- Email: [email protected]
Noradrenergic Response in Vas Deferens from Rats Submitted to Acute and Repeated Stress
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00280-2 All rights reserved
Gen. Pharmac. Vol. 30, No. 3, pp. 417–421, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Noradrenergic Response in Vas Deferens from Rats Submitted to Acute and Repeated Stress Oduvaldo C. M. Pereira,* Mara R. Dega and Maricelma S. S. Souza Department of Pharmacology, Institute of Bioscience-Sa˜o Paulo State University (UNESP), 18618-000, Botucatu, Sa˜o Paulo, Brazil ABSTRACT. 1. This work investigated the effects of androgens on the norepinephrine sensitivity of vasa deferentia from rats submitted to acute or repeated stress, as well as the participation of a1-adrenoceptors in the response of intact and bisected vasa deferentia from adult normal rats submitted to acute or repeated stress. 2. The acute stress produced subsensitivity to norepinephrine only in intact vasa deferentia from adult normal rats, which was prevented by lack of androgens, suggesting that the sensitivity may be dependent on the physiological level of androgen. 3. No change was observed in intact vas deferens sensitivity to norepinephrine in repeated stress, suggesting the occurrence of adaptation to elevated norepinephrine levels or a mild decrease in androgen levels or both. 4. The changes in sensitivity observed in acute and repeated stress may also be due to alterations in a1-adrenergic receptors that are located in the prostatic portion of the vas deferens. gen pharmac 30;3:417–421, 1998. 1998 Elsevier Science Inc. KEY WORDS. Swimming stress, vas deferens, norepinephrine, prazosin, rat INTRODUCTION Previous studies demonstrated the importance of the androgenic levels in the development, size and secretion of sexual male accessory organs of mammals, as well as in the pharmacological reactivity of these tissues to the sympathomimetic agonists (Picarelli and Valle, 1969; Porter and Melampy, 1952; Sjo¨strand and Swedin, 1976). The rat vas deferens is an accessory reproductive organ that is widely used as a model preparation for the study of neurotransmission and of adrenergic drugs and their mechanisms of action (Benge and Jordan, 1993). Organs from rats submitted to castration and cryptorchidism may be androgenic depressed models, where experimental evidence demonstrated spontaneous and rhythmic contractions or alterations in the reactivity to various drugs or both (Pereira et al., 1993; Valle et al., 1982). The vas deferens is also a suitable preparation to evaluate the effect of age or sexual development or both on adrenergic drug neurotransmission and mechanism of action. Therefore, sexual immaturity may alter the pharmacological reactivity of the rat vas deferens to the sympathomimetic agonists through changes in the neuronal uptake system, in the modulation of release of norepinephrine and in the level of postjunctional a1adrenoceptors (Avellar and Markus, 1993; Markus and Avellar, 1992; Soares et al., 1993). Besides, pharmacological reactivity of various organs to the sympathomimetic agonists may be modified subsequent to hormonal alterations, such as those induced by stressinducing situations (Bispo and Pereira, 1992; Carlos et al., 1995; Spadari and De Moraes, 1988; Spadari et al., 1988). Therefore, if these transformations protect life and ensure the maintenance of homeostasis, it might result in damage to the organism or in a pathological state (Bispo and Pereira, 1992). Thus, acute swimming stress induced a major a1-adrenoceptor contractile activity in the rat vas deferens that was contradicted by b2-adrenoceptors, either equally * To whom all correspondence should be addressed. Received 4 November 1996; accepted 6 May 1997.
active (Chies and Pereira, 1995). The forced swimming stress is characterized by emotional and physical components and it is painless. Elevated levels of norepinephrine and epinephrine are present in this experimental model, which are the physical components imposed by the forced swimming, and also the strong emotional components (Ostman-Smith, 1979; Pe´rronnet et al., 1981). Nevertheless, little is known about the role of acute and repeated stress in the sympathoadrenomedullary system in the rat vas deferens. Moreover, there are differences in the relation of receptor occupancy and the evoked response between the epididymal and prostatic halves of the vas deferens (Salle´s and Badia, 1991). Burt et al. (1996) demonstrated that the contraction of the epididymal portion of the rat vas deferens is mediated by a1A-adrenoceptors, which depends on protein kinase C activation by diacylglycerol, resulting in calcium extracellular influx. The aim of the present work was to investigate the effects of androgen deprivation in the norepinephrine sensibility of the rat vas deferens submitted to acute or repeated stress. Furthermore, the participation of a1-adrenoceptors in norepinephrine sensitivity in intact and bisected vasa deferentia from adult rats submitted to acute or repeated stress was investigated. MATERIAL AND METHODS
Animals Male Wistar rats (15, 40 and 105 days old) were divided into four experimental groups (adult, immature, cryptorchid and castrated rats, subdivided into control, acute and repeated stress) housed five rats per cage with food and water freely available and with a 12-hr light–dark cycle. The temperature and humidity were controlled at 25618C and 5565%, respectively.
Swimming stress Animals were submitted to a single forced swimming stress individually for 50 min, according to Bispo and Pereira (1992). Repeated
418
O. C. M. Pereira et al.
swimming stress consisted of one session per day for 3 consecutive days with durations of 5, 15 and 30 min, respectively (Spadari and De Moraes, 1988). Animals from the two stressed groups were submitted to swimming stress in a glass tank containing water at room temperature, each animal carrying a metallic ring of about 2% of its corporal weight on its tail. This tank was 45 cm long, 35 cm wide and 33 cm deep. After the swimming session, the animals were killed by inhalation of an overdose of ethyl ether followed by decapitation.
tatic portions obtained from adult rats (Fig. 1B). Its correspondent pD2 value decreased significantly after the acute stress (Table 2). On the other hand, repeated stress induced a shift to the left on the concentration-response curve for norepinephrine only in prostatic portions obtained from adult rats (Fig. 1C). Its correspondent pD2 value increased significantly after the repeated stress (Table 2). However, these significant alterations observed in both portions disappeared when the concentration-response curves to norepinephrine were made in the presence of 1028 M of prazosin (Table 3).
