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Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming
Comparative Biochemistry and Physiology, Part C 139 (2004) 219 – 224 www.elsevier.com/locate/cbpc
Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming W.L.G. Cavalcantea, M. Dal Pai-Silvab, M. Gallaccia,* a
Department of Pharmacology, Institute of Bioscience, University Estadual Paulista (Unesp), Botucatu, Sa˜o Paulo, CEP 18618-000, Brazil b Department of Morphology, Institute of Bioscience, University Estadual Paulista (Unesp), Botucatu, Sa˜o Paulo, CEP 18618-000, Brazil Received 10 August 2004; received in revised form 10 November 2004; accepted 14 November 2004
Abstract This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 910 7 M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin–eosin (HE); nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA ( pb0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3F4.7% vs. S=75.7F2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81F3.1% vs. Sw=71.0F4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. On the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise. D 2004 Elsevier Inc. All rights reserved. Keywords: Anabolic–androgenic steroids; Swimming; Safety margin; Neuromuscular junction
1. Introduction Anabolic–androgenic steroids (AAS) are synthetic derivatives of testosterone modified to enhance anabolic (myotrophic) rather than androgenic (masculinizing) actions of the original hormone (Kochakian, 1993). Due to these characteristics, suprapharmacologic doses of AAS have been taken by athletes in the expectation to increase
* Corresponding author. Tel.: +55 14 6802 6253; fax: +55 14 3815 3744. E-mail address: [email protected] (M. Gallacci). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.11.004
muscle size and improve performance in sportive activities (Lukas, 1993). Many studies have been focused on the effects of AAS in muscle structure and function in both humans (Fiedl, 2000; Kuhn, 2002) and animals (Bates et al., 1987; Lewis et al., 1999; Joumaa and Leoty, 2001), but their results are controversial. The structure and function of skeletal muscle are largely dependent on the interaction between motor nerves and muscles that occurs in the neuromuscular junction (Herscovich and Gershon, 1987). Previous works have shown that androgen receptors may be expressed in both motoneuron and skeletal muscle (Breedlove and Arnold, 1980; Michel and Baulieu, 1980). In addition, steroid hormones,
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including metabolites of testosterone and their synthetic derivatives, may acts as allosteric modulators of the nicotinic acetylcholine (Ach) receptor family of ligandgated ion channels (Blanton et al., 1999). In this work, we investigated the impact of suprapharmacologic doses of AAS upon the overall safety margin of synaptic transmission of rats submitted to swimming. Contractile properties, fatigue resistance, and fibers characteristics of EDL muscle were also studied.
polygraphic recording of muscle tension was performed by means of an isometric transducer (GM2/GM3-Gould). 2.3. Contractile properties of indirect twitches Peak of twitch force, time to peak, and relaxation time were determined from a series of indirect twitches evoked at a frequency of 0.5 Hz. 2.4. Safety margin of synaptic transmission
2. Materials and methods 2.1. Animals and treatment Male Wistar rats (Rattus norvegicus) 60 days old were used. Animals were housed in groups of four in standard rat cages in a temperature-controlled room (23F2 8C) and exposed to a 12-h light/dark cycle. Food and water were provided ad libitum. All procedures are in accordance with Canadian Council on Animal Care, and had local ethical committee approval. Nandrolone decanoate (ND) was injected subcutaneously at a dose of 3 mg/kg, 5 days/week, for 4 weeks. Rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. Rats swam in groups of 5 or 6, in a heated tank (33F1 8C) with an average water depth of 60 cm. Initially, to familiarize animals with water contact, rats were kept in a tank with a water depth of 7 cm for 15 min daily for 1 week.
The overall safety margin of the synaptic transmission was evaluated from the susceptibility of indirectly evoked contractions of the EDL muscle to the blockade induced by pancuronium. This agent is able to affect the synaptic transmission both by inhibiting the effect of ACh on postsynaptic nicotinic and by reducing the amount of ACh released via inhibition of positive-feedback modulation of ACh release (Vizi et al., 1987; Bowman et al., 1988). Suitable concentrations of pancuronium (710 7 M and 910 7 M) were added to the organ bath, respecting a period of 30 min between them. The ratio of the muscle tension in the presence and absence of pancuronium was used to estimate the safety margin of neuromuscular transmission. 2.5. Fatigue resistance Fatigue resistance protocol consisted of 180 brief tetanic contractions, 0.25 sec long and directly elicited every 5 sec at a frequency of 30 Hz (Fitts and Holloszy, 1977). Fatigue
2.2. Neuromuscular preparation and recording of muscle contractions The sciatic nerve extensor digitorum longus (EDL) muscle preparation was removed and mounted for myographic recording bin vitroQ, according to Gallacci and Oliveira (1994). Briefly, the preparation was mounted vertically in a conventional isolated organ-bath chamber containing 50 mL of physiological solution of the following composition (mmol/L): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; NaHCO3, 15; NaH2PO4, 1, glucose, 11. This solution was gassed with O2 (95%)+CO2 (5%), which kept the pH at 7.4 to 7.5, and maintained at 27 8C. Indirect contractions were evoked by supramaximal strength pulses (0.5 Hz; 0.5 ms), delivered from an electronic stimulator (Narco Bio-System) and applied on the phrenic nerve by means of a suction electrode. Direct contractions were evoked by supramaximal pulses (0.2 Hz, 5 ms) through a bipolar electrode positioned on opposite sides of the muscle. To avoid the interference of indirect contractions, these experiments were performed in the presence of pancuronium bromide (210 6 M). The
Fig. 1. Safety margin of neuromuscular transmission evaluated from the resistance to the indirectly evoked contractions of EDL muscle to pancuronium (710 7 M) in sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Twitch amplitudes were expressed as a percentage from the control situation (without pancuronium). Values are meansFS.E., n=5 animals for groups, at least. *Significant difference ( pb0.05); two-way ANOVA.
