Catecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress

Catecholaminergic responses in vas deferens isolated from rats submitted to acute swimming stress

123 Pharmacological Research. Vol. 32, No. 3, 1995 CATECHOLAMINERGIC RESPONSES SUBMITTED IN VAS DEFERENS TO ACUTE SWIMMING ISOLATED FROM RAT...

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123

Pharmacological Research. Vol. 32, No. 3, 1995

CATECHOLAMINERGIC

RESPONSES

SUBMITTED

IN VAS DEFERENS

TO ACUTE

SWIMMING

ISOLATED

FROM

RATS

STRESS

A. B. CHIES and O. C. M. PEREIRA

Departamemo de Farmacologia, Instituto de BiociOncias, Universidade Estadual Paulista (UNESP), 18618-000, Botucatu-SP, Brasil Accepted 22 August 1995

The study was performed to examine the responses to catecholamines in vas deferens isolated from rats submitted to acute swimming-induced stress. It was demonstrated that acute stress induces a significant subsensitivity of rat vas deferens to norepinephrine. This subsensitivity was inhibited when the experiment was carried out in the presence of either cocaine (10--~M) or timolol (10-sM). On the other hand, the rat vas deferens sensitivity to methoxamine was significantly increased by acute swimming-induced stress. Thus, despite acute swimming stress inducing a reduction in response to norepinephrine, the aradrenoceptor-mediated contractile response was increased. Additionally there were increases in neuronal uptake and fl,_-adrenoceptor activity that opposes the aradrenoceptor activity. Integrated, these phenomena are responsible for the rat vas deferens subsensitivity to norepinephrine which may be involved in body homeostasis in stressogenic situations. © 1995 The Italian Pharmacological Society KEYWORDS:acute stress, catecholamines, adrenoceptor, neuronal uptake.

INTRODUCTION Sensitivity of some tissues to the sympathetic agonists may be modified following hormonal changes induced by stress. Participation of the adrenal gland on response to norepinephrine in rat vas deferens obtained from animals submitted to acute stress has been shown [1]. This mediation may occur via epinephrine and/or glucocorticoids since the adrenal glands are the main source of these substances [1-3]. Moreover, repeated footshock-stress-induced supersensitivity to isoprenaline was also observed in the isolated rat pacemaker [2]. It has been shown that single swimming stress induces supersensitivity of isolated rat right atria to sympathetic agonists such as isoprenaline and epinephrine. Some of these alterations may be mediated, at least partially, by a high level of glucocorticoids [2, 4]. On the other hand, dynamic exercise has also been shown to induce translocations of fl-adrenergic receptors from intracellular sites to the cell surface in human lymphocytes. This physiological change is promoted by high levels of endogenous catecholamines [3]. A similar phenomenon has previously been demonstrated in rat myocardium [5]. These sensitivity alterations participate in a wide variety of mechanism involved in the adaptation of the organism to stressogenic conditions [2, 6]. Correspondenceto Dr O. C. M. Pereira. 1043-6618/95/090123-05/512.00/0

Considering a relationship between stress and adrenergic action of agonists, it is important to study the adrenergic mechanisms involved in stressogenic conditions. In this regard, the rat vas deferens has been commonly considered a suitable preparation to study post-junctional a-adrenoceptors [7]. When this smooth muscle is stimulated by adrenergic agonists, the post-junctional ct~-adrenoceptor population is responsible for the characteristic contraction obtained [8-10], although Vohra [11] observed evidence of inhibitory fl2-adrenoceptors that produce relaxation o f the smooth muscle if stimulated. Regarding norepinephrine, the relaxation effect of fl=-adrenoceptor stimulation is not observed since the contractile action of the aradrenoceptor is greater than the relaxation induced by fl2-adrenoceptor stimulation. On the~other hand, sympathetic nervous terminals also influence the responses of rat vas deferens to norepinephrine. The sympathetic nervous terminals are the principal removal site of catecholamines present in adrenergic synapses through a neuronal uptake process. Thus, any alteration in neuronal uptake may change the responses of vas deferens muscle to the sympathetic agonists [ 12]. The aim of the present study was to investigate the responses of rat vas deferens tO catechoi'ami~eS ~in acute stress situations. The contribution 0'f different adrenoceptor populations present '~n this; smo6~ muscle and neuronal uptake activity were also evali~ ated in these stress conditions. ';

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© 1995The ItalianPharmacologicalSociety

PharmacologicalResearch. Vol. 32. No. 3. 1995

124 M A T E R I A L AND M E T H O D S

100

Animals Adult male Wistar rats (about 3 months old, average weight 250-300 g) were divided into four experimental groups, housed five rats per cage with food and water freely available and with a 12 h light-dark cycle. The temperature and humidity were controlled at 25+1 °C and 55+_5%, respectively. Stress protocol Animals were submitted to a single forced swimming-stress procedure in a glass tank containing water at room temperature, individually for 50 rain, carrying a metallic ring of about 2% of their corporal weight on their tails. The tank measures were 45 cm length, 35 cm width, and with 33 cm depth. After the swimming session, the animals were killed by inhalation of an overdose of ethyl ether, followed by decapitation.