Cryptorchidism and castration
DISCUSSION
Animals were rendered cryptorchid bilaterally when they were 17–20 days old by fixing the gubernaculum testis to the posterior abdominal wall under ether anesthesia (Pereira et al., 1993). Castration consisted in transcrotally bilateral orchiectomies, which were done under ether anesthesia when the rats were 75 days old.
It is known that the rat vas deferens has a high concentration of norepinephrine due to a dense innervation by short adrenergic neurons (Sjo¨strand 1962, 1965). On the other hand, experiments using rats submitted to exercise demonstrated that epinephrine released from adrenal glands during exercises facilitates the output of norepinephrine from adrenergic nerve endings (Scheurink, 1989). In stress conditions, despite the suggestion of Stone (1979) that subsensitivity does not occur after acute stress but only after chronic stress when the animals show clear evidence of resistance to its adverse effects, the data of the present work show that acute swimminginduced stress produces subsensitivity to norepinephrine in vasa deferentia from adult normal rats. Thus, this subsensitivity might be an attempt of the organ to protect itself from high plasma levels of catecholamines released during the stress, which could be injurious to the organism. Previous work also related the importance of gonadal steroids in the reactivity of organs from male reproductive system (Porto et al., 1988; Valle et al., 1982). Androgens are necessary to the functional and trophic development of the male reproductive system smooth muscles (Sjo¨strand and Swedin, 1976). The results of the present study show that a lack of androgens prevented stressinduced subsensitivity to norepinephrine, which could be beneficial to the homeostasis of the organ, when catecholamine levels are high. Thus, testosterone may play an important role in the maintenance of the homeostasis of the rat vas deferens, causing subsensitivity to norepinephrine in acute stress situations (Carlos et al., 1995). Moreover, Charlwood et al. (1979) concluded that adequate testosterone levels are necessary for the maintenance of contractility in the rat vas deferens. On the other hand, there is variation in the responses to stress that depends on the type and duration of stressogenic situations to which the organism is submitted. Thus, shortterm exercise induced an increased plasma level of luteinizing and follicle-stimulating hormones, of catecholamines and of testosterone. Moreover, under prolonged physical stress, such as bicycle competition lasting for several days and psychological stress in military operations, the plasma levels of androgens decreased (Opstad, 1992). In the present work, subsensitivity to norepinephrine was observed in acute stress situations, instead of repeated stress. In repeated stress, the animals are exposed to the same stress stimulation daily, and adaptive changes would appear in the sympathoadrenomedullary system, resulting in an increase in the synthesis and basal levels of catecholamines (McCarty et al., 1988) and corticosteroids (Spadari and De Moraes, 1988). Therefore, repeated stress may result in a decreased androgen level, because this stress model consisted of repeated sessions of intense emotional stress and physical exercise. This suggests that the subsensitivity to norepinephrine observed after acute stress may depend on the androgen physiological level. On the basis of the results obtained with repeated stress that did not show an alteration in sensitivity to norepinephrine, we suggest that this absence of alteration may be a consequence of adaptation to elevated norepinephrine levels, or a mild decrease in andro-
Organ-bath studies For the analysis of their contractile capacity, the vasa deferentia were individually set up in 10-ml organ baths, containing continuously aerated nutritive solution at 308C, according to a technique previously described (Pereira, 1987). Longitudinal contractions were recorded with the aid of an isotonic level with a load of 0.5 or 1.0 g. After an initial resting period of 45 min, concentrationresponse curves to norepinephrine were obtained by cumulative addition of molar concentrations of the drug increasing geometrically (Van Rossum, 1963; Van Rossum and Van den Brink, 1963).
Pharmacological parameter The pD2 values, expressed as the negative logarithm of the agonist concentration producing 50% (ED50) of its maximum effect (Miller et al., 1948), were determined.
Drugs Norepinephrine (l-arterenol bitartrate) was from Sigma (USA); prazosin (prazosin hydrochloride) was from Pfizer (SA). Stock solutions of norepinephrine were performed in 0.01 N HCl and maintained frozen. Working solutions were diluted in saline solution (0.9% Na Cl) plus ascorbic acid (0.005%) immediately before use.
Statistical analysis The mean6SEM of pD2 values were calculated and compared previously by analysis of variance of the 333 factorial experiment in one-way classification. Tukey’s test was made for further comparisons between pairs of means (Ostle, 1973). RESULTS Without previous training, the animals swam vigorously during the stress sessions and showed signs of intense emotional distress such as defecation and vocalization. The mean concentration-response curve to norepinephrine obtained only in vasa deferentia from adult rats was shifted to the right after acute stress condition (Fig. 1A), and its correspondent pD2 value also decreased significantly (Table 1), which indicates subsensitivity. Furthermore, there were no sensitivity alterations when the experiments were carried out in immature, cryptorchid and castrated rats. Moreover, the acute stress induced a shift to the right on the concentration-response curve for norepinephrine only in pros-
Stress versus Vas Deferens Reactivity
419
FIGURE 1. Cumulative concentration-response curves for norepinephrine in isolated intact vas deferens (A), and in isolated prostatic portions of vas deferens (B, C) from adult rat. Abscissa shows the molar concentration of the drugs on a logarithmic scale. Ordinate shows effects as a percentage of the maximal contraction produced by the drug. Vertical bars indicate SEM. Each point represents a mean of at least six experiments. gen levels or both. This adaptation might occur by a prejunctional a2-adrenoceptor alteration as a consequence of high concentrations of norepinephrine in the biophasis. Nevertheless, little is known of the role of acute and repeated stress in the sympathoadrenomedullary system in the rat vas deferens. On the other hand, the two portions of the rat vas deferens also differ not only in their anatomical and histological structures, but also in their postsynaptic a-adreno-
ceptor subtypes (Badia and Salle´s, 1989; Brito et al., 1989; MacDonald and McGrath, 1980; Markus and Avellar, 1992). Our results showed that acute stress induced a decrease in the sensitivity to norepinephrine in the prostatic portion from the adult normal rat vas deferens. In contrast, repeated stress also induced an increase in the sensitivity to norepinephrine in the prostatic portion from the adult normal rat vas deferens. However, these sensitivity alterations dis-
