EFFECTS OF CLENBUTEROL TREATMENT ON THE RELAXANT RESPONSE IN RAT UTERUS

Pharmacological Research, Vol. 41, No. 2, 2000 Article No. phrs.1999.0578, available online at http:rrwww.idealibrary.com on

EFFECTS OF CLENBUTEROL TREATMENT ON THE RELAXANT RESPONSE IN RAT UTERUS GUILLERMO F. BRAMUGLIAU and MODESTO C. RUBIO Catedra de Farmacologıa, Uni¨ ersidad de Buenos Aires, ´ ´ Facultad de Farmacia y Bioquımica, ´ ININFA (CONICET ), Junin 956 5⬚ piso, 1113 Buenos Aires, Argentina Accepted 19 July 1999

Prolonged treatment with the ␤ 2-adrenergic agonist clenbuterol Ž0.25 mg kgy1 s.c. once daily for 10 days. produced a reduction of the relaxant response and cAMP production mediated by stimulation of ␤-adrenoceptors in oestrogen-treated rat uterus. Substantial decreases in the relaxant effect of isoproterenol is observed in uterine rings precontracted with 50 mM KCl from clenbuterol-treated rats. The recovery of the relaxant response was also studied and significant differences were seen between acute and prolonged treatment with clenbuterol Ž P- 0.05 ¨ s control.. In contrast the relaxant effect of forskolin and 3-isobutyl-1-methylxanthine was similar in untreated or treated rats. Sodium fluoride also showed a relaxant response which was not affected by the treatment with clenbuterol. The radioligand studies showed a reduction in the number of ␤-adrenoreceptors after acute and prolonged treatment with clenbuterol in rat uterus. These results suggest that prolonged treatment with clenbuterol caused a desensitization of the relaxant uterine response through ␤ 2-adrenoceptors and also showed differences in the recovery of the relaxant response depending on the duration of treatment. 䊚 2000 Academic Press KEY

WORDS:

␤-adrenoceptor desensitization, clenbuterol, smooth muscle.

INTRODUCTION

␤ 2-adrenoceptor activation leads to relaxation of uterine smooth muscle in several species including humans w1᎐4x. This response may involve the stimulation of the adenylate cyclase and cAMP production w5x. Numerous pharmacological agents such as magnesium sulphate, ␤-adrenergic receptor agonists, and calcium channel blockers have been used to suppress labour and prevent preterm delivery, but in some women ␤-agonists fail to suppress labour or their effect is transient. Desensitization of the ␤-adrenergic system has been studied in different cell types and tissues, such as erythrocytes w6x; lymphocytes w7x; heart w8x; and brain w9x. It has been postulated that the relaxation and desensitization processes in several tissues are elicited via independent pathways following activaU

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tion of the ␤-adrenoceptors w10, 11x, and various theories have been put forward in order to explain the mechanisms of duration of action of the relaxant responses of the ␤ 2-adrenoceptor agonists w12x. Repeated administration of the ␤ 2-adrenoceptor agonist clenbuterol ŽCLEN. to rats leads to ␤ 2adrenoceptor desensitization in vascular smooth muscle w13, 14x. In previous work we have shown that a single dose of CLEN was sufficient to produce a rapid desensitization of the uterine relaxant response through ␤ 2-adrenoceptors w15x. The aim of this paper was to analyse the effect of CLEN treatment on the relaxant response in rat uterus. The results obtained showed that the prolonged treatment with CLEN induced a substantial reduction in the ␤-adrenoceptor relaxant response, which was accompanied by a reduction in the number of ␤adrenergic receptors in rat uterus, and also showed a different pattern in the recovery of the relaxant response after acute or prolonged treatment with CLEN. 䊚 2000 Academic Press

