Genomic and non-genomic effects of steroidal drugs on smooth muscle contraction in vitro

LifeSciences,Vol.55,No.6, pp.437-443,1994 Copyright©1994ElsevierScience Ltd Printed in the USA. All rightsreserved

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GENOMIC AND NON-GENOMIC EFFECTS OF STEROIDAL DRUGS ON SMOOTH MUSCLE CONTRACTION I N VITRO M. Guti6rrez, V. Martfnez, B. Cantabrana and A. Hidalgo* Laboratorio de Farmacologfa, Departamento de Medicina, Facultad de Medicina. C/Julifin Claverfa s/n. 33006 Oviedo. Spain. (Received in final form May 25, 1994) Summary_ The effect of steroidal drugs on KCI (60 mM)-induced tonic contraction in in vitro rat uterus has been assayed. Ouabain had no effect and aldosterone only relaxed the KC1 contraction up to 27_ 7.3 %. However, estradiol, testosterone, progesterone, cortisol and alphaxalone relaxed the contraction in a dose-dependent way, and CaCI 2 (0.1 to 6 mM) counteracted this effect. Cycloheximide (1 and 10 /~g/ml) did not modify the effect of progesterone, testosterone or alphaxalone. Cycloheximide, but not actinomycin D (5 #g/ml), reduced the effect of cortisol. Both cycloheximide and actinomycin D shifted righward the relaxing effect of estradiol. This suggests that the steroidal relaxing effect in smooth muscle is preferably non-genomic andat plasma membrane level. However, cortisol and estradiol also act at intracellular levesl and induce transcriptional (estradiol) or non-transcriptional (cortisol) effects. Key Words: steroids,genomic effect, calcium, uterus

The classical genomic mechanism of steroid hormones supposes interaction with intracellular receptor activation of transcription and induction of protein synthesis. Thus, the effects induced through this genomic mechanism are decreased by inhibitors of transcription or protein synthesis (1). However, in recent years effects of steroid hormones in the plasma membrane have been proposed (2-6) and binding sites of steroids to plasma membrane of different structures have been described (7). In vitro effects of steroids, including modification of cardiac contractility (8-11), smooth muscle contraction (12,13) and membrane excitability of nerve cells (4,5,14) have also been reported. These effects are presumably non-genomic because they are rapid in onset and are not modified by inhibitors of protein synthesis (4,5,14). In a previous paper, we have described the inhibitory effects of the estrogens estradiol (E2) and diethylstilbestrol (13) and the non-steroidal antiestrogens (15) in smooth muscle contraction induced by KC1 and CaC12. A similar effect has been described for gestagens 17-OH progesterone derivatives (16) and 5,,- and 513- dihydrotestosterone (17). The aim of the present work is to study if other steroidal drugs, with or without * Corresponding author.

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hormonal activity have a similar effect in smooth muscle contraction induced by KCI in vitro. To dilucidate if the mechanism involved in their effect is genomic or non-genomic, the modification by a protein synthesis inhibitor (cycloheximide) and a transcription inhibitor (actinomycin D) has also been assayed.

Materials and methods lSxperimental procedure: Female Wistar rats, weighing 220-280 g, estrogenized with 2 mg/Kg i.m. of polyestradiol phosphate (Estradurin R, Abell6) (18) were used. The animals were killed by decapitation 72 h after injection, as the plasma estradiol levels estabilize in this time (18). Both uterine horns were extracted, cleaned of adherences, and cut in two halves. The preparations were vertically placed in 6 mi isolated organ baths and bubbled with carbogen mixture (95% 02 and 5% CO2). Jal6n's solution was the incubation medium and its mM composition was as follows: NaCl, 154; KC1, 5.63; CaCl2, 0.648; NaHCO3, 5.95 and glucose, 2,77. The preload was 1 g and the equilibration period not less than 45 minutes. The contractions were recorded through force-displacement transducers TRI 110 on a polygraph Letica 4006. Dose-response curves of the drugs were performed against the tonic response of KCI (60 mM)-induced contractions as shown in fig. 1. When the tonic component of the response to KC1 was stable (10-15 min) the steroidal hormones and steroids without hormonal activity were added in increasing-and cumulative doses. Each dose was left until its effect was stable (approximately 10 min). 100% relaxation was obtained when the baseline was reached. When the maximum inhibition was reached an attempt was made to recover the contraction with CaCI2 administered in increasing and cumulative doses from 0.1 to 10 mM. Only one dose-response curve of each drug was assayed for each uterine horn. The preparation was washed and disposed of. 5min

1oi

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•~. KCI(60mM)

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LESTRADIOL ( p M )

Fig.1 Recording of estradiol relating effect on KCl-induced contractions and its counteraction with CaCI2.