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resistance was defined as the force developed in the last contraction of a series relative to the force in the first contraction and was expressed as a percentage. Following recordings muscles were dried and weighed.
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ences among group means. Probability values ( P)b0.05 were considered significant.
3. Results 2.6. Histological and histochemical procedures Small fragments of EDL muscle were cooled in nhexane, and then frozen in liquid nitrogen ( 196 8C). Transverse muscle sections (7 to 10 Am) were submitted to haematoxylin and eosin (HE), for morphological analysis, and to nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-TR) for metabolic pattern evaluation (Dubowitz, 1985). 2.7. Statistical analysis All results are expressed as the meanFstandard error (S.E.) in the tables and are indicated by vertical bars in the figures. Data were analyzed by two-factor analysis of variance method (with exercise and vitamin E as factors). When a significant F ratio was found, Student–Newman– Keuls multiple-range test was used to assess the differ-
The overall safety margin of synaptic transmission, evaluated in terms of resistance to neuromuscular blockade induced by suitable concentrations of pancuronium, is shown in Fig. 1. Swimming did not change the susceptibility to the neuromuscular blockade induced by pancuronium in animals not treated with ND (SwS), indicating that the factor swimming alone does not affect the safety margin of synaptic transmission. In contrast, ND reduced the safety margin of sedentary animals. This effect was evident from the observation that the resistance to the blockade induced by pancuronium (710 7 M) was lower in SND than in S rats. However, there was a significant interaction between the factors swimming and ND, since there was no change in pacuronium resistance between Sw and SwND. This result indicates that ND does not affect the safety margin of animals submitted to swimming.
Fig. 2. Contractile properties of indirectly evoked twitches of EDL muscles from sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Values are meansFS.E., n=5 animals for groups, at least. (A) Twitch amplitude was expressed in grams of force by grams of tissue. (B and C) Time to peak and time to fall of twitches were expressed in milliseconds.
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Fig. 3. Fatigue resistance evaluated from the decrease of 180 direct contractions of EDL muscle of sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Values are meansFS.E., n=5 animals for groups, at least. Amplitude of directly evoked twitches were expressed as a percentage to the initial amplitude. *Significant difference ( pb0.05); two-way ANOVA.
The amplitude and the kinetic properties of indirectly evoked twitches (time to peak and time to relaxation) were not changed by swimming, ND or the combination of swimming and ND (Fig. 2). Animals submitted to swimming showed higher resistance to fatigue when compared to sedentary (SwS). However, the treatment with ND did not affect the fatigue resistance of both sedentary (SSND) and swimming rats (SwSwND; Fig 3). Histological examinations of hematoxylin–eosin-stained muscles showed no obvious differences among groups.
Fig. 5. EDL muscles of rats: S (A), SND (B), Sw (C), and SwND (D), submitted to NADHTR histochemical reaction. Muscle fibers SO (1), FOG (2), and FG (3). NADHTR, 50.
Fiber dimensions were not affected by swimming or treatment with ND (Fig. 4). Histochemistry analysis (NADH-TR) did not revealed alterations in the proportions of oxidative and glycolytic fibers between the experimental groups (Fig. 5). During the 4 weeks of nandrolone treatment, there was no change in animals’ body weight. During the experimental period, all groups showed gain of body mass. However, as shown on Fig. 1, the gain of body mass in SND and in Sw were lower that of S groups. Both nandrolone treatment and swimming reduced the gain of body mass (SSND and SSw).
4. Discussion
Fig. 4. EDL muscles of rats: S (A), SND (B), Sw (C), and SwND (D) submitted to HE. Muscle fibers with normal morphology (F). Peripheric nuclei (N). Endomysium (E). HE: 50.
In this work, swimming was chosen as a suitable mode of exercising rats since they are good swimmers by nature and are less stressed than when subjected to treadmill running, wherein the animals are constantly under the fear of electrical stimulation (Russell et al., 1980; Sonne and Galbo, 1980). Rosenheimer (1985) noted that the nerve terminals from sedentary shocked controls were markedly different either from regular sedentary controls or from animals that were exercised with shock for motivation. This suggests that the stress represented by electrical shock is a confounding variable with respect to nerve terminal morphology and, consequently, it could also change the neuromuscular transmission. The present data demonstrate that administration of nandrolone decanoate to young adult rats submitted to swimming did not produce ergogenic effects. First, it should be mentioned that anabolic effects of AAS are influenced by