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METHODS

Physiological procedures Virgin female Wistar rats of 200᎐250 g were used. Animals were housed at constant temperature Ž22⬚C. and relative humidity Ž50%. on a regular light᎐dark schedule Žlight 07:00 h to 19:00 h.. Food and water were freely available. The animals were administered with a single dose of CLEN Žacute treatment: 0.25 mg kgy1 s.c.., or during 10 successive days with CLEN Žprolonged treatment: 0.25 mg kgy1 per day s.c... The animals were killed by stunning 24 h after the single dose Žacute treatment . or the last dose Žprolonged treatment .. Since the level of oestradiol is of importance for the total number of ␤-adrenoceptors oestradiol benzoate in sesame oil vehicle was administered once daily for 2 successive days Ž50 mcg kgy1 per day i.m.. 48 and 24 h before the animals were killed. For the study of the recovery of the relaxant response the animals were killed by stunning after 1, 2, 3 or 5 days following acute or prolonged treatment with CLEN. The two uterine horns were removed and four rings of approximately 2-mm width were dissected from the middle portion of the tissue. The rings were mounted between two L-shaped stainless steel hooks in a 10-ml organ bath. The upper hook was connected to a force-displacement transducer ŽGrass FT 03. to record the isometric tension of the preparation on a Grass polygraph Žmodel 79 D.. The rings were equilibrated for 2 h at 37⬚C under 1 g tension in modified Krebs solution of the following composition Žin mM.: NaCl 118.0; KCl 4.7; CaCl 2 2.6; MgCl 2 1.2; NaH 2 PO4 1.0; NaHCO3 25.0; glucose 11.1; ascorbic acid 0.11. The medium was changed every 20 min during this period. The solution was administered with an O 2rCO 2 mixture Ž95:5. so that its final pH was 7.4. The relaxant effect was studied on rings that had been precontracted with 50 mM KCl in the presence of phentolamine 1 ␮ M, added 10 min before KCl. To maintain the isotonicity of the Krebs solution, the concentration of NaCl was reduced to 73.0 mM during the incubation with KCl. When the rings attained a steady state level of contraction, the relaxant agents were added in a cumulative manner by stepwise increases of the concentration as soon as the response to the previous administration levelled off. Complete relaxation was attained when tension reached baseline.

Measurement of cAMP production The two uterine horns were removed, cleaned and cut lengthwise into four rings. The rings were incubated without tension for 60 min at 37⬚C in vials containing modified Krebs solution. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine ŽIBMX., was then added to the bath at a final concentration of 100 ␮ M for 5 min.

The incubation period with isoproterenol ŽISO. was 2 min, then the reaction was stopped by quickly submerging the tissues in ice-cold trichloroacetic acid ŽTCA. 5%. The tissues were rapidly homogenized in 2 ml of TCA. After centrifugation Ž3000 g for 15 min at 4⬚C., 1.5 ml of the supernatant was thoroughly mixed five times with three volumes of diethylether saturated with distilled water to remove TCA, and cAMP was determined in the supernatant by the method of Brown et al. w16x. Basal cAMP values were obtained in the presence of the appropriate solvent. The pellet was used for the determination of proteins w17x.

Binding assay of [ 3H ]dihydroalprenolol ([ 3H ] DHA ) Miometrial strips were homogenized with an Ultra-Turrax homogenizer in 10 volumes of cold buffer consisting of 0.25 M sucrose, 1 mM MgCl 2 , 5 mM Tris᎐HCl ŽpH 7.4. and centrifuged at 700 g for 5 min. The sediment was washed by resuspension in the buffer and centrifuged at 700 g for 5 min. The pooled 700 g supernatant fractions were centrifuged at 10,000 g for 1 h. The resulting pellet was suspended in cold 50 mM Tris᎐HCl ŽpH 7.4., 5 mM MgCl 2 , 0.5 mM EDTA, and 1 mM dithiothreitol at 4 mg mly1 final concentration w18x. Membrane proteins Ž0.4 mg. were incubated with the indicated concentrations of w 3 HxDHA Ž1᎐15 nM. for 15 min at 30⬚C in 50 mM Tris᎐HCl ŽpH 7.4., 10 mM MgCl 2 , in a 400-␮ l final volume. At the end of the incubation, triplicate aliquots were diluted with 4 ml ice-cold buffer and immediately filtered through Whatman GFrC glass fiber filters under reduced pressure. Filters were washed twice with 4 ml of cold buffer, dried and the bound radioactivity determined by scintillation counting. Specific binding was defined as the difference between the amount of w 3 HxDHA bound in the absence Žtotal binding. and the presence Žnon-specific binding. of 10 ␮ M of unlabelled propranolol, never exceeding 10% of specific binding. Non-linear regression analysis of the data was applied to calculate the parameters Bmax and K d .

