Gen. Pharmac. Vol. 24, No. 3, pp. 769-773, 1993 0306-3623/93 $6.00 + 0.00 Printed in Great Britain Pergamon Press Ltd BETA-ADRENOCEPTOR DESENSITIZATION BY CLENBUTEROL IN RAT UTERUS GUILLERMO F. BRAMUGLIA,MARCELO G. KAZANIETZ, ERNESTO PEZMAN and MARIA AMELIA ESERO~ Catedra de Farmacologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Instituto de Investigaciones Farmacol6gicas--CONICET, Junin 956 5*, Ill3 Buenos Aires, Argentina [Tel. 961-7111; Fax 54-1-962-5341] (Received 30 September 1992) Abstract--1. The relaxant response and cAMP production mediated by stimulation of isoproterenol is reduced in uterine rings from clenbuterol treated rats (0.25mg kg -~ s.c. 24hr before experiments) precontracted with 50 mM KCI. 2. Forskolin, in contrast, showed similar relaxant responses in untreated or clenbuterol treated rats. 3. Isoproterenol produces a biphasic response that is composed of a rapid relaxation followed by a slower regaining of tension, which is considered as desensitization. 4. The kinetic study demonstrates marked changes in the desensitization process of fl-adrenoceptors after clenbuterol administration. INTRODUCTION Stimulation of uterine ~-adrenoceptors induces con- traction whereas activation of fl2-adrenoceptors leads to relaxation of uterine muscle in several species (Miller and Marshall, 1965; Pennefather and Isaac, 1967; Butterworth and Randall, 1970; Riemer et al., 1988) including humans (Garret, 1955). fl-Adreno- ceptor-mediated relaxation results from the concerted effects of both a cAMP-dependent and a cAMP-inde- pendent process (Do Khac et al., 1986). The endocrine state of the uterus is of importance for the fluctuations in number of adrenergic receptors in the uterus (Johansson et al., 1980; Roberts et al., 1981). Prolonged treatment of the myometrium with iso- proterenol resulted in a marked alteration of fl- adrenoceptors with a concomitant desensitization of both fl-adrenoceptor-mediated cAMP and relaxant responses (Tougui et al., 1980; Tanfin-Tougui et al., 1981). It has been postulated that the relaxation and desensitization processes in several tissues are elicited via independent pathways following activation of the fl-adrenoceptors (for reviews see Sibley and Lefkowitz, 1987; Benovic et al., 1986; Fishman and Perkins, 1988). Moreover, Keitz et al. (1990) pro- posed a kinetic characterization of the relaxation and desensitization responses to a fl-adrenoceptor in rabbit aorta. Subehronic administration of the selective fl2- adrenoceptor agonist, clenbuterol (Engelhardt, 1976; ODeeeased. Cohen et al., 1982) to rats induces desensitization of fl2-adrenoceptors in peripheral vascular tissues (Kazanietz and Enero, 1990; Kazanietz et al., 1990) as well as in the central nervous system (Vos et al., 1987). However, the aim of this paper was to analyze a functional study, and to make a kinetic characteriz- ation of the effect of pretreatment with clenbuterol on the relaxant responses in uterine rings isolated from estrogen-treated rats. The results obtained showed that a single close of clenbuterol caused a rapid desensitization of the relaxant uterine response through fl2-adrenoceptors. MATERIALS AND METHODS Physiological procedures Virgin female Wistar rats of 200-250 g were used. Estra- diol benzoate in a sesame oil vehicle was administered once daily for two successive days (50/~g kg -I s.c. per day) and the animals killed by cervical dislocation 24 hr after the last dose. The two uterine horns were removed and four rings of approx. 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 hath. The upper hook was connected to a force-displacement trans- ducer to record the isometric tension of the preparation on a Grass polygraph. The rings were equilibrated for 2 hr at 37°C under I g tension in modified Krebs solution of the following composition (in mM): NaCI 118.0; KCI 4.7; CaC!2 2.6; MgCl2 1.2; Nail2 PO4 1.0; NaHCO3 25.0; glucose 11.1; ascorbic acid 0.11. The solution was gassed with a 95% 02-5% CO: mixture so that its final pH was 7.4. The relaxant effect was studied on rings that had been precontracted with 50 mM KC1 in the presence of phento- lamine l/~M, added 10rain before KCI. To maintain the isotonicity of the Krebs solution, the concentration of NaCl was reduced to 73 mM during the incubation with KCI. When the rings had attained a steady state level of contrac- tion the relaxant agent was added in a cumulative manner 769