In order to elucidate the involvement of intracellular mechanisms on steroids, the modification of their effect with the inhibitor of protein synthesis, cyclohexirnide (1 and 10 #g/ml) (17,19,20) and transcription inhibitor, Actinomycin D (5/zg/ml) (21) added 30 min before KCI (60 mM) was also assayed.

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D__ngg Actinomycin D; Aldosterone (d-aldosterone); Alphaxalone (5a-pregnan-3o -o1-11,20-dione); Cydoheximide (3-[2-(3,5-Dimethyl-2-oxoeyclohexyl)-2-hydroxyethyl] glutarimide); cortisol (Hydrocortisone: llg,17t~-21-Trihydroxypregn-4-ene-3,20-dione); Estradiol (17-g-estradiol benzoate); O u a b a i n (ouabain octahydrate); Progesterone (4Pregnene-3,20-dione); Testosterone (4-androsten-17g-ol-3-one) S I G M A . T h e stock solution was made in dimethylsulfoxide (DMSO), P R O B U S . The effect of D M S O in the experiments is shown in the figure 2 A and C. Statistical analvsis: T h e results are expressed as m e a n value + SEM. Statistical evaluation was m a d e using the Student "t" test for unpaired data, considering the values of p < 0.05 as significant.

Results T h e steroids assayed have a different behaviour on KCI (60 mM)-induced contractions in the isolated rat uterus. T h e sex hormones, estradiol (3 to 300 ~M), progesterone (6 to 60/~M) and testosterone (3 to 60 ~M), induced a dose-dependent relaxation of the tonic contraction to KCI (Fig. 2 A). CaC12 (0,1 to 6 m M ) totally counteracted the inhibitory effect of progesterone, testosterone and of E 2 (Fig. 2 B).

Table i

Concentration of each drug that produces 50% inhibition (ECso) of the contractions induced by the KCI and its modification with cycloheximide (CHX) and actinomycin D (Ac D). Percentage maximal effect (Emax %) DRUG

N

ECf,0 (xl0 pM)

Emax (%)

Estradiol + CHX 1 I~g/ml CHX 10 I~g/ml Ac D 5 I~g/ml

12 7 8 6

2.05-+0.06 2.95+0.05"'* 16.5+0.8 ** 28.0-+6.0 ***

100 100 100 70.8-+2.4

Cortisol + CHX 1 I~g/ml CHX 10 pg/ml Ac D 5 I~g/ml

8 8 9 8

5.2 + 1.0 15-+0.9"** 12.4-+3.0* 5.0-+0.6

99.6 + 0.4 96.4-+1.4 100 98.7-+0.8

Testosterone + CHX 10 ~g/ml

16 12

1.1 + 0.1 1.3_+0.1

95.7 + 1.7 97.5+1.7

Progesterone + CHX 10 I~g/ml

17 12

3.1 + 0.4 2.4-+0.3

91.0-+3.0 96.0+3.2

Alphaxalone + CHX 10 p.g/ml

6 7

1.3 -+0.3 1.0-+0.1

100 100

Each value is the mean _+ s.e.m, for n experiments. * p<0.05; ** p<0.01; *** p<0.001 vs steroid control.

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The corticosteroid cortisol (1 to 300 #M) and the anesthetic alphaxalone (1 to 60 #M) also relaxed, in a dose-dependent way, the tonic contraction induced by KCI(60 mM). However, the mineralcorticoid aldosterone (1 nM to 300/~M) only relaxed the tonic 100

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Fig. 2 Concentration-response curve for A) steroidal sex hormones, C) other steroidal agents on the KCI (60 raM) induced tonic contraction. DMSO represents the effect of the necessary volume of solvent to obtain the dose of the drug. Counteraction of the relaxing effect of B) steroidal sex hormones, D) other steroidal agents, with CaC12. Each point is the mean _+ s.e.m, for at least 6 different experiments.

contraction to KC1 up to 27__.7.3% (Fig. 2 C). The digitalic ouabain (10 to 600/~M) assayed but lacked relaxing effect on KCl-contraction (the effect of ouabain 600/~M less than 10% of relaxation). The relaxing effect of alphaxalone and aldosterone counteracted by CaCIz (Fig. 2 D) similar to the effect of sex hormones. However, cortisol induced relaxation was counteracted up to 53.3-3.8 %.

was was was the

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The inhibitor of protein synthesis cycloheximide (1 and 10/~g/ml) shifted the doseresponse curve of E2 and cortisol rightward in a dose-dependent way (Fig. 3). Thus the ECs0 for E 2 and cortisol were significantly enhanced (Table I). The effect of progesterone, testosterone and alphaxalone were unmodified by cycloheximide (10/~g/ml) (Table I). The incubation with actinomycin D (5 ~g/ml), also shifted the effect of E2 to the right but did not modify the effect of cortisol (Fig. 3, Table I).

