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Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.

Testosterone and its synthetic derivatives anabolic-androgenic steroids have been shown to increase skeletal muscle work capacity and fatigue resistance, but the molecular basis for these effects remains uncertain. Since muscle performance has been related to redox status of exercising muscles, this investigation was aimed at testing whether a treatment with suprapharmacological doses of the anabolic-androgenic steroid stanozolol, (2 mg/kg body weight, 5 days/week, for 8 weeks), either alone or in conjunction with treadmill training (12 weeks), enhanced antioxidant defences in rat muscles. Stanozolol treatment did not modify thiobarbituric acid reactive substances and glutathione content in soleus and extensor digitorum longus (EDL) homogenates. In soleus from sedentary rats, superoxide dismutase and glutathione reductase activities were increased by 25% (P < 0.05) and by 40% (P < 0.01) after stanozolol administration, whereas catalase and glutathione peroxidase activities were not modified. This response was similar to that induced by training alone. In EDL from sedentary rats, stanozolol increased only superoxide dismutase activity (20%, P < 0.05). In no case, the effects of steroid administration and training were additive. HSP72 levels were up-regulated in soleus (1.5-fold, P < 0.01) and EDL (threefold, P < 0.001) following training but remained unchanged after stanozolol treatment. Endurance capacity, assessed in a treadmill endurance test, was similar for treated and control rats. We conclude that stanozolol treatment increases antioxidant capacity in selected skeletal muscles from sedentary rats. However, the steroid was not effective in improving endurance capacity or enhancing the training effects on muscle antioxidant defence systems.

[Background] Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP)., [Methods] The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted., [Results] The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity., [Conclusions] Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site., This work was funded by the Spanish Ministry of Science and Innovation (SAF2002-04057-CO2-01) and the Instituto de Salud Carlos III (ISCIII) as part of project MPY 1404/09, and ISCIII-AESI project MPY 1279/15. Carolina Hurtado and Pamela Campioli were supported by grants from the ISCIII (Fondo de Investigaciones Sanitarias), through VI NP of I + D + I (2008–2011), ISCIII General Sub-Direction of Networks and Centers for Collaborative Research (RETIC-RICET, RD06/0021/0019, RD12/0018/011). Victoria Martínez Sernández holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario).

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37 degrees C, the rate of H2O2 release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03-0.15 mg protein/ml), to decrease (0.2-0.5 mg protein/ml) and to be negligible (over 0.5 mg protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2 mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1 mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.

The present study was conducted to investigate the effects of long-term exercise training on the main components involved in excitation-contraction coupling and relaxation in rat myocardium. Twenty male Wistar rats were divided into sedentary (S) and treadmill-trained (T) groups. Group T was trained for 24 weeks, 5 days/week (25 m/min, 45-60 min, 0% slope). 48 h after the last exercise session, animals were killed and ventricular and soleus muscle homogenates were obtained. The citrate synthase activity in soleus muscle was significantly increased (163%) in T compared with S rats ( P0.01), confirming the exercise training efficacy. Although heart weight and cardiac oxidative capacity were not modified by exercise training, the binding of [(3)H] ryanodine and the dihydropyridine [(3)H]PN200-110 to cardiac homogenates, and sarcoplasmic reticulum Ca(2+)-ATPase activity were increased significantly in the ventricular homogenates from T compared with S animals ( P0.01). Western blot analysis of ventricular homogenates failed to show significant alterations in dihydropyridine receptor and Ca(2+)-ATPase levels in T animals, but revealed an increase of ryanodine receptor density in this group ( P0.01). The activity of the ectoenzymes 5'-nucleotidase and Mg(2+)-ATPase was not affected by training ( P0.05). In conclusion, long-term treadmill training induces adaptive changes in some of the components of myocardial rat excitation-contraction coupling and relaxation systems that could contribute to the improvement of cardiac function.

The purpose of this work was to study whether exercise training induces changes in the lipid profile of rat aorta and nervous system and in the in vitro intrinsic responsiveness of these tissues to endothel in-1 (ET-1) treatment. The exercise program performed successfully produced the characteristic metabolic alterations of the trained state. Exercise training induced a large and significant increase in the levels of both aortic ethanolamine plasmalogens (PlasEtn) and glucosylceramides. In contrast, a decrease of aortic ceramide and cholesterol levels was evoked by exercise training. ET-1 increased PlasEtn content only in sedentary animals. An exercise-induced increase in cerebellum levels of ceramides and ceramide monohexosides was found. The cerebellum ceramide content was increased by ET-1 more noticeably in sedentary rats than in trained animals. In contrast, cerebral cortex was observed to be largely insensitive to both exercise training and ET-1 treatment. It was concluded that exercise training (i) induces changes in both vascular and cerebellar lipid profiles, the former being much more pronounced than the latter, and (ii) diminishes the aortic and cerebellar sensitivity to ET-1 action.

