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Effects of swimming and nandrolone decanoate treatment on vas deferens response to norepinephrine
Life Sciences 85 (2009) 541–545
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Effects of swimming and nandrolone decanoate treatment on vas deferens response to norepinephrine Lidyane R.G. Castor a, Eunice Oba b, Oduvaldo C.M. Pereira a,c,⁎ a b c
Department of Pharmacology, Institute of Biosciences, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil Department of Animal Reproduction and Radiology, School of Veterinary Medicine and Animal Science, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil Department of Internal Medicine, Botucatu Medical School, Sao Paulo State University – UNESP, 18618-000 Botucatu, Sao Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 19 June 2008 Accepted 13 August 2009 Keywords: Swimming Vas deferens Norepinephrine response Nandrolone decanoate Rats
a b s t r a c t Aims: To investigate the response to norepinephrine of vas deferens isolated from intact and castrated rats submitted to swimming and/or treated with nandrolone decanoate. Main methods: Intact and castrated male rats were submitted to swimming for 15 days, 1 session per day, 5 days/week and were either treated or not with 7.5 mg/kg of nandrolone decanoate on days 1, 5, 9, and 13 after the beginning of training. Plasma androgen concentration was measured by radioimmunoassay. Vas deferens was isolated and set up for analysis of its contractile capacity in response to norepinephrine. Key findings: In intact rats, nandrolone, training, and training plus nandrolone did not change body mass or vas deferens weight. In castrated rats, the vas deferens wet weight was decreased in both untrained and trained groups. In castrated rats, nandrolone prevented vas deferens atrophy. In intact animals, nandrolone decreased (P < 0.05) the androgen level in untrained group, while in castrated rats this treatment partially restored the androgen level. An increased sensitivity (P < 0.05) to norepinephrine was observed in vas deferens isolated from intact trained rats, treated or not with nandrolone decanoate, while nandrolone did not alter norepinephrine response in organs from untrained animals. In untrained castrated rats, nandrolone fully restored the sensitivity to norepinephrine in untrained rats, while in trained castrated rats the anabolic steroid only partially restored this response. Significance: The present results indicate that training can increase norepinephrine response of vas deferens in intact rats, while nandrolone decanoate can partially restore the responsiveness to norepinephrine in castrated rats. © 2009 Published by Elsevier Inc.
Introduction Anabolic steroids enhance body-tissue-building processes and retard or reverse tissue catabolism. Based on these properties, some athletes use anabolic steroids in an attempt to improve their athletic performance. However, serious adverse outcomes have been correlated with the use of anabolic steroid (Maravelias et al. 2005; Casavant et al. 2007). Moreover, altered androgenic levels have been found following administration of anabolic steroids as well as during prolonged physical activity (Opstad 1992; Hackney et al. 2003). One potentially useful experimental model of exercise in rats is forced swimming. However, elevated levels of norepinephrine and epinephrine are also found during forced swimming, resulting from the physical imposition of forced exercise and the strong emotional components (Östman-Smith 1979; Perronét et al. 1981). In this
⁎ Corresponding author. Department of Pharmacology, Institute of Biosciences, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil. E-mail address: [email protected] (O.C.M. Pereira). 0024-3205/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.lfs.2009.08.009
exercise model, adaptation to water is necessary to reduce stress to the animals. Changes in testosterone levels can also modulate adrenergic neurotransmission in some reproductive organs, including the vas deferens (MacDonald and McGrath 1980). The rat vas deferens, a smooth muscle of the genital tract, has commonly been considered a suitable preparation for studying α-adrenoceptors (Sallés and Badia 1991). Studies in our laboratory demonstrated reduced sensitivity of vas deferens to norepinephrine in rats submitted to acute swimming. The reduced sensitivity was a consequence of increased neuronal uptake of norepinephrine and β2-adrenoceptor upregulation, which opposes α1-adrenoceptor activity (Chies and Pereira 1995). In androgenic deprivation models, altered responses of isolated seminal vesicles and vas deferens to various drugs have been shown (Porto et al. 1988; Pereira et al. 1993; Souza and Pereira 1999). In addition, castration has been proposed to result in altered response to norepinephrine as a consequence of changes in functional α1-adrenoceptors subtypes involved in the rat vas deferens contractile responses (Pupo 1998). Thus, the physiological level of testosterone, the use of anabolic steroids and exposure to intensive exercise may be involved in the noradrenergic response
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pattern of the smooth muscle of the genital tract. On the basis of these considerations, the present study aimed to investigate the norepinephrine response of vas deferens isolated from rats submitted to swimming and/or treated with nandrolone decanoate, an anabolic steroid.
radioimmunoassay (Coat-A-Count Rat Testosterone kit, DPC, Los Angeles, CA, Lot 40101). All samples were measured in duplicate. Data are reported as nanomol per liter of plasma. The interassay coefficient of variation was 6.0%. Pharmacological analysis
Materials and methods Animals Adult male Wistar rats (about 3 months old, average weight 300– 350 g) were randomly divided into respective experimental groups and housed five rats per polypropylene cage (32 × 40 × 18 cm) with bedding of wood shavings. Food and water were freely available with a 12 h light–dark cycle. The temperature and humidity were controlled at 25 ± 1 °C and 55 ± 5%, respectively. The animals used in this study were maintained in accordance with Ethical Principles in Animal Research, adopted by the Brazilian College of Animal Experimentation and approved by the Bioscience Institute/UNESP Animal Research Ethics Committee, protocol number 93/01. Experimental groups Six experimental groups were included in this study, as defined by whether they were castrated or not, submitted to swimming (referred to as training) or not, and either treated or not with nandrolone decanoate. Control groups were included for each of these groups.