420
O. C. M. Pereira et al.
TABLE 1. Effect of acute or repeated swimming-induced stress on response to norepinephrine in isolated vasa deferentia from adult, immature, cryptorchid and castrated rats pD2 @ values expressed as mean 6 S.E.M. Rats Adult Immature Cryptorchid Castrated
Control 5.27 6 0.05a (6)A 5.09 6 0.08a (6)A 5.18 6 0.05a (6)A 5.09 6 0.07a (6)A
Acute stress 4.95 6 0.03b (6)B 5.21 6 0.08a (6)AB 5.30 6 0.02a (6)A 5.23 6 0.04a (6)AB
Repeated stress 5.40 6 0.09a (6)A 5.32 6 0.06a (7)A 5.41 6 0.08a (5)A 5.33 6 0.15a (4)A
Number of experiments in parentheses. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect. a, b indicate horizontal significant difference (P , 0.05, Tukey’s test). A, B, or AB indicate vertical significant difference (P , 0.05, Tukey’s test).
TABLE 3. Effect of acute or repeated swimming-induced stress on response to norepinephrine in intact and bisected vasa deferentia isolated from adult rats (experiments were performed in the presence of 1028 M prazosin.) pD2 @ values expressed as mean 6 S.E.M. Vas deferens Intact Epididymal Prostatic
Control
Acute stress
Repeated stress
4.78 6 0.05 (8) 4.84 6 0.05 (8) 4.28 6 0.05 (8)
4.67 6 0.06 (10) 4.77 6 0.02 (8) 4.13 6 0.11 (8)
4.65 6 0.04 (6) 4.78 6 0.03 (8) 4.21 6 0.09 (8)
Number of experiments in parentheses. There were no significant differences among the groups. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect.
appeared when experiments were performed in the presence of 1028 M prazosin. This suggests that the subsensitivity to norepinephrine induced by acute stress is also due to alterations in a1-adrenergic receptors located in the prostate portion. This subsensitivity to norepinephrine may also be modulated by androgen levels and, in repeated stress, this alteration is not present.
in sensitivity to norepinephrine, only in the prostatic portion from the adult rat vas deferens. Integrated, these phenomena are responsible for the rat vas deferens subsensitivity to norepinephrine, which may be involved in body homeostasis in stressogenic situations. This subsensitivity is also due to alterations in a1-adrenergic receptors located in the vas deferens prostatic portion, which is physiologically androgen dependent, and it does not appear after repeated stress.
SUMMARY
The authors thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, process 94/4133-2 and 95/2076-4) for financial support.
The study was performed to examine the responses to norepinephrine in vasa deferentia isolated from adult normal, immature, cryptorchid and castrated rats submitted to acute or repeated swimminginduced stress. Additionally, the participation of the postjunctional a1-adrenoceptor in response to norepinephrine in intact or bisected vasa deferentia from adult normal rats submitted to acute or repeated stress was investigated. Acute stress was demonstrated to induce a significant subsensitivity to norepinephrine only in intact vasa deferentia from adult normal rats. This subsensitivity was inhibited when the experiments were carried out in groups with androgenic deprivation and in the repeated-stress model. Moreover, our results showed an acute stress-induced decrease in sensitivity to norepinephrine, which opposes the repeated stress-induced increase TABLE 2. Effect of acute or repeated swimming-induced stress on response to norepinephrine in intact and bisected vasa deferentia isolated from adult rats pD2 @ values expressed as mean 6 S.E.M. Vas deferens Intact Epididymal Prostatic
Control
Acute stress
Repeated stress
5.27 6 0.05a (6)A 5.46 6 0.08a (8)A 4.98 6 0.05b (8)B
4.95 6 0.03b (6)B 5.42 6 0.07a (8)A 4.70 6 0.05c (8)B
5.40 6 0.09a (6)A 5.47 6 0.09a (8)A 5.27 6 0.14a (8)A
Number of experiments in parentheses. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect. a, b, c indicate horizontal significant difference (P , 0.05, Tukey’s test). A, B, or AB indicate vertical significant difference (P , 0.05, Tukey’s test).