The purpose of this investigation was to examine the effects of chronic and acute exercise on the main components involved in excitation-contraction coupling and relaxation in rat heart. Sixty male Wistar rats were divided into a sedentary (S) and three 12-wk treadmill-trained groups (T-1, moderate intensity; T-2, high intensity; T-3, interval running). After 12-wk, 15 rats from the S group and 15 rats from the T-2 group were subjected to a single treadmill-exercise session until exhaustion before being killed at 0, 24, or 48 h (acute exercise). The remaining animals were killed 48 h after the last standard exercise session (chronic exercise). The efficacy of the training programs was confirmed by an increase in treadmill endurance time and in skeletal muscle citrate synthase activity. None of the exercise programs modified heart weight or cardiac oxidative capacity. [3H]PN200–110 and [3H]ryanodine binding to cardiac homogenates indicated that the density of L-type and sarcoplasmic reticulum (SR) Ca2+channels was the same in S and trained rats. The SR Ca2+-ATPase activity was also unmodified. Finally, the activities of the ectoenzymes Mg2+-ATPase and 5′-nucleotidase, which are involved in degradation of extracellular nucleotides, were not affected by either of the running programs. After the acute exercise session, no changes were detected in either of the tested parameters in heart homogenates of S and T-2 animals. We conclude that neither treadmill-exercise training for 12 wk nor exhaustive exercise alters the density of Ca2+channels involved in excitation-contraction coupling or the SR Ca2+-ATPase and the ectonucleotidase activities in rat heart.

Purpose The aim of this study was to examine the separate and combined effects of an 8-wk treatment with high doses of 17alpha-alkylated anabolic-androgenic steroids (AAS) and exercise training on selected lysosomal and mitochondrial enzyme activities in rat liver. Methods Sedentary and treadmill-trained (25 m x min(-1), 45 min x d(-1), 5 d x wk(-1)) male rats were treated with fluoxymesterone, methylandrostanolone, or stanozolol (2 mg x kg body weight(-1), 5 d x wk(-1)) for 8 wk. Results Acid phosphatase, arylsulfatase, beta-glucuronidase, and beta-galactosidase activities were increased in liver homogenates of sedentary and trained AAS-treated rats. The mitochondrial respiratory chain activities rotenone-sensitive NADH-cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome oxidase (COX) showed a significant decrease in steroid-administered rats, whereas citrate synthase (CS), a matrix enzyme, exhibited no changes in activity, pointing to a selective effect of AAS on mitochondrial membrane complexes. In vitro studies in mitochondrial fractions isolated from the liver of control rats showed that COX and CS activities were insensitive to the AAS, whereas NCCR and SCCR activities were partly inhibited. On the other hand, the mean values of serum parameters related to hepatic function were within normal ranges in all the experimental groups of animals. Conclusions The present data show that 8-wk ingestion of three different anabolic-androgenic steroids, either with or without concurrent exercise training, affects lysosomal hydrolases and mitochondrial respiratory chain electron transport in rat liver without modifying classical serum indicators of hepatic function.

Isolated Langendorff-perfused hearts from sedentary and prolonged (24 weeks) treadmill-trained rats were subjected to 30 min of normoxic perfusion either alone or followed by 20 min of global ischaemia, or by 20 min of global ischaemia and 15 min of normoxic reperfusion. Pre-ischaemic values of antioxidant enzyme activities and ecto-5'-nucleotidase activity were not different in sedentary and trained hearts but a 5-fold increase of 72-kDa heat shock protein (HSP72) levels was detected in trained myocardium. After ischaemia and reperfusion (I/R), metabolic recovery was better in trained than in sedentary hearts as indicated by higher ATP and creatine phosphate levels. However, antioxidant enzymatic activities, glutathione reductase, and total and mitochondrial superoxide dismutase decreased in trained rats after I/R, whereas they remained unchanged in the sedentary ones. Ecto-5'-nucleotidase activity was modified by I/R in sedentary as well as in trained hearts while HSP72 content did not change. Ecto-5'-nucleotidase activity and HSP72 content increased in parallel by the 30-min perfusion period. In conclusion, the cardioprotection induced by long-term training could be mediated by the exercise-induced increase in HSP72 levels and is not related to enhanced antioxidant systems or ecto-5'-NT activity.