The left vas deferens was removed, separated from the surrounding tissue, freed of secretions, weighed, and set up for analysis of contractile capacity in a 10 ml organ-bath circulating a nutritive solution aerated with 95% O2 and 5% CO2 and maintained at 30 °C. The composition of the nutritive solution was 136.0 mM NaCl, 5.7 mM KCl, 1.8 mM CaCl2, 0.36 mM NaH2PO4·H2O, 15.0 mM NaHCO3, and 5.5 mM dextrose, prepared in a glass containing distilled water (Picarelli et al. 1962). A resting tension of 1.0 g was applied to the tissue and the change in isometric tension was measured via force displacement transducers. After an initial resting period of 30 min, concentration– response curves for norepinephrine (arterenol bitartrate, Sigma) were obtained. The concentration–response curves for norepinephrine were obtained in the absence and presence of timolol (10−5 M; timolol maleate, Sigma) added 30 min before the norepinephrine. The agonists were added at successively increasing molar concentrations (van Rossum 1963; van Rossum and van Der Brink 1963). The parameter determined was the pD2, estimated as the negative of the logarithm of the agonist concentration producing 50% (ED50) of the maximum effect (Miller et al. 1948). Statistical analysis
Castration Data are presented as mean ± SEM. Data sets were first compared by analysis of variance, and Tukey's test was used for post-hoc comparisons between pairs of means. The results were considered significant at P < 0.05.
Castration, consisting of bilateral removal of the testicles, was carried out via transscrotal access under sodium pentobarbital (40 mg/kg, ip) anesthesia when the rats were at least 75 days old. These rats were utilized 7 days after orchidectomy in the subsequent experiments.
Results
Nandrolone decanoate treatment
Body mass and wet weight of vas deferens
Rats received subcutaneous injections of 7.5 mg/kg of nandrolone decanoate (Deca durabolinR Organon) or vehicle (peanut oil plus benzilic alcohol, 1:16) 1, 5, 9, and 13 days after beginning the exercise protocol.
Intact and castrated rats, untrained or submitted to swimming (trained), treated or not with nandrolone decanoate showed no significant difference in body mass. Vas deferens from intact rats, untrained or submitted to swimming, treated or not with nandrolone decanoate also showed no alteration in wet weight, while vas deferens from castrated rats, untrained or submitted to exercise weighed significantly less than intact rats. In castrated rats treated with nandrolone decanoate, the wet weight of the vas deferens did not differ significantly from those of intact rats (Table 1).
Exercise protocol Rats were submitted to a 7-day period of adaptation to water in order to minimize stress. For 15 min each day, rats were placed in water (27 °C) within a glass tank of increasing depth, starting with 5 cm of water and increasing gradually to a water height of 40 cm. After this process, the animals were individually submitted to 60 min of swimming each day for 15 days, 1 session per day, 5 days per week. For the swimming procedure (training), the rats carried a metallic ring of about 2% of their body weight on their tails. The tank measured 35 cm in length, 17 cm in width, and 50 cm in depth. Immediately after the last swimming session, the animals were anesthetized by volatile anesthetic, blood samples were collected, and the animals were killed by decapitation.
Plasma androgen level Plasma androgen level (total testosterone) in intact rats decreased significantly after nandrolone decanoate treatment for both rats undergoing training as well as those undergoing training plus nandrolone decanoate. In castrated rats, treatment with the synthetic testosterone, nandrolone decanoate, restored the plasma androgen levels to levels observed in intact untrained rats treated with nandrolone decanoate, intact trained rats, and intact trained rats treated with nandrolone decanoate (Table 2).
Plasma androgen level Pharmacological response of the vas deferens Plasma androgen levels represent total testosterone levels in the rat. To measure these levels blood samples (2.0–2.5 ml) were collected from the abdominal aorta into heparinized vials, always between 09:00 and 10:00 am. Immediately after collection, blood samples were centrifuged (700 × g for 20 min, at 2 °C) and the plasma androgen level (total testosterone) was assayed by solid-phase [I125]
All rats swam vigorously during the swimming sessions. The mean concentration–response curves for norepinephrine in vas deferens from intact rats submitted to swimming, treated or not with nandrolone decanoate, was shifted to the left (Fig. 1). In addition, the corresponding pD2 values were significantly increased (Table 3).
L.R.G. Castor et al. / Life Sciences 85 (2009) 541–545
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Table 1 Body mass and wet weight of vas deferens from intact and castrated adult male rats, untrained and submitted to swimming (trained), and either treated or not with nandrolone decanoate (ND). Groups
Body mass (g) Intact rats
349.00 ± 12.89a A Untrained+ ND 348.57 ± 6.35a A Trained 366.30 ± 6.93a A Trained + ND 348.80 ± 8.14a A Untrained
Vas deferens (mg) Castrated rats
Intact rats
Castrated rats
362.28 ± 8.45a A 380.30 ± 13.00a A 371.28 ± 8.54a A 383.00 ± 7.13a A
76.28 ± 6.20a A 72.14 ± 5.80a A 88.85 ± 2.34a A 83.14 ± 5.55a A
35.71 ± 2.22b A 79.14 ± 2.34a B 36.71 ± 2.50b A 77.40 ± 3.10a B
Values expressed as mean ± SEM of six animals per group. A,B Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant differences between castrated rats and their respective control (p < 0.05, Tukey's test).