Analysis of data and statistics Results are expressed as mean values " SEM. The significance of differences between values was determined with a one-way analysis of variance ŽANOVA. followed by the Newman᎐Keuls test or Student’s t-test. P values - 0.05 were considered significant in each case.

Drugs Forskolin, IBMX, ISO, sodium fluoride ŽNaF. propranolol and phentolamine hydrochloride were obtained from Sigma ŽSt. Louis, MO, USA.. w 3 HxcAMP and w 3 HxDHA were obtained from New

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England Nuclear ŽUSA.. Clenbuterol hydrochloride was a generous gift from Dupomar Laboratory ŽBuenos Aires, Argentina.. CLEN was dissolved in 0.9% NaCl and was administered Ž0.25 mg kgy1 s.c.. once daily for 1 or 10 days. ISO was dissolved in modified Krebs solution.

RESULTS

Relaxant studies The contractile response elicited by KCl Ž50 mM. was similar in uterine rings from untreated and treated rats Žuntreated: 1.5" 0.25 g, n s 8; treated with CLEN once daily during 10 days: 1.55" 0.20 g, n s 8; acute treatment with CLEN: 1.49" 0.09 g, n s 8.. Cumulative addition of the non-selective ␤adrenoceptor agonist, ISO, in the presence of phentolamine 1 ␮ M, induced a concentration-dependent relaxation of KCl contracted rings from untreated rats, that reached a maximum effect of 55% relaxation. On the other hand, the concentrationdependent relaxation in rings from CLEN-treated rats for 10 days was substantially reduced reaching a maximum effect of 14% Ž P- 0.05. wFig. 1Ža.x. This

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reduction in the relaxant response was of similar magnitude to the response seen after a single dose of CLEN wFigs 1Ža. and 2Ža.x. In contrast the relaxant effect of NaF Ža nonspecific G protein activator. was similar in uterus from untreated or treated rats during 10 days. At 5 mM, NaF caused a contractile response wFig. 1Žb.x. Forskolin Ža direct activator of the adenylate cyclase. and the phosphodiesterase inhibitor, IBMX, also induced a relaxant response which was not affected by the treatment with CLEN wFig. 1Žc,d.x. In previous work, we showed that the treatment with a single dose of CLEN produced a desensitization of the relaxant response through ␤ adrenoceptors in rat uterus. In this condition, the relaxant response to ISO was completely restored after 48 h wFig. 2Ža.x. On the other hand, the decreased response to ISO after prolonged treatment with CLEN, continued for more than 48 h after the end of the drug administration, and the relaxant response was completely restored after 5 days wFig. 2Žb.x.

cAMP production These experiments were designed to determine

Fig. 1. Concentration᎐response curves for the relaxant effects of ISO Ža., NaF Žb., forskolin Žc., and IBMX Žd. in 50 mM KCl-contracted uterine rings from untreated Ž䢇. and CLEN-treated rats Ž10 days treatment . Ž`.. Ordinates: percent of relaxation expressed as a percentage of relaxation to baseline tension. Abscissa: concentration of the drugs. Each point is the mean " SEM for six rings from different animals. U P- 0.05 ¨ s control.