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a series of conditions, such as studied species, age, circulating synergic hormones, muscle, androgen, dose mode of administration, duration of treatment, and activity level (Bisschop et al., 1997). All of these variables probably play a role in the lack of consistent results found in the literature when anabolic agents are used. In the present study, the body mass was unaltered by concomitant administration of the anabolic steroid and swimming. This is similar to what has been reported for male young rats where body mass is not changed (Prezant et al., 1993; Saborido et al., 1991; Gayan-Ramirez et al., 2000) or stunted (Hervey and Hutchinson, 1973; Kochakian and Endahl, 1959), depending on the dose used. Although the exact mechanism of this effect is not known, it is possible that the basal level of the circulating endogenic androgen will play a role, as previously described (Bisschop et al., 1997). In the present work, the kinetic properties of indirect twitches as well as the fatigue resistance of EDL muscle remained unchanged after nandrolone treatment. Several studies examined effects of anabolic steroids on contractility and fatigue of peripheral muscles and revealed no additional effects of these agents when compared with those obtained with training alone (Egginton, 1987; Exner et al., 1973; Lubek, 1984; Bisschop et al., 1997; Gayan-Ramirez et al., 2000; Tamaki et al., 2001). The EDL muscle fibers distribution also is not changed, indicating that the treatment with suprapharmacologic dose of nandrolone for 4 weeks does not modify muscle phenotype. These results agree well with those of Tsika et al. (1987), Bisschop et al. (1997), and Noirez and Ferry (2000). Suggested mechanisms for the ergogenic effects of AAS include the possibility that these agents: (1) act through the central nervous system, allowing the subjects to train harder (Ariel and Saville, 1972); or (2) may improve skeletal muscle function directly by increasing protein synthesis (Egginton, 1987; Griggs et al., 1989). The major finding of this work was the observation that nandrolone decanoate reduces the safety margin of synaptic transmission in rats sedentary but did not affect this margin in animals submitted to 4 weeks of swimming. The excess of both nicotinic receptor and acetylcholine at the synapse assures the safety margin of the neuromuscular transmission. It is largely known that physical exercise increases the safety margin of synaptic transmission (Dorlo¨chter et al., 1991; Fahim, 1997; Desaulniers et al., 1998) by promoting hypertrophy of the synapses. However, the findings of this work showed that 4 weeks of swimming was not enough to increase this safety margin. On the other hand, nandrolone reduced the safety margin of sedentary animals, but did not affect that margin in rats submitted to swimming. Previous work has shown that suprapharmacological doses of anabolic androgenic steroids may increase choline acetyltransferase (ChAT) mRNA levels in motoneurons (Blanco et al., 1997, 2001). On this basis, it has been suggested that AAS could lead to a greater presynaptic capacity to synthesize Ach. These actions obviously do not explain
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the AAS-induced decrease in the safety margin of neuromuscular transmission observed in this work. However, in vitro studies have shown that steroid hormones, including testosterone and their synthetic derivative may induce blockade of nicotinic receptors (Valera et al., 1992; Bouzat and Barrantes, 1996; Arias, 1997). This blockade seems to be of noncompetitive type and be caused by the interaction of the hormone with the lipid protein interface and/or extracellular domain of the nicotinic receptor (Arias, 1997). Thus, it is possible that the treatment with high dose of nandrolone lead to a residual blockade of the nicotinic receptor, and this reduces the safety margin of neuromuscular transmission of sedentary animal. This effect was not evident in swimming rats due to the interaction between the anabolic androgenic steroid with exercise. Although exercise alone was not able to increase the safety margin, anabolic androgenic steroids may further enhance the myotrophic effect of exercise (Dahlberg et al., 1981). This myotrophic effect was not expressed as an increase in the safety margin in trained animals due to the residual blocking action of anabolic androgenic steroid. In conclusion, this study shows that the treatment of swimming rats with suprapharmacologic dose of nandrolone decanoate during 4 weeks does not promote ergogenic effects. On the contrary, this work revealed that high dose of the steroid hormone may compromise the safety margin of neuromuscular transmission in animals not submitted to exercise. Although the present study was performed in rats, our results may indicate that high doses of nandrolone decanoate would not result in additional benefit to humans submitted to a short training program. On the other hand, untrained individuals should be more susceptible to a fade on the process of the neuromuscular transmission. Nevertheless, care should be taken when one extrapolates data from animal models to humans.
Acknowledgments This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (0201278-8).
References Arias, H.R., 1997. The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor. Biochim. Biophys. Acta 1347, 9 – 22. Ariel, G., Saville, W., 1972. Effect of anabolic steroids on reflex components. J. Appl. Physiol. 32, 795 – 797. Bates, P.C., Chew, L.F., Millward, D.J., 1987. Effects of the anabolic steroid stanozolol on growth and protein metabolism in the rat. J. Endocrinol. 114, 373 – 381. Bisschop, A., Gayan-Ramirez, G., Rollier, H., Dekhuijzen, P.N., Dom, R., De Bock, V., Decramer, M., 1997. Effects of nandrolone decanoate on respiratory and peripheral muscles in male and female rats. J. Appl. Physiol. 82, 112 – 118.