Experiments performed in the presence of the non-selective β1 and β2-antagonist timolol yielded mean concentration–response curves of norepinephrine superimposed on curves obtained from organs of either untrained or trained rats, treated or not with nandrolone decanoate, and consequently their correspondent pD2 values did not differ significantly between the groups (P > 0.05; data not shown). In vas deferens from castrated untrained rats, the norepinephrine curves were shifted to the right compared with intact untrained rats treated or not with nandrolone decanoate, and the anabolic steroid treatment restored the sensitivity in castrated untrained rats (Fig. 2A). In vas deferens from trained castrated rats, the mean concentration response curves for norepinephrine were shifted to the right in comparison with those obtained from intact trained rats treated or not with nandrolone decanoate. Treatment with the anabolic steroid only partially restored the norepinephrine response of vas deferens from castrated trained rats (Fig. 2B). Corresponding pD2 values are shown in Table 3. Discussion The importance of hormonal control has been shown in the development of accessory reproductive organs and in the reactivity of smooth muscle within the male genital organs (Picarelli and Valle 1969; Valle et al. 1982). Thus, androgens are necessary for the development and function of the male genital tract (Sjöstrand and Swedin 1976; Pereira et al. 1991) and androgens may also modulate the noradrenergic response in vas deferens isolated from rats (Pereira et al. 1998). In the present study, treatment of intact rats with Table 2 Plasma androgen levels (total testosterone) in intact and castrated rats, untrained and submitted to swimming (trained), and treated or not with nandrolone decanoate (ND). Groups
Untrained + ND Trained Trained + ND
Table 3 pD2 values of norepinephrine in vas deferens from intact and castrated rats, untrained and submitted to swimming (trained), and treated or not with nandrolone decanoate (ND). pD2 Norepinephrine
Castrated rats a
9.02 ± 1.17 A 3.33 ± 0.23a B 3.82 ± 0.91a B 4.01 ± 0.50a B
b
0.05 ± 0.03 A 3.76 ± 0.73a B 0.07 ± 0.05b A 4.22 ± 0.66a B
Values expressed as mean ± SEM of six animals per group. Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant difference between castrated rats and their respective control (p < 0.05, Tukey's test). A,B
nandrolone decanoate for 2 weeks did not change either the body mass or vas deferens wet weight. These results are consistent with data in the literature showing no change in body weight after two weeks of treatment with anabolic steroids (Blasberg et al. 1997; Tamaki et al. 2001). It is possible that the treatment duration was not sufficient to induce changes in body weight of the animals in this study. In spite of this finding, both the swimming (training) and the treatment with nandrolone decanoate decreased plasma androgens level in intact rats. These results also agree with those showing that exercise training frequently results in a decrease of serum testosterone and may rarely be associated with reduced libido, sperm production and fertility (Eliakim and Nemet 2006). In this exercise protocol, the decreased testosterone was likely due to the repeated exercise plus residual stress, which produced increased corticosterone levels. On the other hand, in the present study, the decreased testosterone level observed after nandrolone decanoate treatment may be a result of hypothalamus–hypophysial feedback mechanisms, which exert a depressive effect on endogenous secretion (Signoret 1976; Hackney et al. 2003). However, in castrated rats treated nandrolone decanoate, androgenic levels were restored to approximately 40% of that observed in intact untrained rats. This result indicates that despite the high dose of synthetic androgen used, by exerting predominantly anabolic activity nandrolone decanoate was not able to restore androgen level to values observed in the control
Androgen nmol/l Intact rats
Untrained
Fig. 1. Cumulative concentration–response curves for norepinephrine in vas deferens from intact untrained rats as well as those submitted to swimming (trained), treated or not with nandrolone decanoate (ND). The X-axis shows the molar concentration of the drug on a logarithmic scale. The Y-axis shows effects of norepinephrine as a percentage of the maximal contraction produced by the agonist. Vertical bars indicate SEM. Each point represents a mean of six experiments.
Groups Intact Intact + ND Castrated Castrated + ND
Untrained
Trained a
5.55 ± 0.05 A 5.46 ± 0.07a A 5.11 ± 0.10a B 5.55 ± 0.11a A
5.96 ± 0.13b A 5.88 ± 0.07b A 5.18 ± 0.01a B 5.40 ± 0.15a C
Values expressed as mean ± SEM of six animals per group. A,B Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant difference between trained and untrained rats (p < 0.05, Tukey's test).
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Fig. 2. Cumulative concentration–response curves for norepinephrine in vas deferens from intact and castrated rats, untrained (A) and submitted to swimming (trained, B), treated or not with nandrolone decanoate (ND). The X-axis shows the molar concentration of norepinephrine on a logarithmic scale. The Y-axis shows effects as a percentage of the maximal contraction produced by norepinephrine. Vertical bars indicate SEM. Each point represents a mean of at least six experiments.