- (A)

50

o

6

5

4

6

5

4

5 -Log [M] of drug

4

100 -(B)

E

50

0

Pharmacological analysis The vasa deferentia were removed, separated from the surrounding tissue, freed of secretions and individually set up for the analysis of their contractile capacity in 10ml organ-baths through which circulated the nutritive solution, aerated with 95% 02 and 5% CO2 maintained at 30°C, according to the methods previously described by Pereira [13]. The composition of the nutritive solution consisted of: NaCI 136.0 mM; KCI 5.7 mM; CaC12 1.8 mM; NaH2PO4.H20 0.36 mM; NaHCO3 15.0 mM; dextrose 5.5 mM, prepared in glass distilled water [14]. Longitudinal contractions were recorded with the aid of an isotonic level with a load of 1.0 g. After an initial resting period of 45 min, complete concentration-response curves to norepinephrine (arterenol bitartrate, Sigma), in the absence and the presence of cocaine 10-5M (cocaine hydrochloride, Merck) or timolol 10-5M (timolol maleate, Sigma), phenylephrine (l-phenylephrine hydrochloride, Sigma), and methoxamine (methoxamine hydrochloride, Sigma) were obtained by cumulative addition of geometrically increasing molar concentrations of the drug [15]. The drug parameters determined were pD2, the negative logarithm of the drug concentration producing 50% (ED50) of its maximum effect [16] and relative responsiveness ratio, estimated as the relationship between the height of the maximal response of total agonist concentration and the height of the maximal response of the organ [17], taken as that induced by the addition of 30 mM of barium (barium chloride, Erich Lowenberg). Statistical analysis The mean.t.SEM of pD2 values and p were calculated and compared previously by analysis of variance of the 2x3 factorial experiment in one-way classifications. Tukey's test was made for further comparisons between pairs of means [18]. Student's t-test was

100

- (C)

50

0

I 6

Fig. 1. Cumulative concentration-response curves for norepinephrine (A), phenylephrine (B) and methoxamine (C) in isolated vasa deferentia from control (O) and acute stressed rats (0). 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 seven experiments. used to compare arithmetic mear~+sEM between just two groups.

RESULTS Without previous training, the animals swam vigorously during the 50 min and showed signs of intense emotional distress, such as defecation. The mean concentration-response curve to norepinephrine was shifted to the right (Fig. 1A) and its correspondent pD2 value decreased significantly following acute stress (Table I), which indicate subsensitivity. Furthermore, there were no stressinduced alterations when the experiments were carried out in presence of cocaine. However, cocaine was

125

Pharmacological Research. Vol. 32. No. 3. 1995

effective in blocking neuronal uptake since the pD2 value and the relative responsiveness ratio values for norepinephrine were increased in presence of this drug (Table I). Despite acute stress inducing a slight displacement to the left on the concentration-response curve for phenylephrine (Fig. IB), no significant alteration in sensitivity was shown in the pD2 and relative responsiveness ratio values (Table II). However, it was demonstrated that acute swimming stress induces supersensitivity of the rat vas deferens to methoxamine expressed by shifts of the concentration-response curves to the left and by a significant increase in pD: value (Fig. IC and Table II). Timolol, a fl-adrenoceptor antagonist was also able to inhibit the stress-induced decrease in rat vas deferens sensitivity to norepinephrine (Table I).

ably helps the body to maintain homeostasis during stressogenic situations [2, 6]. Adjustment mechanisms may be important in stress situations as long as an increased output of norepinephrine in adrenergic synapses may occur. Experiments using rats submitted to exercise demonstrated that epinephrine released from adrenal glands during exercise facilitates the output of norepinephrine [19]. Alterations in hormonal level may be involved in these changes which may modulate the responses of some tissues to sympathetic agonists. In addition, it has been shown that bilateral adrenalectomy performed 2 days before the acute swimming-induced stress increased the sensitivity of the rat vas deferens to norepinephrine [1]. Similar sensitivity alterations have been demonstrated in isolated rat pacemaker [2, 20] and isolated rat right atrium [4]. Neuronal uptake may modulate the smooth muscle sensitivity to catecholamines as it participates in the maintenance of the synaptic concentration of catecholamines. In the longitudinal muscle layer of rat vas deferens, neuronal uptake is an important mechanism to remove catecholamines from adrenergic synapses [21]. Therefore, any alterations in this process

