Gen. Pharmac. Vol. 23, No. 4, pp. 643-647, 1992 Printed in Great Britain. All rights reserved

0306-3623/92 $5.00 + 0.00 Copyright ~ 1992 Pergamon Press Ltd

EFFECTS OF ANTIANDROGENS AND PROGESTERONE ON ISOLATED RAT UTERUS J. A. SANCHEZ APARiCIO, B. CANTABRANA and A. HIDALGO* Laboratorio de Farmacologia, Departamento de Medicina, Facultad de Medicina, C/. Julian Claveria s/n, 33006 Oviedo, Spain [Fax 523-2255]

(Received 2 January 1992) Al~tract--l. The effect of progesterone (P, 6 x 10-6--6 × 10 -5 M) and the antiandrogens cyproterone acetate (CPA, 1 0 - 7 - 1 0 -5 M), flutamide (F, 10-6-6 × 10 -5 M) and spironolactone (S, 1 0 - 6 - 6 × 10 -5 M ) on the KCI-induced tonic contraction of the isolated rat uterus have been assayed. 2. The antiandrogens relaxed, in a dose-dependent way, the KCl-induced contraction (EC~, 2.804 + 0.506 x 10-6; 1.671 + 0.308 × 10-~; and 3.042 + 0.14 × 10 ~ M, respectively for CPA, S and F). P also relaxed the KCl-induced contraction (EC~0, 2.436 + 0.524 × 10 -S M). 3. CaCI 2 (0.1-10 mM) counteracted the relaxing effect of CPA, S, F, and P, respectively, up to 100, 80.63, 60.66 and 90.57%. 4. The 17-OH-progesterone derivative CPA, but not S or F, reduces at small doses (6 × 10 -s M), but not at higher concentrations (6 × 107-6 × 10 -6 M), the relaxing effect of progesterone.

INTRODUCTION

Abell6) (Batra and Bengtsson, 1978) were used. The animals were killed by decapitation and both uterine horns were extracted, cleaned of adherences, and cut in two halves. Only the ovary part was used (I.5-2cm). The preparations were placed in 6ml isolated organ baths, incubated in Jal6n's solution and bubbled with carbogen mixture (95% 02 and 5% CO2). The preload was 1 g and the equilibration period not less than 30-45rain. The contractions were recorded through force--displacement transducers UFI on a polygraph Omniscribe D 5000.

Effects/n vitro of sexual hormones and their antagonists have been described in the last years. These effects are presumably non-genomic, because they are rapid in onset and non-modified by protein synthesis inhibitors (Schumacher, 1990; McEwen, 1991). Several non-genomic effects have been documented on cardiac (Raddino et al., 1986; Garcia Valencia et al., 1989) and smooth muscle (Perusquia et al., 1990; Padilla et al., 1991; Batra and Bengtsson, 1978; Fernb.ndez et al., 1991; Osa and Ogasawara, 1984) pharmacological preparations. In previous papers, there have been described the steroidal and non-steroidal antiandrogens produced negative inotropic effect in left atria of the rat (Garcia Valencia et al., 1991); this effect is direct, presumably non-genomic and related to reduction of calcium level able for contraction. In this paper we have also proposed that the steroidal antiandrogens 17-OHprogesterone derivatives effects (p.e., cyproterone acetate) are related to gestagenic activity. Since the sexual steroids, including estrogens, androgens and progesterone (Perusquia et al., 1990; Fernhndez et al., 1991; Osa and Ogasawara, 1984) produced inhibition of contraction of the rat uterus, for interference with calcium-dependent processes, the purpose of the present paper was to study the effect o f the steroidal (cyproterone acetate and spironolactone) and non-steroidal antiandrogens (flutamide) on rat uterus contraction induced by KCI and its counteraction with calcium.

Solution Jal6n's solution was used. Its composition (raM) was: NaCI, 154; KC1, 5.63; CaCI2, 0.648; NaHCO 3, 5.95; and glucose. 2.77.

MAIZRIALS AND METHODS Animals u.~ed and method of preparation Female Wistar rats, weighing 220-2808, estrogenized with 2 mg/kg i.m. of polyestradiol phosphate (Estradurin '~, *To whom all correspondence should be addressed.

Experimental procedure The effect of antiandrogens was studied on KCl-induced contractions. The preparation remained 30-45min in Jal6n's solution at 32°C. After equilibration, KCI (60 raM) was added. This drug produces tonic contractions which last more than 2 hr. When the contraction was stable (10-15 rain) the antiandrogens cyproterone acetate (10 -710-SM), flutamide ( 6 × 1 0 - 6 - 6 x 1 0 - S M ) or spironolactone (10-7-6 x 10-SM) were added to increasing and cumulative doses. Each dose was left until its effect was stable (approx. 10 rain). When the maximum inhibition had been reached, an attempt was made to recover the contraction with CaCI 2 (0.1-10mM) added to increasing and cumulative doses. In other experiments, the effect of progesterone (6 x 10-6-6 x 10 -5 M) on KCI-induced contraction and the modification of the effects of progesterone by antiandrogens have also been assayed. Drugs The following drugs were used- cyproterone acetate, Schering AG, Berlin; flutamide and progesterone, Sigma; spironolactone, Searle; calcium chloride, Probus (valued solution I M). Stock solution (10-3M) of antiandrogens

643

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J. A.