group. In addition, although nandrolone decanoate was able to partly restore the androgen level in the castrated animals, nandrolone prevented both the vas deferens atrophy and the decreased reactivity of these tissues to norepinephrine. In rats undergoing a single forced swimming trial, a model in which stress is highly important, both functional and endocrinological alterations of the organism have been observed, with the prejunctional α2-adrenoceptors, the postjunctional α1-adrenoceptors, and the neuronal uptake of catecholamines being involved in vas deferens homeostasis (Sanchez and Pereira 2002). In the present study, the swimming, which functioned as repeated exercise (with a period of adaptation to water in order to minimize the stress), resulted in supersensitivity to norepinephrine of vas deferens isolated from intact rats treated or not with nandrolone decanoate. This supersensitivity to norepinephrine in organs from rats submitted to swimming may be correlated with changes in norepinephrine receptors and levels of androgens. A study from our laboratory showed that acute exercise (swimming) induced significant α1-adrenoceptor contractile activity in rat vas deferens due to stressogenic activity, and this effect was antagonized by β2-adrenoceptors, both α1- and β2-adrenoceptors equally active (Chies and Pereira 1995). In contrast, the results of the present study showed that the postjunctional β2-adrenoceptors likely did not participate in the norepinephrine response pattern observed in vas deferens after the repeated exercise because the response to norepinephrine in the presence of timolol was similar for all
experimental groups. Thus, by minimizing stress, the repeated exercise may be responsible for the observed absence of participation of β2-adrenoceptors. In addition, previous studies from our laboratory showed that a one-day swimming protocol produced subsensitivity to norepinephrine of the vas deferens of intact adult rats, while no change in sensitivity was observed for this parameter when the animals were submitted to swimming for three consecutive days, thus suggesting the occurrence of adaptation to elevated norepinephrine levels, a mild decrease in androgen levels or both (Pereira et al. 1998). On the other hand, hormonal deprivation–castration induced subsensitivity to norepinephrine in vas deferens from untrained and trained rats. In castrated untrained rats, nandrolone decanoate fully restored sensitivity to norepinephrine, while in castrated trained rats the norepinephrine sensitivity was only partially restored by nandrolone decanoate relative to intact trained rats. In another study, testosterone and 5α-norethisterone were also shown to restore the sensitivity to methoxamine and serotonin of the epididymal portion of vas deferens from castrated rats (Campos et al. 1999). These data together with the results of the present study support the effectiveness of androgens in modulating the autonomic-receptor response in smooth muscle of the reproductive tract. Finally, despite the indication of reduced (minimized) stress in the swimming-exercise model, we suggest that the effects of “training” are actually different from those previously described as an effect of stress. The results of the current study indicate that in intact rats swimming (training) could increase the norepinephrine response pattern in the vas deferens, while nandrolone decanoate did not modify this response. That said, the possibility that other mechanisms may be involved in increased sensitivity to norepinephrine cannot be discarded. However, in castrated rats, swimming was not effective in altering the response to norepinephrine, while nandrolone decanoate did partly modify this response. To summarize, through using castration and exercise training in rats, we have found that in the absence of androgens anabolic androgenic steroid treatment restored at least partially the response of the isolated vas deferens to norepinephrine, confirming findings that the response of the smooth muscles of male organs to norepinephrine are not only hormonesensitive, but also androgen-dependent. In conclusion, training can increase the norepinephrine response in the vas deferens of intact rats, while nandrolone decanoate can be effective only in conditions of androgen deficiency. Acknowledgement Research was supported by CAPES. This study is part of the M.Sc. thesis presented to Sao Paulo State University (UNESP) by Lidyane R.G. Castor. References Blasberg ME, Langan CJ, Clark AS. The effects of 17α-methyltestosterone, methandrostenolone, and nandrolone decanoate on the rat estrous cycle. Physiology and Behavior 61, 265–272, 1997. Campos MG, Oropeza MV, Lemus AE, Garcia GA, Reynoso ME, Campos P, Ponce-Monte H. The androgenic effect of norethisterone and 5α-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin. Life Sciences 64, 227–233, 1999. Casavant MJ, Blake K, Griffith J, Yates A, Copley LM. Consequences of use of anabolic androgenic steroids. Pediatric Clinics of North America 54, 677–690, 2007. Chies AB, Pereira OCM. Cathecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress. Pharmacological Research 32 (3), 123–127, 1995. Eliakim A, Nemet D. Exercise and the male reproductive system. Harefuah 145 (9), 677–681 702,701, 2006. Hackney AC, Szczepanowska E, Viru AM. Basal testicular testosterone production in endurance-trained men is suppressed. European Journal of Applied Physiology 89, 198–201, 2003. MacDonald A, McGrath JC. The effects of castration on neurotransmission in the rat vas deferens. British Journal of Pharmacology 69, 49–58, 1980.
L.R.G. Castor et al. / Life Sciences 85 (2009) 541–545 Maravelias C, Dona A, Stefanidou M, Spiliopoulou C. Adverse effects of anabolic in athletes. A constant threat. Toxicology Letters 158 (3), 167–175, 2005. Miller LC, Becker TJ, Tainter ML. Quantitative evaluation of spasmolytic drugs in vitro. Journal of Pharmacology and Experimental Therapeutics 92, 260–268, 1948. Opstad PK. Androgenic hormones during prolonged physical stress, sleep, and energy deficiency. Journal of Clinical Endocrinology and Metabolism 74, 1176–1183, 1992. Östman-Smith I. Adaptive changes in the sympathetic nervous system and some effector organs of the rat following long-term exercise and cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hypertrophy. Acta Physiologica Scandinavica 477, 1–118 Suppl, 1979. Pereira OCM, Dega MR, Souza MSS. Noradrenergic response in vas deferens from rats submitted to acute and repeated stress. General Pharmacology 30 (3), 417–421, 1998. Pereira OCM, Picarelli ZP, Picarelli ZP, Abreu LC. Cryptorchidism versus morphological aspects of the reproductive organs. Arquivos de Medicina 4 (3), 199–203, 1991. Pereira OCM, Picarelli ZP, Abreu LC. Experimental cryptorchidism in prepubertal rats: some effects on reactivity of the seminal vesicle to catecholamines. Pharmacological Research 28 (2), 175–181, 1993. Perronét F, Cléroux J, Perrault H, Cousineau D, De Champlain J, Nadeau R. Plasma norepinephrine response to exercise before and after training in humans. Journal of Applied Physiology 51, 812–815, 1981. Picarelli ZP, Valle JR. Hormonal regulation of the rat seminal vesicle sensitivity to adrenaline, acetylcholine and acetyl-beta-methylcholine. British Journal of Pharmacology 35, 468–475, 1969. Picarelli ZP, Hyppolito N, Valle JR. Synergistic effect of 5-hydroxytryptamine on the response of rats seminal vesicles to adrenaline and noradrenaline. Archives Internationales de Pharmacodynamie et de Thérapie 138, 354–363, 1962. Porto CS, Abreu LC, Picarelli ZP. Influence of castration and of the kind of sympathomimetic drug used upon the reactivity of rat seminal vesicles. Archives Internationales de Pharmacodynamie et de Thérapie 292, 101–111, 1988.