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Fig. 2. Concentration᎐response curves for the relaxant effect of ISO showing the recovery of the relaxant response in uterine rings after the treatment with CLEN. Ža. single dose of CLEN; Žb. 10 days treatment with CLEN. Control Ž䢇., relaxant response after 1 Ž`., 2 ŽI., 3 Ž'. or 5 Ž䉫. days of the single Žacute. or the last dose Ž10 days treatment . of CLEN. Ordinates: percent of relaxation expressed as a percentage of relaxation to baseline tension. Abscissa: concentration of U ISO. Each point is the mean " SEM for six rings from different animals. P - 0.05 ¨ s control.

Table I Parameters obtained from the radioligand binding assay

Control CLEN Žsingle dose. CLEN Ž10 days treatment .

Bma x

Kd

50.3" 12.2

3.2" 0.5

30.0" 7.4U

3.3" 0.9

24.7" 1.5U

2.7" 0.8

Equilibrium dissociation constant Ž K d . and the maximal numbers of binding sites Ž Bmax . were expressed in nanomoles and fentomoles of w 3 HxDHA bound per milligram of protein, respectively. Note: values are mean " U SEM. P- 0.05 ¨ s control Ž n s 5..

Fig. 3. Effect of ISO on cAMP production in uterine rings from untreated Žopen bars. and clenbuterol-treated rats Ž10 days treatment . Žhatched bars.. Basal values were obtained by incubating with medium. Cyclic AMP was measured by the method of Brown et al. w16x. The values are expressed as the mean " SEM for five tissues. U P - 0.05 ¨ s control.

related to a reduced cAMP production. ISO Ž10y8 ᎐10y6 M. induced a concentration-dependent increase in cAMP levels from control rats. On the other hand, prolonged treatment with CLEN produced a lower cAMP increase after ISO stimulation ŽFig. 3..

Binding assay of [ 3H ]DHA whether the reduced ␤-adrenoceptor-mediated relaxation in uterus from CLEN-treated rats could be

CLEN treatment Ž1 or 10 days. produced a reduction of the relaxant response in uterine smooth muscle. This phenomenon was also accompanied by

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Fig. 4. Scatchard plot of the radioligand binding assay using w 3 Hxdihydroalprenolol. Membrane proteins Ž0.4 mg. were incubated with the indicated concentrations of w 3 HxDHA Ž1᎐15 nM. for 15 min at 30⬚C in 50 mM Tris᎐HCl ŽpH 7.4., 10 mM MgCl 2 , in 400 ␮ l final volume control Ž䢇., single dose of CLEN Ž'., 10 days treatment with CLEN Ž`..

a reduction in the number of ␤-adrenergic receptors in the membranes. The mean total number of w 3 HxDHA binding sites in the membranes prepared from CLEN-treated rats represent only 59% Žacute treatment ., and 50% Žprolonged treatment ., of the initial number of binding sites in membranes from control animals, showing no significant differences between acute and prolonged treatment in the reduction of the number of ␤-adrenergic receptors in uterine tissues ŽTable I.. Non-linear regression analysis of the data indicated that K d of w 3 HxDHA for binding to control and desensitized membranes are virtually identical showing a one site fit ŽFig. 4, Table I..

DISCUSSION The present study showed that prolonged treatment with the ␤ 2-agonist CLEN produced a reduction in the relaxant response and cAMP production mediated by stimulation of ␤-adrenoceptors in rat uterus. The relaxant effect of the non-specific ␤adrenoceptor ISO markedly diminished in KCl precontracted uterine rings from rats treated with CLEN for 10 days. The radioligand study also showed a reduction in the number of ␤-adrenergic receptors in the membranes after the treatment with CLEN. In previous work we demonstrated that a single dose of CLEN caused a rapid desensitization of the