SANCH~ZAPARiCIOet al.

- t

tt 0.6 0.1 0.3 CaCl • 2 (mM)

•

KCI

(60 raM)

ICyproterone

Acetate

t 1

g

I

10"5

(M)

t /

10

6xl 0 - 5

% KCl

tt3

(60 mM)

I

I lSpironolactone

CaCI 2 (mM)

(M)

l,g 15 min

KCl

(60 mM)

Or.t31

[ [ Flutamide

CaC12 (mM) (M)

Fig. 1. Recording of the effect of cyproterone acetate, spironolactone and flutamide on KCI (60 mM)induced tonic contraction and their counteraction with CaCI 2. and progesterone was made in dimethylsulfoxide (DMSO, Probus).

RESULTS

Statistical analysis

Effects o f antiandrogens and progesterone on the KClinduced contraction

The results are expressed as mean value + SE. Statistical evaluation was carried out by using the Student t-test for paired and unpaired data, considering the values of P ~g0.05 as significant.

T h e c o n t r a c t i o n induced by KCI ( 6 0 m M ) was 2 . 4 9 + 0 . 0 7 (n = 16). A n t i a n d r o g e n s , cyproterone acetate 0 0 - 7 - 6 x l0 -5 M), flutamide

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Antiandrogens and progesterone on rat uterus

A

A--A

PROGESTERONE

O--O

CYPROTERONE ACETATE

A - - A SPIRONOLACTONE 1 O0

O--O

FLUTAMIDE

I--I

DMSO

B 1 O0

/ A

Z

o_ I-
/6.

( 6 x 10-6-6 x 10-SM) and spironolactone (10 - L 6 x 10 -5 M) relaxed, in a dose-dependent way, the tonic contraction induced by KCI (60 mM) in the rat uterus incubated in Jal6n's solution [Figs 1 and 2(A)]. Progesterone (6 x 10-6-6 x 10 -5 M) also relaxed the KCi-induced contraction. The maximal relaxation produced by all these drugs is higher than 90%. The concentrations of antiandrogens and progesterone that produces a relaxation of 50% (ECs0) are shown in Table 1. The relaxation of the tonic contraction of KCI by cyproterone acetate was counteracted by CaCI2 (0.1-3raM) up to 100%. CaC12 counteracts up to 60.66, 80.63 and 90.57% the relaxation produced by flutamide, spironolactone and progesterone respectively [Figs 1 and 2(B)]. Modifications by antiandrogens on the relaxant effect of progesterone The effect of progesterone is significantly reduced by the steroidal 17-OH-progesterone derivative antiandrogen cyproterone acetate at small doses (6 x 10 -s M), but superior doses (10-7-6 x 10 -6 M) do not modify the effect of progesterone [Fig. 3(A), Table 2]. The steroidal antiandrogen spironolactone (6 x 10-~M) and the non-steroidal antiandrogen flutamide (10 -6 and 10-SM) do not significantly modify the effect of progesterone, but flutamide induces a small increase on the effect of progesterone [Fig. 3(B), Table 2].

DISCUSSION

The antiandrogens assayed as well as progesterone relaxed, in a dose-dependent way, the KCl-induced tonic contraction. The three antiandrogens have a similar effect on left atria in vitro (Garcia et al., 1991). This effect is, obviously, not due to their antiandrogenic activity and non-mediated by intracellular androgen receptors but related to their steroidal or non-steroidal structure. This relaxant effect of antiandrogens and progesterone could be a non-genomic effect, because it is rapid in onset and the genomic effects of steroids are characterized by a slow onset (Moss and Dudley, 1984; Haukkamaa, 1987; Batra, 1980; Mironneau et ai., 1990; Schumacher, 1990; Perusquia et al., 1990; Fern/mdez et al., 1991). The relaxing effect is reverted by CaCl2. It suggests that it is probably related to calcium dependent processes, in agreement with previous papers (Batra and Sj6gren, 1983; Batra, 1980; Batra and Bcngtsson, 1978; Fernfindez et al., 1991), that suggest a calcium Table I. Concentration of drug that produces 50% relaxation (EC~0) on the KCI-induced contraction in rat uterus and percent of maximal inhibition obtained Drug

n

Progtsterone Cyproterone acetate Spironolactone Flutamide

8 8 6 8

Maximal inhibition (%)

EC~0 (M) 2.745 ± 0.601 2.804 ± 0.506 1.671 ±0.308 3.042 ± 0.140

Values are expressed as the mean ± SE.

x x x x

10-s 10 -6 10 -s 10 -s

95.360 ± 95.050 ± 95.467 ± 94.322 ±

2.570 1.985 1.932 1.455

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J. A. SAr4CH~ZAPARiCIO et al. •

0 Spironolactone 6x10 - 6 M

Control

• CPA 6x10 - 8 M • 100