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Pupo AS. Functional effects of castration on α1-adrenoceptors in rat vas deferens. European Journal of Pharmacology 351, 217–223, 1998. Sallés J, Badia A. Mechanisms underlying the differential sensitivity to α1-adrenoceptor activation in the bisected rat vas deferens. British Journal of Pharmacology 102, 439–445, 1991. Sanchez A, Pereira OCM. Acute swimming stress induces changes in noradrenergic mechanisms in order to maintain the response pattern of the rat vas deferens to norepinephrine. Pharmacological Research 46 (1), 55–60, 2002. Signoret JP. Influence of anabolic agents on behavior. Environmental Quality and Safety 5, 143–150 Supplement, 1976. Sjöstrand NO, Swedin G. Influence of age, growth, castration and testosterone treatment on the noradrenaline levels of ductus deferens and the auxiliary male reproductive glands of the rat. Acta Physiologica Scandinavica 98, 323–338, 1976. Souza MSS, Pereira OCM. The importance of androgen on noradrenergic responses of vas deferens isolated from acutely stressed rats. Pharmacological Research 40 (4), 307–311, 1999. Tamaki T, Uchiyama S, Uchiyama Y, Akatsuka A, Roy RR, Edgerton VR. Anabolic steroids increase exercise tolerance. American Journal of Physiology. Endocrinology and Metabolism 280, E973–E981, 2001. Valle RMR, Abreu LC, Picarelli ZP, Valle JR. Influence of castration upon trophism and reactivity of rat seminal vesicles. Brazilian Journal of Medical and Biological Research 15, 49–53, 1982. van Rossum JM. Cumulative dose–response curves II: Technique for the making of dose–response curves in isolated organs and the evaluation of drug parameters. Archives Internationales de Pharmacodynamie et de Thérapie 143, 299–330, 1963. van Rossum JM, van Den Brink FG. Cumulative dose–response curves. I: Introduction to the technique. Archives Internationales de Pharmacodynamie et de Thérapie 143, 240–246, 1963.
Contents lists available at ScienceDirect
Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Effects of swimming and nandrolone decanoate treatment on vas deferens response to norepinephrine Lidyane R.G. Castor a, Eunice Oba b, Oduvaldo C.M. Pereira a,c,⁎ a b c
Department of Pharmacology, Institute of Biosciences, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil Department of Animal Reproduction and Radiology, School of Veterinary Medicine and Animal Science, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil Department of Internal Medicine, Botucatu Medical School, Sao Paulo State University – UNESP, 18618-000 Botucatu, Sao Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 19 June 2008 Accepted 13 August 2009 Keywords: Swimming Vas deferens Norepinephrine response Nandrolone decanoate Rats
a b s t r a c t Aims: To investigate the response to norepinephrine of vas deferens isolated from intact and castrated rats submitted to swimming and/or treated with nandrolone decanoate. Main methods: Intact and castrated male rats were submitted to swimming for 15 days, 1 session per day, 5 days/week and were either treated or not with 7.5 mg/kg of nandrolone decanoate on days 1, 5, 9, and 13 after the beginning of training. Plasma androgen concentration was measured by radioimmunoassay. Vas deferens was isolated and set up for analysis of its contractile capacity in response to norepinephrine. Key findings: In intact rats, nandrolone, training, and training plus nandrolone did not change body mass or vas deferens weight. In castrated rats, the vas deferens wet weight was decreased in both untrained and trained groups. In castrated rats, nandrolone prevented vas deferens atrophy. In intact animals, nandrolone decreased (P < 0.05) the androgen level in untrained group, while in castrated rats this treatment partially restored the androgen level. An increased sensitivity (P < 0.05) to norepinephrine was observed in vas deferens isolated from intact trained rats, treated or not with nandrolone decanoate, while nandrolone did not alter norepinephrine response in organs from untrained animals. In untrained castrated rats, nandrolone fully restored the sensitivity to norepinephrine in untrained rats, while in trained castrated rats the anabolic steroid only partially restored this response. Significance: The present results indicate that training can increase norepinephrine response of vas deferens in intact rats, while nandrolone decanoate can partially restore the responsiveness to norepinephrine in castrated rats. © 2009 Published by Elsevier Inc.
Introduction Anabolic steroids enhance body-tissue-building processes and retard or reverse tissue catabolism. Based on these properties, some athletes use anabolic steroids in an attempt to improve their athletic performance. However, serious adverse outcomes have been correlated with the use of anabolic steroid (Maravelias et al. 2005; Casavant et al. 2007). Moreover, altered androgenic levels have been found following administration of anabolic steroids as well as during prolonged physical activity (Opstad 1992; Hackney et al. 2003). One potentially useful experimental model of exercise in rats is forced swimming. However, elevated levels of norepinephrine and epinephrine are also found during forced swimming, resulting from the physical imposition of forced exercise and the strong emotional components (Östman-Smith 1979; Perronét et al. 1981). In this
⁎ Corresponding author. Department of Pharmacology, Institute of Biosciences, Sao Paulo State University – UNESP, 18610-000 Botucatu, Sao Paulo, Brazil. E-mail address: [email protected] (O.C.M. Pereira). 0024-3205/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.lfs.2009.08.009
exercise model, adaptation to water is necessary to reduce stress to the animals. Changes in testosterone levels can also modulate adrenergic neurotransmission in some reproductive organs, including the vas deferens (MacDonald and McGrath 1980). The rat vas deferens, a smooth muscle of the genital tract, has commonly been considered a suitable preparation for studying α-adrenoceptors (Sallés and Badia 1991). Studies in our laboratory demonstrated reduced sensitivity of vas deferens to norepinephrine in rats submitted to acute swimming. The reduced sensitivity was a consequence of increased neuronal uptake of norepinephrine and β2-adrenoceptor upregulation, which opposes α1-adrenoceptor activity (Chies and Pereira 1995). In androgenic deprivation models, altered responses of isolated seminal vesicles and vas deferens to various drugs have been shown (Porto et al. 1988; Pereira et al. 1993; Souza and Pereira 1999). In addition, castration has been proposed to result in altered response to norepinephrine as a consequence of changes in functional α1-adrenoceptors subtypes involved in the rat vas deferens contractile responses (Pupo 1998). Thus, the physiological level of testosterone, the use of anabolic steroids and exposure to intensive exercise may be involved in the noradrenergic response
542
L.R.G. Castor et al. / Life Sciences 85 (2009) 541–545
pattern of the smooth muscle of the genital tract. On the basis of these considerations, the present study aimed to investigate the norepinephrine response of vas deferens isolated from rats submitted to swimming and/or treated with nandrolone decanoate, an anabolic steroid.