224
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Blanco, C.E., Popper, P., Micevych, P., 1997. Anabolic–androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat. Neuroscience 78, 873 – 882. Blanco, C.E., Zhan, W.Z, Fang, Y.H., Sieck, G., 2001. Exogenous testosterone treatment decreases diaphragm neuromuscular transmission failure in male rats. J. Appl. Physiol. 90, 850 – 856. Blanton, M.P., Xie, Y., Dangott, L.J., Cohen, 1999. The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid-protein interface. Mol. Pharmacol. 55, 269 – 278. Bouzat, C., Barrantes, F.J., 1996. Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone. Possible allosteric mechanism of channel blockade. J. Biol. Chem. 271, 25835 – 25841. Bowman, W.C., Marshall, I.G., Gibb, A.J., Harborne, A.J., 1988. Feedback control of transmitter release at the neuromuscular junction. Trends Pharmacol. Sci. 9, 16 – 20. Breedlove, S.M., Arnold, A.P., 1980. Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science 210, 564 – 566. Dahlberg, E., Snochowski, M., Gustafsson, J.A., 1981. Regulation of the androgen and glucocorticoid receptors in rat and mouse skeletal muscle cytosol. Endocrinology 108, 1431 – 1440. Desaulniers, P., Lavoie, P.A., Gardiner, P.F., 1998. Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle. NeuroReport 9, 3549 – 3552. Dorlfchter, M., Irintcchev, A., Brinkers, M., Wernig, A., 1991. Effects of enhanced activity on synaptic transmission in mouse extensor digitorum longus muscle. J. Physiol. 436, 283 – 292. Dubowitz, V., 1985. Normal muscle. Muscle Biopsy: A Practical Approach, 2 edn. Bailliere Tindall, London, pp. 41 – 48. Egginton, S., 1987. Effects of an anabolic hormone on striated muscle growth and performance. Pflugers Arch. 410, 349 – 355. Exner, G.U., Staudte, H.W., Pette, D., 1973. Isometric training of rats— effects upon fast and slow muscle and modification by an anabolic hormone (nandrolone decanoate): II. Male rats. Pflugers Arch. 345, 15 – 22. Fahim, M.A., 1997. Endurance exercise modulates neuromuscular junction of C57BL/6NNia aging mice. J. Appl. Physiol. 83, 59 – 66. Fiedl, K.E., 2000. Effect of anabolic steroid use on body composition and physical performance. In: Yesalis, C.E. (Ed.), Anabolic Steroids in Sport and Exercise. Human Kinetics, Champaign, IL, pp. 139 – 174. Fitts, R.H., Holloszy, J.O., 1977. Contractile properties of rat soleus muscle: effects of training and fatigue. Am. J. Physiol. 233, 86 – 91. Gallacci, M., Oliveira, A.C., 1994. Pre- and postsynaptic mechanisms involved in tetanic fade induced by pancuronium in the isolated rat muscle. Pharmacology 49, 265 – 270. Gayan-Ramirez, G., Rollier, H., Vanderhoydonc, F., Verhoeven, G., Gosselink, R., Decramer, M., 2000. Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm. J. Appl. Physiol. 88, 26 – 34. Griggs, R.C., Kingston, W., Jozefowicz, R.F., Herr, B.E., Forbes, G., Halliday, D., 1989. Effect of testosterone on muscle mass and muscle protein synthesis. J. Appl. Physiol. 66, 498 – 503.
Herscovich, S., Gershon, D., 1987. Effects of aging and physical training on the neuromuscular junction of the mouse. Gerontology 33, 7 – 13. Hervey, G.R., Hutchinson, I., 1973. The effects of testosterone on body weight and composition in the rat. J. Endocrinol. 57, 24. Joumaa, W.H., Leoty, C., 2001. Differential effects of nandrolone decanoate in fast and slow rat skeletal muscles. Med. Sci. Sports Exerc. 33, 397 – 403. Kochakian, C.D., 1993. History, chemistry and pharmacodynamics of anabolic–androgenic steroids. Wien. Med. Wochenschr. 143, 359 – 363. Kochakian, C.D., Endahl, B.R., 1959. Changes in body weight of normal and castrated rats by different doses of testosterone propionate. Proc. Soc. Exp. Biol. Med. 100, 520 – 522. Kuhn, C.M., 2002. Anabolic steroids. Recent Prog. Horm. Res. 57, 411 – 434. Lewis, M.I., Fournier, M., Yeh, A.Y., Micevych, P.E., Sieck, G.C., 1999. Alterations in diaphragm contractility after nandrolone administration: an analysis of potential mechanisms. J. Appl. Physiol. 86, 985 – 992. Lubek, B.M., 1984. Contractile responses of rat lateral gastrocnemius and soleus to dianabol (17 beta-hydroxy-17-methyl-1,4-androstadien-3-one) and exercise. Steroids 44, 485 – 495. Lukas, S.E., 1993. Current perspectives on anabolic–androgenic steroid abuse. Trends Pharmacol. Sci. 14, 61 – 68. Michel, G., Baulieu, E.E., 1980. Androgen receptor in rat skeletal muscle: characterization and physiological variations. Endocrinology 107, 2088 – 2098. Noirez, P., Ferry, A., 2000. Effect of anabolic–androgenic steroids on myosin heavy chain expression in hindlimb muscles of male rats. Eur. J. Appl. Physiol. 81, 155 – 158. Prezant, D.J., Valentine, D.E., Gentry, E.I., Richner, B., Cahill, J., Freeman, K., 1993. Effects of short-term and long-term androgen treatment on the diaphragm in male and female rats. J. Appl. Physiol. 75, 1140 – 1149. Rosenheimer, J., 1985. Effects of chronic stress and exercise on age-related changes in end-plate architecture. J. Neurophysiol. 53, 1582 – 1589. Russell, J.C., Campagna, P.D., Wenger, H.A., 1980. Small-animal ergometer. J. Appl. Physiol. 48, 394 – 398. Saborido, A., Vila, J., Molano, F., Megı´as, A., 1991. Effect of anabolic steroids on mitochondria and sarcotubular system of skeletal muscle. J. Appl. Physiol. 70, 1038 – 1043. Sonne, B., Galbo, H., 1980. Simultaneous determinations of metabolic and hormonal responses, heart rate, temperature and oxygen uptake in running rats. Acta Physiol. Scand. 109, 201 – 209. Tamaki, T., Uchiyama, S., Uchiyama, Y., Akatsuka, A., Roy, R.R., Edgerton, V.R., 2001. Anabolic steroids increase exercise tolerance. Am. J. Physiol: Endocrinol. Metab. 280, E973 – E981. Tsika, R.W., Herrick, R.E., Baldwin, K.M., 1987. Effect of anabolic steroids on skeletal muscle mass during hindlimb suspension. J. Appl. Physiol. 63, 2122 – 2127. Valera, S., Ballivet, M., Bertrand, D., 1992. Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U. S. A. 89, 9949 – 9953. Vizi, E.S., Somogyi, G.T., Nagashima, H., Duncalf, D., Chaudhry, I.A., Kobayashi, O., Goldiner, P.L., Foldes, F.F., 1987. Tubocurarine and pancuronium inhibit evoked release of acetylcholine from the mouse hemidiaphragm preparation. Br. J. Anaesth. 59, 226 – 231.
Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming W.L.G. Cavalcantea, M. Dal Pai-Silvab, M. Gallaccia,* a
Department of Pharmacology, Institute of Bioscience, University Estadual Paulista (Unesp), Botucatu, Sa˜o Paulo, CEP 18618-000, Brazil b Department of Morphology, Institute of Bioscience, University Estadual Paulista (Unesp), Botucatu, Sa˜o Paulo, CEP 18618-000, Brazil Received 10 August 2004; received in revised form 10 November 2004; accepted 14 November 2004
Abstract This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 910 7 M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin–eosin (HE); nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA ( pb0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3F4.7% vs. S=75.7F2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81F3.1% vs. Sw=71.0F4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. On the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise. D 2004 Elsevier Inc. All rights reserved. Keywords: Anabolic–androgenic steroids; Swimming; Safety margin; Neuromuscular junction
1. Introduction Anabolic–androgenic steroids (AAS) are synthetic derivatives of testosterone modified to enhance anabolic (myotrophic) rather than androgenic (masculinizing) actions of the original hormone (Kochakian, 1993). Due to these characteristics, suprapharmacologic doses of AAS have been taken by athletes in the expectation to increase
* Corresponding author. Tel.: +55 14 6802 6253; fax: +55 14 3815 3744. E-mail address: [email protected] (M. Gallacci). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.11.004
muscle size and improve performance in sportive activities (Lukas, 1993). Many studies have been focused on the effects of AAS in muscle structure and function in both humans (Fiedl, 2000; Kuhn, 2002) and animals (Bates et al., 1987; Lewis et al., 1999; Joumaa and Leoty, 2001), but their results are controversial. The structure and function of skeletal muscle are largely dependent on the interaction between motor nerves and muscles that occurs in the neuromuscular junction (Herscovich and Gershon, 1987). Previous works have shown that androgen receptors may be expressed in both motoneuron and skeletal muscle (Breedlove and Arnold, 1980; Michel and Baulieu, 1980). In addition, steroid hormones,
220
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including metabolites of testosterone and their synthetic derivatives, may acts as allosteric modulators of the nicotinic acetylcholine (Ach) receptor family of ligandgated ion channels (Blanton et al., 1999). In this work, we investigated the impact of suprapharmacologic doses of AAS upon the overall safety margin of synaptic transmission of rats submitted to swimming. Contractile properties, fatigue resistance, and fibers characteristics of EDL muscle were also studied.
polygraphic recording of muscle tension was performed by means of an isometric transducer (GM2/GM3-Gould). 2.3. Contractile properties of indirect twitches Peak of twitch force, time to peak, and relaxation time were determined from a series of indirect twitches evoked at a frequency of 0.5 Hz. 2.4. Safety margin of synaptic transmission
2. Materials and methods 2.1. Animals and treatment Male Wistar rats (Rattus norvegicus) 60 days old were used. Animals were housed in groups of four in standard rat cages in a temperature-controlled room (23F2 8C) and exposed to a 12-h light/dark cycle. Food and water were provided ad libitum. All procedures are in accordance with Canadian Council on Animal Care, and had local ethical committee approval. Nandrolone decanoate (ND) was injected subcutaneously at a dose of 3 mg/kg, 5 days/week, for 4 weeks. Rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. Rats swam in groups of 5 or 6, in a heated tank (33F1 8C) with an average water depth of 60 cm. Initially, to familiarize animals with water contact, rats were kept in a tank with a water depth of 7 cm for 15 min daily for 1 week.
The overall safety margin of the synaptic transmission was evaluated from the susceptibility of indirectly evoked contractions of the EDL muscle to the blockade induced by pancuronium. This agent is able to affect the synaptic transmission both by inhibiting the effect of ACh on postsynaptic nicotinic and by reducing the amount of ACh released via inhibition of positive-feedback modulation of ACh release (Vizi et al., 1987; Bowman et al., 1988). Suitable concentrations of pancuronium (710 7 M and 910 7 M) were added to the organ bath, respecting a period of 30 min between them. The ratio of the muscle tension in the presence and absence of pancuronium was used to estimate the safety margin of neuromuscular transmission. 2.5. Fatigue resistance Fatigue resistance protocol consisted of 180 brief tetanic contractions, 0.25 sec long and directly elicited every 5 sec at a frequency of 30 Hz (Fitts and Holloszy, 1977). Fatigue
2.2. Neuromuscular preparation and recording of muscle contractions The sciatic nerve extensor digitorum longus (EDL) muscle preparation was removed and mounted for myographic recording bin vitroQ, according to Gallacci and Oliveira (1994). Briefly, the preparation was mounted vertically in a conventional isolated organ-bath chamber containing 50 mL of physiological solution of the following composition (mmol/L): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; NaHCO3, 15; NaH2PO4, 1, glucose, 11. This solution was gassed with O2 (95%)+CO2 (5%), which kept the pH at 7.4 to 7.5, and maintained at 27 8C. Indirect contractions were evoked by supramaximal strength pulses (0.5 Hz; 0.5 ms), delivered from an electronic stimulator (Narco Bio-System) and applied on the phrenic nerve by means of a suction electrode. Direct contractions were evoked by supramaximal pulses (0.2 Hz, 5 ms) through a bipolar electrode positioned on opposite sides of the muscle. To avoid the interference of indirect contractions, these experiments were performed in the presence of pancuronium bromide (210 6 M). The
Fig. 1. Safety margin of neuromuscular transmission evaluated from the resistance to the indirectly evoked contractions of EDL muscle to pancuronium (710 7 M) in sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Twitch amplitudes were expressed as a percentage from the control situation (without pancuronium). Values are meansFS.E., n=5 animals for groups, at least. *Significant difference ( pb0.05); two-way ANOVA.