DISCUSSION The present study shows that acute swimminginduced stress produces subsensitivity in the rat vas deferens to norepinephrine. This phenomenon prob-

Table I Effect of acute swimming-induced stress on the response of the isolated rat vas deferens to norepinephrine (NE). Experiments were performed in the absence or presence of cocaine (10 -s M) or timolol (10 -s M) (mean+s~M; eight rats per group) Rat vas deferens

Relative responsiveness ratio~: N E

pD2~" NE No stress

Stress

No stress

Stress

Control

5.24+0.03 A

4.94+0.03* A

0.36+0.03 A

0.36+0.02 A

I n presence of cocaine (10 -5 M)

6.45+0.06 B

6.51 +0.07 C

0.51 +0.02 B

0.57+0.03 B

I n presence of timolol ( 10-5 u)

5.18+0.05 A

5.21 +0.03 B

0.39+0.02 A

0.43+0.02 A

Different capital letters indicate vertical significant difference (P<0.05 by Tukey's test). *P<0.05, compared to the no stress situation (Tukey's test). tNegative logarithm of the drug concentration producing 50% (EDso) of its maximum effect. :~Relationship between the height of the maximal response to the total agonist concentration and the height of the maximal concentration of the organ taken as that induced by the addition of barium chloride (30 n~0.

Table II Effect of acute swimming-induced stress on the response of the isolated rat vas deferens to phenylephrine or methoxamine (mean---seM; number of experiments in parentheses) Drugs

Relative responsiveness ratio~t

pD2 ~' No stress

Stress

No stress

Stress

Phenylephrine

4.97+0.06 (8)

5.15+0.06 (8)

0.42+0.04 (8)

0.41+0.04 (8)

Methoxamine

4.92+0.08 (8)

5.16_+0.01 * (7)

0.34+0.04 (8)

0.32.~0,03 (7)

*/:'<0.05, compared to the no stress situation (Student's t-test). . tNegative logarithm of the drug concentration producing 50% of its maximum effect. ~l :~Relationship between the height of the maximal response to the total agonist concentration and the height of the m~imal contraction of the organ taken as that induced by the addition of barium chloride (30 rnM). :

126 m a y change the response pattern o f this smooth muscle to the catecholamines [12]. The results of the present study have shown that cocaine was able to prevent the acute stress-induced subsensitivity to norepinephrine. These results also suggest that an increase in neuronal uptake may be involved in the stress-induced subsensitivity to norepinephrine. On the other hand, alterations in post-junctional adrenoceptor activity in stress situations may occur. The results o f studies on the reactivity to phenylephrine, a selective a~radrenoceptor agonist [22], have shown a non-significant increase in a t - a d r e n o c e p t o r contractile activity. Increases in neuronal uptake and in the ot~-adrenoceptor mediated contractile activity following the stress situation were corroborated by the increase in the vas deferens sensitivity to m e t h o x a m ine, a selective a t - a d r e n o c e p t o r agonist that is not taken up by adrenergic nervous terminals [23]. Moreover, participation of other post-junctional adrenoceptors subtypes in this phenomenon of subsensitivity to norepinephrine can not be ruled out. Furthermore, the results obtained in presence o f timolol 10 -s M have shown that alteration in sensitivity to norepinephrine induced by stress did not occur. Previous studies have demonstrated an acute exerciseinduced translocation of the fl-adrenergic receptors in rat m y o c a r d i u m [5]. Acute exercise is thought to promote the translocation o f fl-adrenoceptor from a presumably intracellular site to functional cellular membrane fractions [3, 5]. As the acute s w i m m i n g stress contains a physical component (exercise), a similar phenomenon may occur regarding the fl_,-adrenoceptors present in the rat vas deferens. Therefore, the hypothesis o f stress-induced fl2-adrenoceptor translocation was corroborated. The rat vas deferens sensitivity to norepinephrine may also be decreased through this fl_,-adrenoceptor up-regulation. In conclusion, acute stress induces subsensitivity to norepinephrine in rat vas deferens. In spite of this subsensitivity, the a t - a d r e n o c e p t o r mediated contractile response was increased during acute stress. However, this increase in the o~-adrenoceptor contractile response was not evidenced since neuronal uptake and fl2-adrenoceptor population activities are increased following acute stress. Moreover, increase in neuronal uptake and fl,-adrenoceptor activity may also induce the stress-dependent subsensitivity to norepinephrine. T h e s e p h e n o m e n a may be involved in the maintenance o f b o d y homeostasis in stressogenic situations.

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ACKNOWLEDGEMENTS The authors are grateful to Dr C. R. Padovani for statistical assistance.

17.

18.

REFERENCES 19.

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