radioimmunoassay (Coat-A-Count Rat Testosterone kit, DPC, Los Angeles, CA, Lot 40101). All samples were measured in duplicate. Data are reported as nanomol per liter of plasma. The interassay coefficient of variation was 6.0%. Pharmacological analysis
Materials and methods Animals Adult male Wistar rats (about 3 months old, average weight 300– 350 g) were randomly divided into respective experimental groups and housed five rats per polypropylene cage (32 × 40 × 18 cm) with bedding of wood shavings. Food and water were freely available with a 12 h light–dark cycle. The temperature and humidity were controlled at 25 ± 1 °C and 55 ± 5%, respectively. The animals used in this study were maintained in accordance with Ethical Principles in Animal Research, adopted by the Brazilian College of Animal Experimentation and approved by the Bioscience Institute/UNESP Animal Research Ethics Committee, protocol number 93/01. Experimental groups Six experimental groups were included in this study, as defined by whether they were castrated or not, submitted to swimming (referred to as training) or not, and either treated or not with nandrolone decanoate. Control groups were included for each of these groups.
The left vas deferens was removed, separated from the surrounding tissue, freed of secretions, weighed, and set up for analysis of contractile capacity in a 10 ml organ-bath circulating a nutritive solution aerated with 95% O2 and 5% CO2 and maintained at 30 °C. The composition of the nutritive solution was 136.0 mM NaCl, 5.7 mM KCl, 1.8 mM CaCl2, 0.36 mM NaH2PO4·H2O, 15.0 mM NaHCO3, and 5.5 mM dextrose, prepared in a glass containing distilled water (Picarelli et al. 1962). A resting tension of 1.0 g was applied to the tissue and the change in isometric tension was measured via force displacement transducers. After an initial resting period of 30 min, concentration– response curves for norepinephrine (arterenol bitartrate, Sigma) were obtained. The concentration–response curves for norepinephrine were obtained in the absence and presence of timolol (10−5 M; timolol maleate, Sigma) added 30 min before the norepinephrine. The agonists were added at successively increasing molar concentrations (van Rossum 1963; van Rossum and van Der Brink 1963). The parameter determined was the pD2, estimated as the negative of the logarithm of the agonist concentration producing 50% (ED50) of the maximum effect (Miller et al. 1948). Statistical analysis
Castration Data are presented as mean ± SEM. Data sets were first compared by analysis of variance, and Tukey's test was used for post-hoc comparisons between pairs of means. The results were considered significant at P < 0.05.
Castration, consisting of bilateral removal of the testicles, was carried out via transscrotal access under sodium pentobarbital (40 mg/kg, ip) anesthesia when the rats were at least 75 days old. These rats were utilized 7 days after orchidectomy in the subsequent experiments.
Results
Nandrolone decanoate treatment
Body mass and wet weight of vas deferens
Rats received subcutaneous injections of 7.5 mg/kg of nandrolone decanoate (Deca durabolinR Organon) or vehicle (peanut oil plus benzilic alcohol, 1:16) 1, 5, 9, and 13 days after beginning the exercise protocol.
Intact and castrated rats, untrained or submitted to swimming (trained), treated or not with nandrolone decanoate showed no significant difference in body mass. Vas deferens from intact rats, untrained or submitted to swimming, treated or not with nandrolone decanoate also showed no alteration in wet weight, while vas deferens from castrated rats, untrained or submitted to exercise weighed significantly less than intact rats. In castrated rats treated with nandrolone decanoate, the wet weight of the vas deferens did not differ significantly from those of intact rats (Table 1).
Exercise protocol Rats were submitted to a 7-day period of adaptation to water in order to minimize stress. For 15 min each day, rats were placed in water (27 °C) within a glass tank of increasing depth, starting with 5 cm of water and increasing gradually to a water height of 40 cm. After this process, the animals were individually submitted to 60 min of swimming each day for 15 days, 1 session per day, 5 days per week. For the swimming procedure (training), the rats carried a metallic ring of about 2% of their body weight on their tails. The tank measured 35 cm in length, 17 cm in width, and 50 cm in depth. Immediately after the last swimming session, the animals were anesthetized by volatile anesthetic, blood samples were collected, and the animals were killed by decapitation.
Plasma androgen level Plasma androgen level (total testosterone) in intact rats decreased significantly after nandrolone decanoate treatment for both rats undergoing training as well as those undergoing training plus nandrolone decanoate. In castrated rats, treatment with the synthetic testosterone, nandrolone decanoate, restored the plasma androgen levels to levels observed in intact untrained rats treated with nandrolone decanoate, intact trained rats, and intact trained rats treated with nandrolone decanoate (Table 2).