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resistance was defined as the force developed in the last contraction of a series relative to the force in the first contraction and was expressed as a percentage. Following recordings muscles were dried and weighed.
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ences among group means. Probability values ( P)b0.05 were considered significant.
3. Results 2.6. Histological and histochemical procedures Small fragments of EDL muscle were cooled in nhexane, and then frozen in liquid nitrogen ( 196 8C). Transverse muscle sections (7 to 10 Am) were submitted to haematoxylin and eosin (HE), for morphological analysis, and to nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-TR) for metabolic pattern evaluation (Dubowitz, 1985). 2.7. Statistical analysis All results are expressed as the meanFstandard error (S.E.) in the tables and are indicated by vertical bars in the figures. Data were analyzed by two-factor analysis of variance method (with exercise and vitamin E as factors). When a significant F ratio was found, Student–Newman– Keuls multiple-range test was used to assess the differ-
The overall safety margin of synaptic transmission, evaluated in terms of resistance to neuromuscular blockade induced by suitable concentrations of pancuronium, is shown in Fig. 1. Swimming did not change the susceptibility to the neuromuscular blockade induced by pancuronium in animals not treated with ND (SwS), indicating that the factor swimming alone does not affect the safety margin of synaptic transmission. In contrast, ND reduced the safety margin of sedentary animals. This effect was evident from the observation that the resistance to the blockade induced by pancuronium (710 7 M) was lower in SND than in S rats. However, there was a significant interaction between the factors swimming and ND, since there was no change in pacuronium resistance between Sw and SwND. This result indicates that ND does not affect the safety margin of animals submitted to swimming.
Fig. 2. Contractile properties of indirectly evoked twitches of EDL muscles from sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Values are meansFS.E., n=5 animals for groups, at least. (A) Twitch amplitude was expressed in grams of force by grams of tissue. (B and C) Time to peak and time to fall of twitches were expressed in milliseconds.
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Fig. 3. Fatigue resistance evaluated from the decrease of 180 direct contractions of EDL muscle of sedentary (S), sedentary plus nandrolone (SND), swimming (Sw), and swimming plus nandrolone (SwND) rats. Values are meansFS.E., n=5 animals for groups, at least. Amplitude of directly evoked twitches were expressed as a percentage to the initial amplitude. *Significant difference ( pb0.05); two-way ANOVA.
The amplitude and the kinetic properties of indirectly evoked twitches (time to peak and time to relaxation) were not changed by swimming, ND or the combination of swimming and ND (Fig. 2). Animals submitted to swimming showed higher resistance to fatigue when compared to sedentary (SwS). However, the treatment with ND did not affect the fatigue resistance of both sedentary (SSND) and swimming rats (SwSwND; Fig 3). Histological examinations of hematoxylin–eosin-stained muscles showed no obvious differences among groups.
Fig. 5. EDL muscles of rats: S (A), SND (B), Sw (C), and SwND (D), submitted to NADHTR histochemical reaction. Muscle fibers SO (1), FOG (2), and FG (3). NADHTR, 50.
Fiber dimensions were not affected by swimming or treatment with ND (Fig. 4). Histochemistry analysis (NADH-TR) did not revealed alterations in the proportions of oxidative and glycolytic fibers between the experimental groups (Fig. 5). During the 4 weeks of nandrolone treatment, there was no change in animals’ body weight. During the experimental period, all groups showed gain of body mass. However, as shown on Fig. 1, the gain of body mass in SND and in Sw were lower that of S groups. Both nandrolone treatment and swimming reduced the gain of body mass (SSND and SSw).
4. Discussion
Fig. 4. EDL muscles of rats: S (A), SND (B), Sw (C), and SwND (D) submitted to HE. Muscle fibers with normal morphology (F). Peripheric nuclei (N). Endomysium (E). HE: 50.
In this work, swimming was chosen as a suitable mode of exercising rats since they are good swimmers by nature and are less stressed than when subjected to treadmill running, wherein the animals are constantly under the fear of electrical stimulation (Russell et al., 1980; Sonne and Galbo, 1980). Rosenheimer (1985) noted that the nerve terminals from sedentary shocked controls were markedly different either from regular sedentary controls or from animals that were exercised with shock for motivation. This suggests that the stress represented by electrical shock is a confounding variable with respect to nerve terminal morphology and, consequently, it could also change the neuromuscular transmission. The present data demonstrate that administration of nandrolone decanoate to young adult rats submitted to swimming did not produce ergogenic effects. First, it should be mentioned that anabolic effects of AAS are influenced by