The abuse of anabolic-androgenic steroids (AAS) to enhance physical performance is widespread in sport communities despite their reported side effects. Since the biochemical bases for the hepatotoxic effects of these compounds are largely unknown, this investigation was aimed at testing whether prolonged (8 weeks) treatment with high doses (2 mg kg(-1) body weight; 5 d wk(-1)) of stanozolol (ST), either alone or in conjunction with treadmill-exercise training, induced changes in oxidative stress biomarker levels and antioxidant defence systems in rat liver. After ST oral administration, the mean values of serum parameters related to hepatic function were within normal ranges. No changes in protein carbonyl content and in the reduced to oxidized glutathione (GSH/GSSG) ratio were detected in liver homogenates of ST-treated rats, whereas thiobarbituric acid-reactive substances (TBARS) levels resulted increased (P

Treatment of male rats with the anabolic steroids fluoxymesterone or methylandrostanolone increased the activity of the outer carnitine palmitoyltransferase in liver and fast-twitch muscle mitochondria. This effect was not potentiated by physical exercise and was not observed in heart and slow-twitch muscle mitochondria. Anabolic steroids did not affect the sensitivity of the liver enzyme to inhibition by malonyl-CoA. The data presented herein suggest that androgens may have an important physiological role in the regulation of fatty acid oxidation in liver and fast-twitch muscle mitochondria. In addition, our results are at odds with the notion that (most of) the metabolic effects of anabolic steroids on muscle are only evident when physical training is parallely performed.

The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990, Anal. Biochem. 191, 321-331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 microM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 +/- 2.0 micromol. min-1 x g-1 tissue wet weight (n = 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation, Km for ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.

The chicken T-tubule Mg2+-ATPase is an integral membrane glycoprotein that presents properties different from those of other ATPases located in skeletal muscle cells and exhibits ATP-hydrolysing activity on the extracellular side of the transverse tubule (TT) membranes. In this study we demonstrate that TT vesicles purified from chicken skeletal muscle possess ecto-ADPase and ecto-5ʹ-nucleotidase activities that, along with ecto-ATPase, are able to sequentially degrade extracellular ATP to ADP, AMP and adenosine. Characterization studies of these TT ectonucleotidases revealed remarkable differences between ecto-ATPase and ecto-ADPase activities with respect to thermal stability, temperature dependence of the hydrolytic activity, effect of ionic strength, kinetic behaviour, divalent cation preference and responses to azide, N-ethylmaleimide, NaSCN, Triton X-100 and concanavalin A. Ecto-ATPase, but not ecto-ADPase, was inhibited by a polyclonal antibody against the chicken TT ecto-ATPase. On the basis of these results we propose that ATP and ADP hydrolysis are accomplished by two distinct enzymes and therefore the TT ecto-ATPase is not an apyrase. 5ʹ-Nucleotidase activity was inhibited by adenosine 5ʹ-[α,β-methylene]diphosphate and concanavalin A, followed simple Michaelis-Menten kinetics and was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that AMP hydrolysis in T-tubules is catalysed by a typical ecto-5ʹ-nucleotidase. Results obtained from electrophoresis experiments under native conditions suggest that ecto-ATPase, ecto-ADPase and 5ʹ-nucleotidase might be associated, forming functional complexes in the T-tubule membranes. The TT ectonucleotidases constitute an enzymic cascade for the degradation of extracellular ATP that might be involved in the regulation of purinergic signalling in the muscle fibre.

Calcium overload is a fundamental pathogenic event associated with chronic muscle degeneration in muscular dystrophies. The possibility that L-type voltage-dependent calcium channels were involved in the etiology of chicken muscular dystrophy was investigated by studying the dihydropyridine receptors in transverse tubule membranes isolated from skeletal muscle of normal (line 412) and dystrophic (line 413) chickens. The yield of T-tubular protein from dystrophic muscle was considerably increased compared with that from normal muscle (2.51 +/- 0.18 vs 1.04 +/- 0.31 mg protein x 100 g muscle-1). The binding of the calcium channel antagonist (+) [3H]PN200-110 to the dihydropyridine receptor in transverse tubule preparations was relatively slow, markedly affected by temperature and required divalent cations. (+) [3H]PN200-110 equilibrium binding assays revealed a single class of high-affinity sites and showed that maximum binding capacity (Bmax) (3.17 +/- 0.47 for normal and 3.51 +/- 0.52 pmol x mg protein-1 for dystrophic transverse tubules) and dissociation constant (Kd) (0.32 +/- 0.07 and 0.26 +/- 0.09 nM, respectively) were not significantly different in normal and dystrophic membranes. Kinetic studies indicated that normal and dystrophic transverse tubules did not differ significantly in association (2.54 x 10(6) and 2.27 x 10(6) M(-1)s(-1), respectively) and dissociation (8.5 x 10(-4) and 9.3 x 10(-4)s(-1), respectively) rate constants. Since dissociation kinetics for both preparations were monoexponential under all the experimental conditions employed, no low-affinity binding sites for (+) [3H]PN200-110 could be detected in chicken transverse tubules membranes. However, immunoblot assay, using a monoclonal antibody, revealed that dystrophic transverse tubules as compared with normal membranes were enriched twofold with the alpha 1-subunit of the dihydropyridine receptor. Therefore, although dihydropyridine-binding sites were not altered in transverse tubule membranes from dystrophic chicken skeletal muscle, both the increased yield in T-tubule vesicles and the enhanced immunodetection of the alpha 1-subunit of the dihydropyridine receptor, suggest that total content in dihydropyridine receptor is higher in dystrophic than in normal muscle.