Plasma androgen level Pharmacological response of the vas deferens Plasma androgen levels represent total testosterone levels in the rat. To measure these levels blood samples (2.0–2.5 ml) were collected from the abdominal aorta into heparinized vials, always between 09:00 and 10:00 am. Immediately after collection, blood samples were centrifuged (700 × g for 20 min, at 2 °C) and the plasma androgen level (total testosterone) was assayed by solid-phase [I125]
All rats swam vigorously during the swimming sessions. The mean concentration–response curves for norepinephrine in vas deferens from intact rats submitted to swimming, treated or not with nandrolone decanoate, was shifted to the left (Fig. 1). In addition, the corresponding pD2 values were significantly increased (Table 3).
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Table 1 Body mass and wet weight of vas deferens from intact and castrated adult male rats, untrained and submitted to swimming (trained), and either treated or not with nandrolone decanoate (ND). Groups
Body mass (g) Intact rats
349.00 ± 12.89a A Untrained+ ND 348.57 ± 6.35a A Trained 366.30 ± 6.93a A Trained + ND 348.80 ± 8.14a A Untrained
Vas deferens (mg) Castrated rats
Intact rats
Castrated rats
362.28 ± 8.45a A 380.30 ± 13.00a A 371.28 ± 8.54a A 383.00 ± 7.13a A
76.28 ± 6.20a A 72.14 ± 5.80a A 88.85 ± 2.34a A 83.14 ± 5.55a A
35.71 ± 2.22b A 79.14 ± 2.34a B 36.71 ± 2.50b A 77.40 ± 3.10a B
Values expressed as mean ± SEM of six animals per group. A,B Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant differences between castrated rats and their respective control (p < 0.05, Tukey's test).
Experiments performed in the presence of the non-selective β1 and β2-antagonist timolol yielded mean concentration–response curves of norepinephrine superimposed on curves obtained from organs of either untrained or trained rats, treated or not with nandrolone decanoate, and consequently their correspondent pD2 values did not differ significantly between the groups (P > 0.05; data not shown). In vas deferens from castrated untrained rats, the norepinephrine curves were shifted to the right compared with intact untrained rats treated or not with nandrolone decanoate, and the anabolic steroid treatment restored the sensitivity in castrated untrained rats (Fig. 2A). In vas deferens from trained castrated rats, the mean concentration response curves for norepinephrine were shifted to the right in comparison with those obtained from intact trained rats treated or not with nandrolone decanoate. Treatment with the anabolic steroid only partially restored the norepinephrine response of vas deferens from castrated trained rats (Fig. 2B). Corresponding pD2 values are shown in Table 3. Discussion The importance of hormonal control has been shown in the development of accessory reproductive organs and in the reactivity of smooth muscle within the male genital organs (Picarelli and Valle 1969; Valle et al. 1982). Thus, androgens are necessary for the development and function of the male genital tract (Sjöstrand and Swedin 1976; Pereira et al. 1991) and androgens may also modulate the noradrenergic response in vas deferens isolated from rats (Pereira et al. 1998). In the present study, treatment of intact rats with Table 2 Plasma androgen levels (total testosterone) in intact and castrated rats, untrained and submitted to swimming (trained), and treated or not with nandrolone decanoate (ND). Groups
Untrained + ND Trained Trained + ND
Table 3 pD2 values of norepinephrine in vas deferens from intact and castrated rats, untrained and submitted to swimming (trained), and treated or not with nandrolone decanoate (ND). pD2 Norepinephrine
Castrated rats a
9.02 ± 1.17 A 3.33 ± 0.23a B 3.82 ± 0.91a B 4.01 ± 0.50a B
b
0.05 ± 0.03 A 3.76 ± 0.73a B 0.07 ± 0.05b A 4.22 ± 0.66a B
Values expressed as mean ± SEM of six animals per group. Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant difference between castrated rats and their respective control (p < 0.05, Tukey's test). A,B
nandrolone decanoate for 2 weeks did not change either the body mass or vas deferens wet weight. These results are consistent with data in the literature showing no change in body weight after two weeks of treatment with anabolic steroids (Blasberg et al. 1997; Tamaki et al. 2001). It is possible that the treatment duration was not sufficient to induce changes in body weight of the animals in this study. In spite of this finding, both the swimming (training) and the treatment with nandrolone decanoate decreased plasma androgens level in intact rats. These results also agree with those showing that exercise training frequently results in a decrease of serum testosterone and may rarely be associated with reduced libido, sperm production and fertility (Eliakim and Nemet 2006). In this exercise protocol, the decreased testosterone was likely due to the repeated exercise plus residual stress, which produced increased corticosterone levels. On the other hand, in the present study, the decreased testosterone level observed after nandrolone decanoate treatment may be a result of hypothalamus–hypophysial feedback mechanisms, which exert a depressive effect on endogenous secretion (Signoret 1976; Hackney et al. 2003). However, in castrated rats treated nandrolone decanoate, androgenic levels were restored to approximately 40% of that observed in intact untrained rats. This result indicates that despite the high dose of synthetic androgen used, by exerting predominantly anabolic activity nandrolone decanoate was not able to restore androgen level to values observed in the control
Androgen nmol/l Intact rats
Untrained
Fig. 1. Cumulative concentration–response curves for norepinephrine in vas deferens from intact untrained rats as well as those submitted to swimming (trained), treated or not with nandrolone decanoate (ND). The X-axis shows the molar concentration of the drug on a logarithmic scale. The Y-axis shows effects of norepinephrine as a percentage of the maximal contraction produced by the agonist. Vertical bars indicate SEM. Each point represents a mean of six experiments.