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a series of conditions, such as studied species, age, circulating synergic hormones, muscle, androgen, dose mode of administration, duration of treatment, and activity level (Bisschop et al., 1997). All of these variables probably play a role in the lack of consistent results found in the literature when anabolic agents are used. In the present study, the body mass was unaltered by concomitant administration of the anabolic steroid and swimming. This is similar to what has been reported for male young rats where body mass is not changed (Prezant et al., 1993; Saborido et al., 1991; Gayan-Ramirez et al., 2000) or stunted (Hervey and Hutchinson, 1973; Kochakian and Endahl, 1959), depending on the dose used. Although the exact mechanism of this effect is not known, it is possible that the basal level of the circulating endogenic androgen will play a role, as previously described (Bisschop et al., 1997). In the present work, the kinetic properties of indirect twitches as well as the fatigue resistance of EDL muscle remained unchanged after nandrolone treatment. Several studies examined effects of anabolic steroids on contractility and fatigue of peripheral muscles and revealed no additional effects of these agents when compared with those obtained with training alone (Egginton, 1987; Exner et al., 1973; Lubek, 1984; Bisschop et al., 1997; Gayan-Ramirez et al., 2000; Tamaki et al., 2001). The EDL muscle fibers distribution also is not changed, indicating that the treatment with suprapharmacologic dose of nandrolone for 4 weeks does not modify muscle phenotype. These results agree well with those of Tsika et al. (1987), Bisschop et al. (1997), and Noirez and Ferry (2000). Suggested mechanisms for the ergogenic effects of AAS include the possibility that these agents: (1) act through the central nervous system, allowing the subjects to train harder (Ariel and Saville, 1972); or (2) may improve skeletal muscle function directly by increasing protein synthesis (Egginton, 1987; Griggs et al., 1989). The major finding of this work was the observation that nandrolone decanoate reduces the safety margin of synaptic transmission in rats sedentary but did not affect this margin in animals submitted to 4 weeks of swimming. The excess of both nicotinic receptor and acetylcholine at the synapse assures the safety margin of the neuromuscular transmission. It is largely known that physical exercise increases the safety margin of synaptic transmission (Dorlo¨chter et al., 1991; Fahim, 1997; Desaulniers et al., 1998) by promoting hypertrophy of the synapses. However, the findings of this work showed that 4 weeks of swimming was not enough to increase this safety margin. On the other hand, nandrolone reduced the safety margin of sedentary animals, but did not affect that margin in rats submitted to swimming. Previous work has shown that suprapharmacological doses of anabolic androgenic steroids may increase choline acetyltransferase (ChAT) mRNA levels in motoneurons (Blanco et al., 1997, 2001). On this basis, it has been suggested that AAS could lead to a greater presynaptic capacity to synthesize Ach. These actions obviously do not explain
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the AAS-induced decrease in the safety margin of neuromuscular transmission observed in this work. However, in vitro studies have shown that steroid hormones, including testosterone and their synthetic derivative may induce blockade of nicotinic receptors (Valera et al., 1992; Bouzat and Barrantes, 1996; Arias, 1997). This blockade seems to be of noncompetitive type and be caused by the interaction of the hormone with the lipid protein interface and/or extracellular domain of the nicotinic receptor (Arias, 1997). Thus, it is possible that the treatment with high dose of nandrolone lead to a residual blockade of the nicotinic receptor, and this reduces the safety margin of neuromuscular transmission of sedentary animal. This effect was not evident in swimming rats due to the interaction between the anabolic androgenic steroid with exercise. Although exercise alone was not able to increase the safety margin, anabolic androgenic steroids may further enhance the myotrophic effect of exercise (Dahlberg et al., 1981). This myotrophic effect was not expressed as an increase in the safety margin in trained animals due to the residual blocking action of anabolic androgenic steroid. In conclusion, this study shows that the treatment of swimming rats with suprapharmacologic dose of nandrolone decanoate during 4 weeks does not promote ergogenic effects. On the contrary, this work revealed that high dose of the steroid hormone may compromise the safety margin of neuromuscular transmission in animals not submitted to exercise. Although the present study was performed in rats, our results may indicate that high doses of nandrolone decanoate would not result in additional benefit to humans submitted to a short training program. On the other hand, untrained individuals should be more susceptible to a fade on the process of the neuromuscular transmission. Nevertheless, care should be taken when one extrapolates data from animal models to humans.
Acknowledgments This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (0201278-8).
References Arias, H.R., 1997. The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor. Biochim. Biophys. Acta 1347, 9 – 22. Ariel, G., Saville, W., 1972. Effect of anabolic steroids on reflex components. J. Appl. Physiol. 32, 795 – 797. Bates, P.C., Chew, L.F., Millward, D.J., 1987. Effects of the anabolic steroid stanozolol on growth and protein metabolism in the rat. J. Endocrinol. 114, 373 – 381. Bisschop, A., Gayan-Ramirez, G., Rollier, H., Dekhuijzen, P.N., Dom, R., De Bock, V., Decramer, M., 1997. Effects of nandrolone decanoate on respiratory and peripheral muscles in male and female rats. J. Appl. Physiol. 82, 112 – 118.
224