References Avellar M. C. W. and Markus R. P. (1993) Age related changes in norepinephrine release and its modulation by presynaptic alpha-2 adrenoceptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 267, 38–44. Badia A. and Salle´s J. (1989) Effects of St-587 on the a-adrenoceptors in the bisected rat vas deferens. J. Pharm. Pharmac. 41, 612–616. Benge B. N. and Jordan G. H. (1993) Prepubertal vasal injury: its effect on postpubertal vas deferens. J. Urol. 149, 906–907. Bispo D. L. N. and Pereira O. C. M. (1992) Involvement of swimminginduced acute stress in the sensitivity of rat vas deferens to norepinephrine. Gen. Pharmac. 23, 1125–1128. Brito A. R. M., Medeiros I. A. and Markus R. P. (1989) Do differences in innervation result in different post-synaptic responses to exogenous agonists? Gen. Pharmac. 20, 65–69. Burt R. P., Chapple C. R. and Marshall I. (1996) The role of diacylglycerol and activation of protein Kinase C in a1a-adrenoceptor–mediated contraction to noradrenaline of rat isolated epididymal vas deferens. Br. J. Pharmac. 117, 224–230. Carlos C. P., Padovani C. R. and Pereira O. C. M. (1995) Role of androgenic deprivation on the contractile response to norepinephrine in vas deferens isolated from rats submitted to swimming-stress. Rev. Cieˆnc. Biome´d. 16, 7–15. Charlwood R. A., Handberg G. M. and Pennefather J. N. (1979) A study of the effects of steroids on a-adrenoceptor–mediated contraction of the rat vas deferens. Clin. Exp. Pharmac. Physiol. 7, 11–21. Chies A. B. and Pereira O. C. M. (1995) Catecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress. Pharmac. Res. 32, 123–127. MacDonald A. and McGrath J. C. (1980) The effects of castration on neurotransmission in the rat vas deferens. Br. J. Pharmac. 69, 49–58. Markus R. P. and Avellar M. C. W. (1992) Effect of ageing on the number of neuronal noradrenaline uptake sites in the rat deferens. NaunynSchmiedeberg’s Arch Pharmac. 346, 405–409. McCarty R., Howa T. T. K. and Konarska M. (1988) Chronic stress and sympathetic-adrenal medullary responsiveness. Soc. Sci. Med. 26, 333– 341. Miller L. C., Becker T. J. and Trainter M. L. (1948) The quantitative evaluation of spasmolytic drugs “in vitro.” J. Pharmac. Exp. Ther. 92, 260–268.
Stress versus Vas Deferens Reactivity Opstad P. K. (1992) Androgenic hormones during prolonged physical stress, sleep, and energy deficiency. J. Clin. Endocrinol. Metabol. 74, 1176– 1183. Ostle B. (1973) Analisis de un disen˜o completamente azarizado que involucra conbinaciones de tratamiento factorial. In Estatı´stica aplicada. pp. 350–371. Limusa-Willey, Me´xico. ¨ stman-Smith I. (1979) Adaptative changes in the sympathetic nervous O system and some effector organs of the rat following long-term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hypertrophy. Acta Physiol. Scand. Suppl. 477, 1–118. Pereira O. C. M. (1987) Some effects of cimetidine on the reproductive organs of rats. Gen. Pharmac. 18, 197–199. Pereira O. C. M., Picarelli Z. P. and Abreu L. C. (1993) Experimental cryptorchidism in prepubertal rats: some effects on reactivity of the seminal vesicle to catecholamines. Pharmac. Res. 28, 175–181. Perronne´t F., Cle´roux J., Perrault H., Cousineau D., De Champlain J. and Nadeau R. (1981) Plasma norepinephrine response to exercise before and after training in humans. J. Appl. Physiol. 51, 812–815. Picarelli Z. P. and Valle J. R. (1969) Hormonal regulation of the rat seminal vesicle sensitivity to adrenaline, acetylcholine and acetyl-beta-methylcoline. Br. J. Pharmac. 35, 468–475. Porter J. C. and Melampy R. M. (1952) Effects of testosterone propionate on the seminal vesicle response to parasympathomimetic drugs. Endocrinology 51, 412–420. Porto C. S., Abreu M. L. C. and Picarelli Z. P. (1988) Influence of castration and of the kind of sympathomimetic drug used upon the reactivity of rat seminal vesicles. Arch. Int. Pharmacodyn. Ther. 292, 101–111. Salles J. and Badia A. (1991) Mechanisms underlying the differencial sensitivity to a1-adrenoceptor activation in the bisected rat vas deferens. Br. J. Pharmac. 102, 439–445. Scheurink A. J. W., Steffens A. B., Bouritius H., Dreteler G. H., Bruntink
421 R., Remie R. and Zaagsma J. (1989) Adrenal and sympathetic catecholamines in exercising rats. Am. J. Physiol. 256, r155–r160. Sjostrand N. O. (1962) Effect of reserpine and hypogastric denervation on the noradrenaline content of the vas deferens and the seminal vesicle of guinea-pig. Acta Physiol. Scand. 56, 376–380. Sjostrand N. O. (1965) The adrenergic innervation of the vas deferens and the accessory male genital glands. Acta Physiol. Scand. 65(Suppl. 257), 82P. Sjostrand N. O. and Swedin G. (1976) Influence of age, growth, castration and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands of the rat. Acta Physiol. Scand. 98, 323–338. Soares R. D. F., Abreu M. L. C. and Porto C. S. (1993) Post-natal development modulates the rat seminal vesicle sensitivity to sympathomimetic agonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 348, 53–57. Spadari R. C. and De Moraes S. (1988) Repeated swimming stress and responsiveness of the isolated rat pacemaker to the chronotropic effect of noradrenaline and isoprenaline: role of adrenal corticosteroids. Gen. Pharmac. 19, 553–557. Spadari R. C., Bassani R. A. and De Moraes S. (1988) Supersensitivity to isoprenaline and epinephrine in right atria isolated from rats submitted to a single swimming session. Gen. Pharmac. 19, 129–135. Stone E. A. (1981) Mechanism of stress-induced subsensitivity to norepinephrine. Pharmac. Biochem. Behav. 14, 719–723. Valle R. M. R., Abreu L. C., Picarelli Z. P. and Valle J. R. (1982) Influence of castration upon trophism and reactivity of rat seminal vesicles. Braz. J. Med. Biol. Res. 15, 49–53. Van Rossum J. M. (1963) Cumulative dose-response curves II: technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. Int. Pharmacodyn. Ther. 143, 299–330. Van Rossum J. M. and Van Den Brink F. G. (1963) Cumulative doseresponse curves I: introduction to the technique. Arch. Int. Pharmacodyn. Ther. 143, 240–246.