Groups Intact Intact + ND Castrated Castrated + ND
Untrained
Trained a
5.55 ± 0.05 A 5.46 ± 0.07a A 5.11 ± 0.10a B 5.55 ± 0.11a A
5.96 ± 0.13b A 5.88 ± 0.07b A 5.18 ± 0.01a B 5.40 ± 0.15a C
Values expressed as mean ± SEM of six animals per group. A,B Different capital letters indicate significant difference within rows (p < 0.05, Tukey's test). a,b Different superscripted small letters indicate horizontal significant difference between trained and untrained rats (p < 0.05, Tukey's test).
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Fig. 2. Cumulative concentration–response curves for norepinephrine in vas deferens from intact and castrated rats, untrained (A) and submitted to swimming (trained, B), treated or not with nandrolone decanoate (ND). The X-axis shows the molar concentration of norepinephrine on a logarithmic scale. The Y-axis shows effects as a percentage of the maximal contraction produced by norepinephrine. Vertical bars indicate SEM. Each point represents a mean of at least six experiments.
group. In addition, although nandrolone decanoate was able to partly restore the androgen level in the castrated animals, nandrolone prevented both the vas deferens atrophy and the decreased reactivity of these tissues to norepinephrine. In rats undergoing a single forced swimming trial, a model in which stress is highly important, both functional and endocrinological alterations of the organism have been observed, with the prejunctional α2-adrenoceptors, the postjunctional α1-adrenoceptors, and the neuronal uptake of catecholamines being involved in vas deferens homeostasis (Sanchez and Pereira 2002). In the present study, the swimming, which functioned as repeated exercise (with a period of adaptation to water in order to minimize the stress), resulted in supersensitivity to norepinephrine of vas deferens isolated from intact rats treated or not with nandrolone decanoate. This supersensitivity to norepinephrine in organs from rats submitted to swimming may be correlated with changes in norepinephrine receptors and levels of androgens. A study from our laboratory showed that acute exercise (swimming) induced significant α1-adrenoceptor contractile activity in rat vas deferens due to stressogenic activity, and this effect was antagonized by β2-adrenoceptors, both α1- and β2-adrenoceptors equally active (Chies and Pereira 1995). In contrast, the results of the present study showed that the postjunctional β2-adrenoceptors likely did not participate in the norepinephrine response pattern observed in vas deferens after the repeated exercise because the response to norepinephrine in the presence of timolol was similar for all
experimental groups. Thus, by minimizing stress, the repeated exercise may be responsible for the observed absence of participation of β2-adrenoceptors. In addition, previous studies from our laboratory showed that a one-day swimming protocol produced subsensitivity to norepinephrine of the vas deferens of intact adult rats, while no change in sensitivity was observed for this parameter when the animals were submitted to swimming for three consecutive days, thus suggesting the occurrence of adaptation to elevated norepinephrine levels, a mild decrease in androgen levels or both (Pereira et al. 1998). On the other hand, hormonal deprivation–castration induced subsensitivity to norepinephrine in vas deferens from untrained and trained rats. In castrated untrained rats, nandrolone decanoate fully restored sensitivity to norepinephrine, while in castrated trained rats the norepinephrine sensitivity was only partially restored by nandrolone decanoate relative to intact trained rats. In another study, testosterone and 5α-norethisterone were also shown to restore the sensitivity to methoxamine and serotonin of the epididymal portion of vas deferens from castrated rats (Campos et al. 1999). These data together with the results of the present study support the effectiveness of androgens in modulating the autonomic-receptor response in smooth muscle of the reproductive tract. Finally, despite the indication of reduced (minimized) stress in the swimming-exercise model, we suggest that the effects of “training” are actually different from those previously described as an effect of stress. The results of the current study indicate that in intact rats swimming (training) could increase the norepinephrine response pattern in the vas deferens, while nandrolone decanoate did not modify this response. That said, the possibility that other mechanisms may be involved in increased sensitivity to norepinephrine cannot be discarded. However, in castrated rats, swimming was not effective in altering the response to norepinephrine, while nandrolone decanoate did partly modify this response. To summarize, through using castration and exercise training in rats, we have found that in the absence of androgens anabolic androgenic steroid treatment restored at least partially the response of the isolated vas deferens to norepinephrine, confirming findings that the response of the smooth muscles of male organs to norepinephrine are not only hormonesensitive, but also androgen-dependent. In conclusion, training can increase the norepinephrine response in the vas deferens of intact rats, while nandrolone decanoate can be effective only in conditions of androgen deficiency. Acknowledgement Research was supported by CAPES. This study is part of the M.Sc. thesis presented to Sao Paulo State University (UNESP) by Lidyane R.G. Castor. References Blasberg ME, Langan CJ, Clark AS. The effects of 17α-methyltestosterone, methandrostenolone, and nandrolone decanoate on the rat estrous cycle. Physiology and Behavior 61, 265–272, 1997. Campos MG, Oropeza MV, Lemus AE, Garcia GA, Reynoso ME, Campos P, Ponce-Monte H. The androgenic effect of norethisterone and 5α-norethisterone on the contractile response of the rat vas deferens to methoxamine and serotonin. Life Sciences 64, 227–233, 1999. Casavant MJ, Blake K, Griffith J, Yates A, Copley LM. Consequences of use of anabolic androgenic steroids. Pediatric Clinics of North America 54, 677–690, 2007. Chies AB, Pereira OCM. Cathecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress. Pharmacological Research 32 (3), 123–127, 1995. Eliakim A, Nemet D. Exercise and the male reproductive system. Harefuah 145 (9), 677–681 702,701, 2006. Hackney AC, Szczepanowska E, Viru AM. Basal testicular testosterone production in endurance-trained men is suppressed. European Journal of Applied Physiology 89, 198–201, 2003. MacDonald A, McGrath JC. The effects of castration on neurotransmission in the rat vas deferens. British Journal of Pharmacology 69, 49–58, 1980.
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