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Effect of Cycloheximide and Actinomycin D (5 pg/ml) on the relaxation produced by: A) estradiol (control) and B) cortisol (control) in the KCI (60 mM)-induced tonic contraction. Each point is the mean -+ s.e.m, for n>._6.

Discussion It is known that the tonic contraction of rat uterus induced by KCI depends on the extracellular calcium entry. On the other hand, drugs that interfere with the influx of calcium ions have a relaxing effect on KCl-induced contraction (22). The drugs assayed in this work a dose-dependent relaxing effect on KCl-induced tonic contraction, except for ouabain and aldosterone. This suggests a reduction of extracellular calcium influx by E 2, testosterone, progesterone and alphaxalone. This relation to calcium is also supported by the fact that CaC12 counteracted, either totally or partially, their inhibitory effect. The possible involvement of B2-adrenergic receptor in the steroid relaxing effect has also been discarded (12,13).

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The inhibitory effect of these drugs was similar to other steroidal drugs such as 17-OHprogesterone antiandrogen derivatives (16), 5a and 513 dihydrotestosterone (17), and estrogens (13,15). However, this is not totally related to their basic steroidal structure because steroidal drugs such as aldosterone and ouabain lack relaxing effect. The inhibitory action was also unrelated to non-specific modification of membrane fluidity described for F_,2 (23), since there was no correlation between lyposolubility and the relaxing effect. Therefore, the counteraction by CaC12 is not related to their liposolubility or the degree of inhibition. Thus, the effect was specific to the drug. On the other hand, the relaxant effect occu rs at concentrations of steroids that inhibit calcium uptake in rat myometrium (24) and mouse thymocites in vitro (25). The site of action is unknown and could involve extra and intracellular events. The counteraction by CaC12 suggests that the effect occurs at membrane level. However, this site of action could be used for alphaxalone, progesterone and testosterone since their effects were not modified by the protein synthesis inhibitor, cycloheximide. The effects of these three drugs are probably non-genomic, because they are rapid in onset, /~M dose is necessary, they are not modified by inhibition of protein synthesis (14) and, arise from plasma membrane level. The site of action of E 2 is presumably different from the other drugs and involves intracellular events because its effect is antagonized, in a dose-dependent way, by cycloheximide and by the transcription inhibitor, actinomycin D. This suggests that when cumulative dose-response curve is performed, E 2 is incorporated into cells at effective concentrations to induce a genomic effect. We think this is possible because first, E 2 uptake is extremely rapid (26), secondly, the concentrations of cycloheximide and actinomycin D are effective in inhibiting the protein synthesis (19,20) and mRNA release (21) at the incubation time assayed and, thirdly, it has been suggested that the transcription could be actived in minutes (27) by steroidal drugs. However, a non-genomic effect similar to progesterone, testosterone and alphaxalone could be not excluded because cycloheximide and actinomycin D (20,21) did not totally inhibit the effect of E 2. The fact that cycloheximide and actinomycin D modify the effect of some steroidal but not all of them suggested that inhibition of estrogen and cortisol effect by inhibitor protein synthesis and actinomycin D are not artefactual. The relaxing effect of cortisol was modified by cycloheximide but not by actinomycin D (5 ~g/ml). This suggests that its effect was non-genomic, presumably at plasma membrane level and involved an intracellular but not transcriptional effect. In summary, our results suggest that sex steroids, cortisol and alphaxalone induce inhibition of smooth muscle contraction by a decrease in the influx of extracellular calcium ions. Although, all of them produce the same effect, the site and mechanism of action is presumably different. The effect is preferably non-genomic and at plasma membrane level but cortisol and E 2 also act at intracellular level and induce transcriptional (estradiol) or non-transcriptional (cortisol) effects. The signalling pathway (mediator involvement) in the intracellular effect of E 2 and cortisol have not been studied in this paper. Acknowledgements This work was supported by grant PB 92-1128. M. Guti6rrez is the recipient of a grant from FYCIT.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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