Gen. Pharmac. Vol. 30, No. 3, pp. 417–421, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Noradrenergic Response in Vas Deferens from Rats Submitted to Acute and Repeated Stress Oduvaldo C. M. Pereira,* Mara R. Dega and Maricelma S. S. Souza Department of Pharmacology, Institute of Bioscience-Sa˜o Paulo State University (UNESP), 18618-000, Botucatu, Sa˜o Paulo, Brazil ABSTRACT. 1. This work investigated the effects of androgens on the norepinephrine sensitivity of vasa deferentia from rats submitted to acute or repeated stress, as well as the participation of a1-adrenoceptors in the response of intact and bisected vasa deferentia from adult normal rats submitted to acute or repeated stress. 2. The acute stress produced subsensitivity to norepinephrine only in intact vasa deferentia from adult normal rats, which was prevented by lack of androgens, suggesting that the sensitivity may be dependent on the physiological level of androgen. 3. No change was observed in intact vas deferens sensitivity to norepinephrine in repeated stress, suggesting the occurrence of adaptation to elevated norepinephrine levels or a mild decrease in androgen levels or both. 4. The changes in sensitivity observed in acute and repeated stress may also be due to alterations in a1-adrenergic receptors that are located in the prostatic portion of the vas deferens. gen pharmac 30;3:417–421, 1998. 1998 Elsevier Science Inc. KEY WORDS. Swimming stress, vas deferens, norepinephrine, prazosin, rat INTRODUCTION Previous studies demonstrated the importance of the androgenic levels in the development, size and secretion of sexual male accessory organs of mammals, as well as in the pharmacological reactivity of these tissues to the sympathomimetic agonists (Picarelli and Valle, 1969; Porter and Melampy, 1952; Sjo¨strand and Swedin, 1976). The rat vas deferens is an accessory reproductive organ that is widely used as a model preparation for the study of neurotransmission and of adrenergic drugs and their mechanisms of action (Benge and Jordan, 1993). Organs from rats submitted to castration and cryptorchidism may be androgenic depressed models, where experimental evidence demonstrated spontaneous and rhythmic contractions or alterations in the reactivity to various drugs or both (Pereira et al., 1993; Valle et al., 1982). The vas deferens is also a suitable preparation to evaluate the effect of age or sexual development or both on adrenergic drug neurotransmission and mechanism of action. Therefore, sexual immaturity may alter the pharmacological reactivity of the rat vas deferens to the sympathomimetic agonists through changes in the neuronal uptake system, in the modulation of release of norepinephrine and in the level of postjunctional a1adrenoceptors (Avellar and Markus, 1993; Markus and Avellar, 1992; Soares et al., 1993). Besides, pharmacological reactivity of various organs to the sympathomimetic agonists may be modified subsequent to hormonal alterations, such as those induced by stressinducing situations (Bispo and Pereira, 1992; Carlos et al., 1995; Spadari and De Moraes, 1988; Spadari et al., 1988). Therefore, if these transformations protect life and ensure the maintenance of homeostasis, it might result in damage to the organism or in a pathological state (Bispo and Pereira, 1992). Thus, acute swimming stress induced a major a1-adrenoceptor contractile activity in the rat vas deferens that was contradicted by b2-adrenoceptors, either equally * To whom all correspondence should be addressed. Received 4 November 1996; accepted 6 May 1997.
active (Chies and Pereira, 1995). The forced swimming stress is characterized by emotional and physical components and it is painless. Elevated levels of norepinephrine and epinephrine are present in this experimental model, which are the physical components imposed by the forced swimming, and also the strong emotional components (Ostman-Smith, 1979; Pe´rronnet et al., 1981). Nevertheless, little is known about the role of acute and repeated stress in the sympathoadrenomedullary system in the rat vas deferens. Moreover, there are differences in the relation of receptor occupancy and the evoked response between the epididymal and prostatic halves of the vas deferens (Salle´s and Badia, 1991). Burt et al. (1996) demonstrated that the contraction of the epididymal portion of the rat vas deferens is mediated by a1A-adrenoceptors, which depends on protein kinase C activation by diacylglycerol, resulting in calcium extracellular influx. The aim of the present work was to investigate the effects of androgen deprivation in the norepinephrine sensibility of the rat vas deferens submitted to acute or repeated stress. Furthermore, the participation of a1-adrenoceptors in norepinephrine sensitivity in intact and bisected vasa deferentia from adult rats submitted to acute or repeated stress was investigated. MATERIAL AND METHODS
Animals Male Wistar rats (15, 40 and 105 days old) were divided into four experimental groups (adult, immature, cryptorchid and castrated rats, subdivided into control, acute and repeated stress) housed five rats per cage with food and water freely available and with a 12-hr light–dark cycle. The temperature and humidity were controlled at 25618C and 5565%, respectively.
Swimming stress Animals were submitted to a single forced swimming stress individually for 50 min, according to Bispo and Pereira (1992). Repeated
418
O. C. M. Pereira et al.
swimming stress consisted of one session per day for 3 consecutive days with durations of 5, 15 and 30 min, respectively (Spadari and De Moraes, 1988). Animals from the two stressed groups were submitted to swimming stress in a glass tank containing water at room temperature, each animal carrying a metallic ring of about 2% of its corporal weight on its tail. This tank was 45 cm long, 35 cm wide and 33 cm deep. After the swimming session, the animals were killed by inhalation of an overdose of ethyl ether followed by decapitation.
tatic portions obtained from adult rats (Fig. 1B). Its correspondent pD2 value decreased significantly after the acute stress (Table 2). On the other hand, repeated stress induced a shift to the left on the concentration-response curve for norepinephrine only in prostatic portions obtained from adult rats (Fig. 1C). Its correspondent pD2 value increased significantly after the repeated stress (Table 2). However, these significant alterations observed in both portions disappeared when the concentration-response curves to norepinephrine were made in the presence of 1028 M of prazosin (Table 3).
