference between total ATPase activity (Na+,K+,Mg2+-dependent ATPase) and Mg2+-dependent ATPase activity. Total ATPase activity was assayed in an incubation medium consisting of 50 mM Tris-HCl, ph 7.4, 120 mM NaCl, 20 mM Kcl, 4 mM MgCl2, 240 mM sucrose, 1 mM ethylenediamine tetraacetic acid K2-salt (K+-EDTA), 3 mM disodium ATP, and 80 – 100 μg protein from the homogenate in a final volume of 1 ml. Ouabain (1 mM) was added in order to determine the activity of Mg2+-ATPase. The reaction was started by adding ATP and stopped after an incubation period of 20 min by the addition of 2 ml of mixtyre consisting of 1% lubroil and 1% ammonium molybdate in 0.9 M H2SO4 [46]. The yellow colour which developed was read at 390 mM. Statistical Analysis Data were analyzed by ANOVA or Student's t-test. Multiple regression analysis was utilized in order to evaluate the correlation coefficients between TAS and protein concentrations as well as the enzyme activities. P values of < 0.05 were considered statistically insignificant. RESULTS As presented in Table 1, protein concentration in the homogenized whole brain was found significantly decreased especially after 3h forced swimming whereas it remained unaltered in the group with L-C administration at the same time of study. As shown in Table 2, both TAS and AChE activity were remarkably decreased in the rat brain after 3h forced exercise. 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Georgala S, Schulpis KH, Georgala C, Michas T. L-Carnitine supplementation in patients with cystic acne on isotretinoin therapy. J Eur Acad Dermatol Venereol 1999; 13:205-209. 43. Schulpis KH, Tsakiris S, Karikas GA, Moukas M, Behrakis P. Effect of diet on plasma total antioxidant status in phenylketonuric patients. Eur J Clun Nutr 2006; 57: 383-387 44. Ellman GL, Courtney D, Andres D, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88-95 45. Tsakiris S. Effects of L-phenylalanine on acetylcholinesterase and Na+,K+-ATPase activities in adult and aged rat brain. Mech Ageing Dev 2001; 122: 491-501 46. Tsakiris S, Angelogianni P, Schulpis KH, Behrakis P. Protective effect of L-cysteine and glutathione on rat brain Na+,K+-ATPase inhibition induced by free radicals. Z Naturforsch 2000; 55C:271-277 Table 1. Changes in protein concentration in homogenized whole brain of rats (n = 7) exposed to forced swimming for various periods with 300 mg/kg (group D) or without (group B) L-Carnitine (L-C) administration. Treatment A-3h A0h A2h A3h B0h B2h B3h C-3h C0h C2h C3h D0h D2h D3h Protein concentration (mg protein/ml) 5.80 0.40 6.00 0.24 6.15 0.50 5.70 0.42 5.90 0.38 (-2%) 5.22 0.20** (-15%) 3.99 0.15*** (-30%) 5.95 0.30 6.05 0.40 5.82 0.35 5.75 0.45 5.80 0.32 (-4%) 6.00 0.42 (+3%) 5.85 0.30 (+2%) Each value indicates the mean SD of seven independent experiments (seven rats per group). The average of each experiment arose from three evaluations of the homogenated brain of each animal. Group A represents “nontreatment” control group and group C controls administered the L-C dose subcutaneously . Values of controls with NaCl 0.9% (SC-3h and SC3h) were not statistically different to that C-3h. Note: Only the statistically significant values are presented. **: p < 0.01 (vs all controls), ***: p < 0.001 (vs all controls) Table 2: Changes in total antioxidant status (TAS) and acetylcholinesterase (AChE) activity in homogenized whole brain of rats (n = 7) exposed to forced swimming for various periods with 300 mg/kg (group D) or without (group B) L-Carnitine (L-C) administration. Treatment A-3h A0h A2h A3h B0h B2h B3h C-3h C0h C2h C3h D0h D2h D3h TAS (mmol/l) 55.15 2.15n 53.85 1.19a 54.50 1.64b 52.15 2.05c 54.00 1.35d (+0.2%) 43.60 1.00e, ** (-20%) 39.11 