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Blanco, C.E., Popper, P., Micevych, P., 1997. Anabolic–androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat. Neuroscience 78, 873 – 882. Blanco, C.E., Zhan, W.Z, Fang, Y.H., Sieck, G., 2001. Exogenous testosterone treatment decreases diaphragm neuromuscular transmission failure in male rats. J. Appl. Physiol. 90, 850 – 856. Blanton, M.P., Xie, Y., Dangott, L.J., Cohen, 1999. The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid-protein interface. Mol. Pharmacol. 55, 269 – 278. Bouzat, C., Barrantes, F.J., 1996. Modulation of muscle nicotinic acetylcholine receptors by the glucocorticoid hydrocortisone. Possible allosteric mechanism of channel blockade. J. Biol. Chem. 271, 25835 – 25841. Bowman, W.C., Marshall, I.G., Gibb, A.J., Harborne, A.J., 1988. Feedback control of transmitter release at the neuromuscular junction. Trends Pharmacol. Sci. 9, 16 – 20. Breedlove, S.M., Arnold, A.P., 1980. Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science 210, 564 – 566. Dahlberg, E., Snochowski, M., Gustafsson, J.A., 1981. Regulation of the androgen and glucocorticoid receptors in rat and mouse skeletal muscle cytosol. Endocrinology 108, 1431 – 1440. Desaulniers, P., Lavoie, P.A., Gardiner, P.F., 1998. Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle. NeuroReport 9, 3549 – 3552. Dorlfchter, M., Irintcchev, A., Brinkers, M., Wernig, A., 1991. Effects of enhanced activity on synaptic transmission in mouse extensor digitorum longus muscle. J. Physiol. 436, 283 – 292. Dubowitz, V., 1985. Normal muscle. Muscle Biopsy: A Practical Approach, 2 edn. Bailliere Tindall, London, pp. 41 – 48. Egginton, S., 1987. Effects of an anabolic hormone on striated muscle growth and performance. Pflugers Arch. 410, 349 – 355. Exner, G.U., Staudte, H.W., Pette, D., 1973. Isometric training of rats— effects upon fast and slow muscle and modification by an anabolic hormone (nandrolone decanoate): II. Male rats. Pflugers Arch. 345, 15 – 22. Fahim, M.A., 1997. Endurance exercise modulates neuromuscular junction of C57BL/6NNia aging mice. J. Appl. Physiol. 83, 59 – 66. Fiedl, K.E., 2000. Effect of anabolic steroid use on body composition and physical performance. In: Yesalis, C.E. (Ed.), Anabolic Steroids in Sport and Exercise. Human Kinetics, Champaign, IL, pp. 139 – 174. Fitts, R.H., Holloszy, J.O., 1977. Contractile properties of rat soleus muscle: effects of training and fatigue. Am. J. Physiol. 233, 86 – 91. Gallacci, M., Oliveira, A.C., 1994. Pre- and postsynaptic mechanisms involved in tetanic fade induced by pancuronium in the isolated rat muscle. Pharmacology 49, 265 – 270. Gayan-Ramirez, G., Rollier, H., Vanderhoydonc, F., Verhoeven, G., Gosselink, R., Decramer, M., 2000. Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm. J. Appl. Physiol. 88, 26 – 34. Griggs, R.C., Kingston, W., Jozefowicz, R.F., Herr, B.E., Forbes, G., Halliday, D., 1989. Effect of testosterone on muscle mass and muscle protein synthesis. J. Appl. Physiol. 66, 498 – 503.
Herscovich, S., Gershon, D., 1987. Effects of aging and physical training on the neuromuscular junction of the mouse. Gerontology 33, 7 – 13. Hervey, G.R., Hutchinson, I., 1973. The effects of testosterone on body weight and composition in the rat. J. Endocrinol. 57, 24. Joumaa, W.H., Leoty, C., 2001. Differential effects of nandrolone decanoate in fast and slow rat skeletal muscles. Med. Sci. Sports Exerc. 33, 397 – 403. Kochakian, C.D., 1993. History, chemistry and pharmacodynamics of anabolic–androgenic steroids. Wien. Med. Wochenschr. 143, 359 – 363. Kochakian, C.D., Endahl, B.R., 1959. Changes in body weight of normal and castrated rats by different doses of testosterone propionate. Proc. Soc. Exp. Biol. Med. 100, 520 – 522. Kuhn, C.M., 2002. Anabolic steroids. Recent Prog. Horm. Res. 57, 411 – 434. Lewis, M.I., Fournier, M., Yeh, A.Y., Micevych, P.E., Sieck, G.C., 1999. Alterations in diaphragm contractility after nandrolone administration: an analysis of potential mechanisms. J. Appl. Physiol. 86, 985 – 992. Lubek, B.M., 1984. Contractile responses of rat lateral gastrocnemius and soleus to dianabol (17 beta-hydroxy-17-methyl-1,4-androstadien-3-one) and exercise. Steroids 44, 485 – 495. Lukas, S.E., 1993. Current perspectives on anabolic–androgenic steroid abuse. Trends Pharmacol. Sci. 14, 61 – 68. Michel, G., Baulieu, E.E., 1980. Androgen receptor in rat skeletal muscle: characterization and physiological variations. Endocrinology 107, 2088 – 2098. Noirez, P., Ferry, A., 2000. Effect of anabolic–androgenic steroids on myosin heavy chain expression in hindlimb muscles of male rats. Eur. J. Appl. Physiol. 81, 155 – 158. Prezant, D.J., Valentine, D.E., Gentry, E.I., Richner, B., Cahill, J., Freeman, K., 1993. Effects of short-term and long-term androgen treatment on the diaphragm in male and female rats. J. Appl. Physiol. 75, 1140 – 1149. Rosenheimer, J., 1985. Effects of chronic stress and exercise on age-related changes in end-plate architecture. J. Neurophysiol. 53, 1582 – 1589. Russell, J.C., Campagna, P.D., Wenger, H.A., 1980. Small-animal ergometer. J. Appl. Physiol. 48, 394 – 398. Saborido, A., Vila, J., Molano, F., Megı´as, A., 1991. Effect of anabolic steroids on mitochondria and sarcotubular system of skeletal muscle. J. Appl. Physiol. 70, 1038 – 1043. Sonne, B., Galbo, H., 1980. Simultaneous determinations of metabolic and hormonal responses, heart rate, temperature and oxygen uptake in running rats. Acta Physiol. Scand. 109, 201 – 209. Tamaki, T., Uchiyama, S., Uchiyama, Y., Akatsuka, A., Roy, R.R., Edgerton, V.R., 2001. Anabolic steroids increase exercise tolerance. Am. J. Physiol: Endocrinol. Metab. 280, E973 – E981. Tsika, R.W., Herrick, R.E., Baldwin, K.M., 1987. Effect of anabolic steroids on skeletal muscle mass during hindlimb suspension. J. Appl. Physiol. 63, 2122 – 2127. Valera, S., Ballivet, M., Bertrand, D., 1992. Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U. S. A. 89, 9949 – 9953. Vizi, E.S., Somogyi, G.T., Nagashima, H., Duncalf, D., Chaudhry, I.A., Kobayashi, O., Goldiner, P.L., Foldes, F.F., 1987. Tubocurarine and pancuronium inhibit evoked release of acetylcholine from the mouse hemidiaphragm preparation. Br. J. Anaesth. 59, 226 – 231.