Cryptorchidism and castration
DISCUSSION
Animals were rendered cryptorchid bilaterally when they were 17–20 days old by fixing the gubernaculum testis to the posterior abdominal wall under ether anesthesia (Pereira et al., 1993). Castration consisted in transcrotally bilateral orchiectomies, which were done under ether anesthesia when the rats were 75 days old.
It is known that the rat vas deferens has a high concentration of norepinephrine due to a dense innervation by short adrenergic neurons (Sjo¨strand 1962, 1965). On the other hand, experiments using rats submitted to exercise demonstrated that epinephrine released from adrenal glands during exercises facilitates the output of norepinephrine from adrenergic nerve endings (Scheurink, 1989). In stress conditions, despite the suggestion of Stone (1979) that subsensitivity does not occur after acute stress but only after chronic stress when the animals show clear evidence of resistance to its adverse effects, the data of the present work show that acute swimminginduced stress produces subsensitivity to norepinephrine in vasa deferentia from adult normal rats. Thus, this subsensitivity might be an attempt of the organ to protect itself from high plasma levels of catecholamines released during the stress, which could be injurious to the organism. Previous work also related the importance of gonadal steroids in the reactivity of organs from male reproductive system (Porto et al., 1988; Valle et al., 1982). Androgens are necessary to the functional and trophic development of the male reproductive system smooth muscles (Sjo¨strand and Swedin, 1976). The results of the present study show that a lack of androgens prevented stressinduced subsensitivity to norepinephrine, which could be beneficial to the homeostasis of the organ, when catecholamine levels are high. Thus, testosterone may play an important role in the maintenance of the homeostasis of the rat vas deferens, causing subsensitivity to norepinephrine in acute stress situations (Carlos et al., 1995). Moreover, Charlwood et al. (1979) concluded that adequate testosterone levels are necessary for the maintenance of contractility in the rat vas deferens. On the other hand, there is variation in the responses to stress that depends on the type and duration of stressogenic situations to which the organism is submitted. Thus, shortterm exercise induced an increased plasma level of luteinizing and follicle-stimulating hormones, of catecholamines and of testosterone. Moreover, under prolonged physical stress, such as bicycle competition lasting for several days and psychological stress in military operations, the plasma levels of androgens decreased (Opstad, 1992). In the present work, subsensitivity to norepinephrine was observed in acute stress situations, instead of repeated stress. In repeated stress, the animals are exposed to the same stress stimulation daily, and adaptive changes would appear in the sympathoadrenomedullary system, resulting in an increase in the synthesis and basal levels of catecholamines (McCarty et al., 1988) and corticosteroids (Spadari and De Moraes, 1988). Therefore, repeated stress may result in a decreased androgen level, because this stress model consisted of repeated sessions of intense emotional stress and physical exercise. This suggests that the subsensitivity to norepinephrine observed after acute stress may depend on the androgen physiological level. On the basis of the results obtained with repeated stress that did not show an alteration in sensitivity to norepinephrine, we suggest that this absence of alteration may be a consequence of adaptation to elevated norepinephrine levels, or a mild decrease in andro-
Organ-bath studies For the analysis of their contractile capacity, the vasa deferentia were individually set up in 10-ml organ baths, containing continuously aerated nutritive solution at 308C, according to a technique previously described (Pereira, 1987). Longitudinal contractions were recorded with the aid of an isotonic level with a load of 0.5 or 1.0 g. After an initial resting period of 45 min, concentrationresponse curves to norepinephrine were obtained by cumulative addition of molar concentrations of the drug increasing geometrically (Van Rossum, 1963; Van Rossum and Van den Brink, 1963).
Pharmacological parameter The pD2 values, expressed as the negative logarithm of the agonist concentration producing 50% (ED50) of its maximum effect (Miller et al., 1948), were determined.
Drugs Norepinephrine (l-arterenol bitartrate) was from Sigma (USA); prazosin (prazosin hydrochloride) was from Pfizer (SA). Stock solutions of norepinephrine were performed in 0.01 N HCl and maintained frozen. Working solutions were diluted in saline solution (0.9% Na Cl) plus ascorbic acid (0.005%) immediately before use.
Statistical analysis The mean6SEM of pD2 values were calculated and compared previously by analysis of variance of the 333 factorial experiment in one-way classification. Tukey’s test was made for further comparisons between pairs of means (Ostle, 1973). RESULTS Without previous training, the animals swam vigorously during the stress sessions and showed signs of intense emotional distress such as defecation and vocalization. The mean concentration-response curve to norepinephrine obtained only in vasa deferentia from adult rats was shifted to the right after acute stress condition (Fig. 1A), and its correspondent pD2 value also decreased significantly (Table 1), which indicates subsensitivity. Furthermore, there were no sensitivity alterations when the experiments were carried out in immature, cryptorchid and castrated rats. Moreover, the acute stress induced a shift to the right on the concentration-response curve for norepinephrine only in pros-
Stress versus Vas Deferens Reactivity
419
FIGURE 1. Cumulative concentration-response curves for norepinephrine in isolated intact vas deferens (A), and in isolated prostatic portions of vas deferens (B, C) from adult rat. Abscissa shows the molar concentration of the drugs on a logarithmic scale. Ordinate shows effects as a percentage of the maximal contraction produced by the drug. Vertical bars indicate SEM. Each point represents a mean of at least six experiments. gen levels or both. This adaptation might occur by a prejunctional a2-adrenoceptor alteration as a consequence of high concentrations of norepinephrine in the biophasis. Nevertheless, little is known of the role of acute and repeated stress in the sympathoadrenomedullary system in the rat vas deferens. On the other hand, the two portions of the rat vas deferens also differ not only in their anatomical and histological structures, but also in their postsynaptic a-adreno-
ceptor subtypes (Badia and Salle´s, 1989; Brito et al., 1989; MacDonald and McGrath, 1980; Markus and Avellar, 1992). Our results showed that acute stress induced a decrease in the sensitivity to norepinephrine in the prostatic portion from the adult normal rat vas deferens. In contrast, repeated stress also induced an increase in the sensitivity to norepinephrine in the prostatic portion from the adult normal rat vas deferens. However, these sensitivity alterations dis-