1.17f, *** (-25%) 55.50 1.80h 56.70 2.40g (+5%) 63.34 1.90i, ** (+16%) 64.62 2.10j, *** (+24%) 54.00 2.00k (-5%) 52.25 2.15l, ** (-18%) 54.51 1.80m, ** (-16%) AChE (ΔOD/min x mg protein) 0.906 0.045b 0.926 0.051a 0.916 0.035c 0.886 0.035d 0.888 0.045e (-4%) 0.645 0.038f, ** (-30%) 0.576 0.032g , *** (-45%) 0.600 0.030i 0.636 0.038h (+6%) 0.696 0.031j, * (+16%) 0.726 0.043k, ** (+21%) 0.629 0.029l (-1%) 0.668 0.040m (-4%) 0.600 0.031n, * (-17%) Each value indicates the mean SD of seven independent experiments (seven rats per group). The average of each experiment arose from three evaluations of the homogenated brain of each animal. Group A represents “nontreatment” controls and group C controls administered the LC dose subcutaneously. Values of saline controls (SC-3h and SC3h) were not statistically different as compared to SC3h. Note: Only statistically significant values are presented. *: p < 0.05 (vs control) , **: p < 0.01 (vs control), ***: p < 0.001 (vs control) TAS: a/e, a/f, a/i, a/j, a/m, n/e, n/j, n/l, n/m, b/e, b/j, b/i, b/f, c/f, b/c, c/e, c/j, c/h, c/g, c/i = p < 0.01 AChE: a/f, a/g, a/h, a/i, a/j, a/k, a/l, a/m, a/n, b/f, b/g, b/n, b/i, b/j, b/k, b/l, b/m, b/n, c/f, c/g, c/i, c/h, c/j, c/k, c/l, c/m, c/n, d/f, d/g, d/i, d/h, d/j, d/k, d/l, d/m, d/n = p <0.01 Table 3: Changes in Na+, K+-ATPase and Mg2+-ATPase activities in homogenized whole brain of rats (n = 7) exposed to forced swimming for various periods with 300 mg/kg (group D) or without (group B) L-Carnitine (L-C) administration. Treatment A-3h A0h A2h A3h B0h B2h B3h C-3h C0h C2h C3h D0h D2h D3h Na+, K+-ATPase activity Mg2+-ATPase activity (μmol Pi / h x mg protein) 5.72 0.51a 7.30 0.78a 5.80 0.48b 7.44 0.66b 6.05 0.55c 7.52 0.60c d 5.92 0.59 7.00 0.58d 6.00 0.36e (+3.4%) 7.20 0.65 (-3%) f, 10.89 1.08 *** (+80%) 10.53 1.05e, *** (+40%) g, 11.84 1.12 *** (+100%) 11.20 1.18f, *** (+60%) 3.08 0.24h 6.95 0.70 i 3.76 0.31 (+22%) 6.80 0.61 (-2%) j, 4.19 0.40 ** (+36%) 6.83 0.65 (-2%) 5.11 0.56k, *** (+66%) 6.54 0.72 (-6%) l 2.98 0.28 (-21%) 6.55 0.62 (-4%) m, 2.80 0.28 ** (-33%) 6.70 0.60 (-2%) n, 2.77 0.22 ** (-46%) 6.66 0.53 (+2%) Each value indicates the mean SD of seven independent experiments (seven rats per group). The average of each experiment arose from three evaluations of the homogenated brain of each animal. Group A represents “nontreatment” control and group C controls administered the LC dose subcutaneously. Values of saline controls (SC-3h and SC3h) were not statistically different as compared to that C-3h. Note: Only statistically significant values are presented. **: p < 0.01 (vs control), ***: p < 0.001 (vs control) Na+, K+-ATPase: a/f, a/g, a/h, a/i, a/j, a/l, a/m, a/n, b/f, b/g, b/h, b/i, b/j, b/l, b/m, b/n, c/f, c/g, c/h, c/i, c/j, c/l, c/m, c/n, d/f, d/g, d/h, d/i, d/j, d/l, d/m, d/n = p < 0.01 Mg2+-ATPase: a/e, a/f, b/e, b/f, c/e, c/f, d/e, d/f = p < 0.001 Table 4: The effect of L-C (5 mM) incubation on the swimming rat brain AChE and Na+, K+-ATPase activities with L-C (group D) or without L-C (group B) administration (see Materials and Methods). AChE activity (ΔOD/min x mg prot) Na+, K+-ATPase activity (μmol Pi/h x mg prot) AChE activity (ΔOD/min x mg prot) Na+, K+-ATPase activity (μmol Pi/h x mg prot) Control 3h (group A) 0.886 0.035a Swimming 3h (group B) 0.576 0.032b Group B 3h + L-C incubation 0.592 0.040e 5.92 0.59a 11.84 1.12b 11.14 0.95e L-C Control 3h (group C) 0. 726 0.043c Swimming 3h (group D) 0.600 0.031d Group D 3h + L-C incubation 0.730 0.038f 5.11 0.56c 2.77 0.22d 5.00 0.48f Values are expressed as mean SD, for four different experiments. AChE: a/b, a/e, c/d, d/f, = p < 0.001; b/e, c/f = NS Na+, K+-ATPase: a/b, a/e, c/d, d/f, = p < 0.001; b/e, c/f = NS