To examine the influence of exercise training on the expression of dihydropyridine (DHP)-sensitive Ca2+ channels in skeletal and cardiac muscle, we have determined DHP receptor levels by [3H]PN200-110-binding and immunoblot analysis in homogenates and microsomal fractions of slow- and fast-twitch muscles and heart from rats subjected to a 12-week programme of moderate endurance training. We found that exercise increases the amount of DHP receptor in homogenates of the slow-twitch soleus (42%) and the fast-twitch extensor digitorum longus (60%). Comparable increases in DHP receptor density with training were also observed in the microsomal fractions isolated from both skeletal muscles; these increases were not due to differences in the membrane composition of the microsomal fractions, since the relative proportion of specific enzyme markers was not affected by exercise training. Levels of DHP receptor were not modified in cardiac muscle as a result of the exercise programme. These data suggest an up-regulation of the DHP receptor in the skeletal muscle as a consequence of exercise training, which may play a role in the adaptation of skeletal muscle to increased contractile activity.

The effects of anabolic-androgenic steroid administration and exercise training on various aspects of hepatic function were investigated in sedentary and trained (treadmill for 12 wk) male and female rats treated orally with fluoxymesterone or methylandrostanolone (2 mg.kg-1 body weight, 5 d.wk-1 for 8 wk). The mean values of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total and direct bilirubin, and total- and high-density lipoprotein-cholesterol remained within normal range in all groups of male animals. The same is true for female rats, except for an increase in alkaline phosphatase activity in the steroid-treated groups. Hepatic microsomal aniline p-hydroxylase activity was reduced in male and increased in female rats by either steroid, whereas no significant effect was detected on 7-ethoxycoumarin deethylase activity. The levels of cytochrome P-450 and cytochrome b5 were markedly decreased by the anabolic-androgenic steroid treatment in male rat microsomes, but neither the steroid administration nor exercise training induced significant changes in the cytochrome levels of female rat livers. Taking into account the significant increase in microsomal protein yield elicited by fluoxymesterone or methylandrostanolone treatment both in males and females, it is noteworthy that the total monooxygenase activities and cytochrome P-450 content, expressed on a per gram liver basis, were significantly increased in female whereas they were apparently unchanged in male rats. In conclusion, the present data show that the prolonged ingestion of high doses of anabolic-androgenic steroids, either with or without concurrent exercise training, can modify in a sex-dependent manner the capacity of rat liver to metabolize drugs without affecting classical serum indicators of hepatic function.

Soleus and extensor digitorum longus (EDL) mitochondria and sarcotubular system were examined in sedentary and trained (treadmill for 12 wk) male rats that were treated with fluoxymesterone or methandrostanolone (2 mg/kg, 5 days/wk, for 8 wk). Neither physical exercise nor anabolic/androgenic steroid administration resulted in a significant change in muscle wet weight. Treatment with the anabolizing androgens increased succinate dehydrogenase activity in fast-twitch muscle mitochondria; this effect was not enhanced by training and was not observed in soleus mitochondria. On the other hand, the content of the slow-twitch muscle in sarcotubular fraction was increased in sedentary rats by fluoxymesterone or methandrostanolone treatment, whereas no significant changes were found in EDL. The training program affected adenosinetriphosphatase (ATPase) activities in the sarcotubular fraction; Mg2(+)-ATPase was increased in both soleus and EDL, but Ca2(+)-ATPase was decreased only in soleus. However, in sedentary animals only the Mg2(+)-dependent activity of EDL was increased by anabolizing androgen treatment, and this change was not potentiated by additional training. The present data indicate that anabolic/androgenic steroids can affect mitochondrial and sarcotubular enzymes in skeletal muscle. The effects are muscle-type specific.

Purified transverse tubule membranes from normal and dystrophic chicken skeletal muscle were isolated by a calcium-loading procedure. Normal and dystrophic T-tubules were similar in cholesterol content and (Na + ,K + )-ATPase and 5′-nucleotidase activities but a significant decrease of Mg 2+ -ATPase activity was observed in dystrophic membranes. A comparative analysis of the enzyme properties revealed that the kinetic parameters were altered in dystrophic T-tubules and the ATP-hydrolyzing activity was differently affected by the ionic strength. However, the influence of temperature and the regulatory effect of concanavalin A were the same as in normal T-tubules. Membrane fluidity was similar in both preparations as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium diphenylhexatriene. These results point to an impairment in the function of Mg 2+ -ATPase due to structural alterations of the enzyme.