420
O. C. M. Pereira et al.
TABLE 1. Effect of acute or repeated swimming-induced stress on response to norepinephrine in isolated vasa deferentia from adult, immature, cryptorchid and castrated rats pD2 @ values expressed as mean 6 S.E.M. Rats Adult Immature Cryptorchid Castrated
Control 5.27 6 0.05a (6)A 5.09 6 0.08a (6)A 5.18 6 0.05a (6)A 5.09 6 0.07a (6)A
Acute stress 4.95 6 0.03b (6)B 5.21 6 0.08a (6)AB 5.30 6 0.02a (6)A 5.23 6 0.04a (6)AB
Repeated stress 5.40 6 0.09a (6)A 5.32 6 0.06a (7)A 5.41 6 0.08a (5)A 5.33 6 0.15a (4)A
Number of experiments in parentheses. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect. a, b indicate horizontal significant difference (P , 0.05, Tukey’s test). A, B, or AB indicate vertical significant difference (P , 0.05, Tukey’s test).
TABLE 3. Effect of acute or repeated swimming-induced stress on response to norepinephrine in intact and bisected vasa deferentia isolated from adult rats (experiments were performed in the presence of 1028 M prazosin.) pD2 @ values expressed as mean 6 S.E.M. Vas deferens Intact Epididymal Prostatic
Control
Acute stress
Repeated stress
4.78 6 0.05 (8) 4.84 6 0.05 (8) 4.28 6 0.05 (8)
4.67 6 0.06 (10) 4.77 6 0.02 (8) 4.13 6 0.11 (8)
4.65 6 0.04 (6) 4.78 6 0.03 (8) 4.21 6 0.09 (8)
Number of experiments in parentheses. There were no significant differences among the groups. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect.
appeared when experiments were performed in the presence of 1028 M prazosin. This suggests that the subsensitivity to norepinephrine induced by acute stress is also due to alterations in a1-adrenergic receptors located in the prostate portion. This subsensitivity to norepinephrine may also be modulated by androgen levels and, in repeated stress, this alteration is not present.
in sensitivity to norepinephrine, only in the prostatic portion from the adult rat vas deferens. Integrated, these phenomena are responsible for the rat vas deferens subsensitivity to norepinephrine, which may be involved in body homeostasis in stressogenic situations. This subsensitivity is also due to alterations in a1-adrenergic receptors located in the vas deferens prostatic portion, which is physiologically androgen dependent, and it does not appear after repeated stress.
SUMMARY
The authors thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, process 94/4133-2 and 95/2076-4) for financial support.
The study was performed to examine the responses to norepinephrine in vasa deferentia isolated from adult normal, immature, cryptorchid and castrated rats submitted to acute or repeated swimminginduced stress. Additionally, the participation of the postjunctional a1-adrenoceptor in response to norepinephrine in intact or bisected vasa deferentia from adult normal rats submitted to acute or repeated stress was investigated. Acute stress was demonstrated to induce a significant subsensitivity to norepinephrine only in intact vasa deferentia from adult normal rats. This subsensitivity was inhibited when the experiments were carried out in groups with androgenic deprivation and in the repeated-stress model. Moreover, our results showed an acute stress-induced decrease in sensitivity to norepinephrine, which opposes the repeated stress-induced increase TABLE 2. Effect of acute or repeated swimming-induced stress on response to norepinephrine in intact and bisected vasa deferentia isolated from adult rats pD2 @ values expressed as mean 6 S.E.M. Vas deferens Intact Epididymal Prostatic
Control
Acute stress
Repeated stress
5.27 6 0.05a (6)A 5.46 6 0.08a (8)A 4.98 6 0.05b (8)B
4.95 6 0.03b (6)B 5.42 6 0.07a (8)A 4.70 6 0.05c (8)B
5.40 6 0.09a (6)A 5.47 6 0.09a (8)A 5.27 6 0.14a (8)A
Number of experiments in parentheses. @. Negative logarithm of the norepinephrine concentration producing 50% of its maximum effect. a, b, c indicate horizontal significant difference (P , 0.05, Tukey’s test). A, B, or AB indicate vertical significant difference (P , 0.05, Tukey’s test).