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relaxant uterine response through ␤-adrenoceptors w15x, while only prolonged administration Ž7 or 14 days. of CLEN produced a desensitization of ISOinduced vasodilatation and the hypotensive effect in anaesthetized rats w13, 14x. The results may suggest differences in the desensitization processes between tissues that could be referred to the selective distribution of the ␤-adrenergic receptors or to the molecular mechanism that involves the receptors sequestration and down regulation w11, 19x. Although cAMP does not have an exclusive role in mediating uterine relaxation by adrenergic agonists w20x, the reduced cAMP production mediated by ISO stimulation observed after CLEN treatment is related to the diminished ISO mediated relaxant response. Thus, the binding assay showed that clenbuterol treatment modified the density Ž Bmax . of ␤-adrenoceptors in rat uterus suggesting a downregulation mechanism to explain the altered relaxant response. The fact that the relaxant effects of forskolin and IBMX were unchanged in uterine tissues from CLEN-treated rats indicates that the function of the catalytic subunit of the adenylate cyclase as well as the mechanisms subsequent to cAMP production Ži.e. cAMP-dependent protein kinase, phosphodiesterase, etc.., are not affected. In accordance with the hypothesis that the possible locus of the impairment may be the ␤adrenoceptor or its coupling with the adenylate cyclase through the GTP regulatory protein Gs, we studied the effect of NaF in rat uterus. In our experiments the relaxant response of NaF was not modified by the treatment with CLEN. The modest contraction response observed at 5 mM could be the result of non-specific G protein activation that leads to the contractile response w21, 22x. Desensitization may occur at different levels of the ␤-adrenergic receptor system. Thus, EuropeFinner et al. w23x, suggested that in human miometrium the differences in contractility observed in labour or during the stage of uterine quiescence may be due to changes at the level of G ␣ s. ␤ 2-adrenergic agonists are widely used in obstetrical practice, mainly to prevent premature labour for a short time. However, previous work showed the return of uterine contractions in women treated with ␤ 2-selective agonists w24x. Caritis et al. w25x, demonstrated that the labour inhibiting effect of the ␤ 2adrenergic agent, ritodrine, is reduced after 24-h infusion. Moreover, Abel and Hollingsworth w26x also showed that the long-term infusion of salbutamol produced tolerance to the inhibitory action on uterine contraction in rat. In the present work, the differences observed in the relaxant response after CLEN treatment were not due to changes either in the adenylate cyclase activity, or in the GTP regulatory protein coupled to

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␤-adrenoceptor. These results are consistent with a localization of the desensitization to the ␤adrenoceptor after the treatment with ␤ 2 agonists. Clenbuterol is a highly lipophilic long acting ␤ 2adrenoceptor agonist. Recently, Coleman et al. w12x, proposed the existence of additional sites of binding, either on the ␤ 2-adrenoceptor itself or in the vicinity of the receptor, to explain the duration of action of salmeterol, another long acting ␤-adrenergic agent. Thus, salmeterol could induce down-regulation of ␤-adrenoceptors that persisted for at least 24 h after a brief exposure to the agonist w27x. Our results also showed that a single dose of CLEN caused a rapid desensitization of the relaxant uterine response through ␤-adrenoceptors that was maintained 24 h after the administration. Although the desensitization of the relaxant response due to the acute treatment with CLEN reversed 48 h after the exposure, on the other hand, after prolonged treatment with CLEN, the relaxant response was not completely recovered up to 5 days after the last dose. The radioligand studies showed no significant differences in the reduction of the number of ␤adrenoreceptor after acute or prolonged treatment with CLEN in rat uterus that could explain the differences observed in the recovery of the relaxant response. In this way, although CLEN is resistant to plasma clearance Žbeta agonists having halogenated aromatic ring systems are mainly metabolized by oxidative and conjugative pathways and have long plasma half-lives., and this may be one pharmacokinetic theory to explain the prolonged duration of adrenoceptor desensitization, it could also indicate differences in the molecular determinants of the down-regulation process related to the duration of the stimulus or to individual tissues w11, 19, 28x that could influence the recovery of the relaxant response. Although it is well known that continuous stimulation of ␤-adrenoceptors leads to receptor inactivation in ¨ itro, these results provide a functional support to study the desensitization and resensitization processes after in ¨ i¨ o administration of ␤-agonists and could form the basis of future experiments to elucidate differences that exist in the mechanism of prolonged duration of action and ␤-adrenoceptors desensitization of CLEN and other ␤-agonists.

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