References Avellar M. C. W. and Markus R. P. (1993) Age related changes in norepinephrine release and its modulation by presynaptic alpha-2 adrenoceptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 267, 38–44. Badia A. and Salle´s J. (1989) Effects of St-587 on the a-adrenoceptors in the bisected rat vas deferens. J. Pharm. Pharmac. 41, 612–616. Benge B. N. and Jordan G. H. (1993) Prepubertal vasal injury: its effect on postpubertal vas deferens. J. Urol. 149, 906–907. Bispo D. L. N. and Pereira O. C. M. (1992) Involvement of swimminginduced acute stress in the sensitivity of rat vas deferens to norepinephrine. Gen. Pharmac. 23, 1125–1128. Brito A. R. M., Medeiros I. A. and Markus R. P. (1989) Do differences in innervation result in different post-synaptic responses to exogenous agonists? Gen. Pharmac. 20, 65–69. Burt R. P., Chapple C. R. and Marshall I. (1996) The role of diacylglycerol and activation of protein Kinase C in a1a-adrenoceptor–mediated contraction to noradrenaline of rat isolated epididymal vas deferens. Br. J. Pharmac. 117, 224–230. Carlos C. P., Padovani C. R. and Pereira O. C. M. (1995) Role of androgenic deprivation on the contractile response to norepinephrine in vas deferens isolated from rats submitted to swimming-stress. Rev. Cieˆnc. Biome´d. 16, 7–15. Charlwood R. A., Handberg G. M. and Pennefather J. N. (1979) A study of the effects of steroids on a-adrenoceptor–mediated contraction of the rat vas deferens. Clin. Exp. Pharmac. Physiol. 7, 11–21. Chies A. B. and Pereira O. C. M. (1995) Catecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress. Pharmac. Res. 32, 123–127. MacDonald A. and McGrath J. C. (1980) The effects of castration on neurotransmission in the rat vas deferens. Br. J. Pharmac. 69, 49–58. Markus R. P. and Avellar M. C. W. (1992) Effect of ageing on the number of neuronal noradrenaline uptake sites in the rat deferens. NaunynSchmiedeberg’s Arch Pharmac. 346, 405–409. McCarty R., Howa T. T. K. and Konarska M. (1988) Chronic stress and sympathetic-adrenal medullary responsiveness. Soc. Sci. Med. 26, 333– 341. Miller L. C., Becker T. J. and Trainter M. L. (1948) The quantitative evaluation of spasmolytic drugs “in vitro.” J. Pharmac. Exp. Ther. 92, 260–268.
Stress versus Vas Deferens Reactivity Opstad P. K. (1992) Androgenic hormones during prolonged physical stress, sleep, and energy deficiency. J. Clin. Endocrinol. Metabol. 74, 1176– 1183. Ostle B. (1973) Analisis de un disen˜o completamente azarizado que involucra conbinaciones de tratamiento factorial. In Estatı´stica aplicada. pp. 350–371. Limusa-Willey, Me´xico. ¨ stman-Smith I. (1979) Adaptative changes in the sympathetic nervous O system and some effector organs of the rat following long-term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hypertrophy. Acta Physiol. Scand. Suppl. 477, 1–118. Pereira O. C. M. (1987) Some effects of cimetidine on the reproductive organs of rats. Gen. Pharmac. 18, 197–199. Pereira O. C. M., Picarelli Z. P. and Abreu L. C. (1993) Experimental cryptorchidism in prepubertal rats: some effects on reactivity of the seminal vesicle to catecholamines. Pharmac. Res. 28, 175–181. Perronne´t F., Cle´roux J., Perrault H., Cousineau D., De Champlain J. and Nadeau R. (1981) Plasma norepinephrine response to exercise before and after training in humans. J. Appl. Physiol. 51, 812–815. Picarelli Z. P. and Valle J. R. (1969) Hormonal regulation of the rat seminal vesicle sensitivity to adrenaline, acetylcholine and acetyl-beta-methylcoline. Br. J. Pharmac. 35, 468–475. Porter J. C. and Melampy R. M. (1952) Effects of testosterone propionate on the seminal vesicle response to parasympathomimetic drugs. Endocrinology 51, 412–420. Porto C. S., Abreu M. L. C. and Picarelli Z. P. (1988) Influence of castration and of the kind of sympathomimetic drug used upon the reactivity of rat seminal vesicles. Arch. Int. Pharmacodyn. Ther. 292, 101–111. Salles J. and Badia A. (1991) Mechanisms underlying the differencial sensitivity to a1-adrenoceptor activation in the bisected rat vas deferens. Br. J. Pharmac. 102, 439–445. Scheurink A. J. W., Steffens A. B., Bouritius H., Dreteler G. H., Bruntink
421 R., Remie R. and Zaagsma J. (1989) Adrenal and sympathetic catecholamines in exercising rats. Am. J. Physiol. 256, r155–r160. Sjostrand N. O. (1962) Effect of reserpine and hypogastric denervation on the noradrenaline content of the vas deferens and the seminal vesicle of guinea-pig. Acta Physiol. Scand. 56, 376–380. Sjostrand N. O. (1965) The adrenergic innervation of the vas deferens and the accessory male genital glands. Acta Physiol. Scand. 65(Suppl. 257), 82P. Sjostrand N. O. and Swedin G. (1976) Influence of age, growth, castration and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands of the rat. Acta Physiol. Scand. 98, 323–338. Soares R. D. F., Abreu M. L. C. and Porto C. S. (1993) Post-natal development modulates the rat seminal vesicle sensitivity to sympathomimetic agonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 348, 53–57. Spadari R. C. and De Moraes S. (1988) Repeated swimming stress and responsiveness of the isolated rat pacemaker to the chronotropic effect of noradrenaline and isoprenaline: role of adrenal corticosteroids. Gen. Pharmac. 19, 553–557. Spadari R. C., Bassani R. A. and De Moraes S. (1988) Supersensitivity to isoprenaline and epinephrine in right atria isolated from rats submitted to a single swimming session. Gen. Pharmac. 19, 129–135. Stone E. A. (1981) Mechanism of stress-induced subsensitivity to norepinephrine. Pharmac. Biochem. Behav. 14, 719–723. Valle R. M. R., Abreu L. C., Picarelli Z. P. and Valle J. R. (1982) Influence of castration upon trophism and reactivity of rat seminal vesicles. Braz. J. Med. Biol. Res. 15, 49–53. Van Rossum J. M. (1963) Cumulative dose-response curves II: technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. Int. Pharmacodyn. Ther. 143, 299–330. Van Rossum J. M. and Van Den Brink F. G. (1963) Cumulative doseresponse curves I: introduction to the technique. Arch. Int. Pharmacodyn. Ther. 143, 240–246.