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Non-genomic mechanism of action of Δ-4 and 5-reduced androgens and progestins on the contractility of the isolated rat myometrium
Life Sciences, Vol. 47, pp. 1547-1553 Printed in the U.S.A.
Pergamon Press
NON-GENOMIC MECHANISM OF ACTION OF A-4 AND 5-REDUCED ANDROGENS AND PROGESTINS ON THE CONTRACTILITYOF THE ISOLATED RAT MYOMETRIUM. Mercedes Perusqufa*, Elvia Garcia-Yafiez, Rafael Ib~fiez and Carlos Kubli-Garfias. Unidad de Investigaci6n Biom~dica del CMN, IMSS., *Unidad de Investigaci6n Cient~fica, Escuela Superior de Medicina, IPN., Savona 52, M~xico D.F. 14300, MEXICO. (Received in final form August 15, 1990) Summary
Effective concentrations50 of androgens, i.e. testosterone, androsterone, androstanediol, 5B-dihydrotestosterone and progestins: progesterone, pregnanolone, pregnanedione, epipregnanolone, allopregnanolone and allopregnanedione were assayed on the tonic contractions of the isolated rat myometrium induced by calcium in high-potassium calcium-free depolarizant solutions. Steroids showed their relaxant effect by fadding the sustained contraction induced by calcium in a depolarized state. Also, the addition of the calcium ionophores A-23187 and X-537A reversed the steroid relaxant effect by increasing sharply the tonic contraction. The possibility of steroid-induced relaxation through release of noradrenaline or histamine was discarded by blocking their specific receptors. From the results it is concluded that A-4 and 5-reduced androgens and progestins produce relaxation by a myogenic mechanism acting on the smooth muscle cell, most likely by directly blocking the calcium channels they causing modulation of: the contraction-relaxation cycle. Modulation of uterine contractility is achieved mainly by nervous and humoral influx. For rat myometrial relaxation, some neurotransmitters are quite important. Thus, noradrenaline through its B2-receptors produces uterine relaxation (1), while histamine has the same effect by activation of its H2-receptors (2). With regard to steroids, the progesterone blocking effect in the myometrium has been reported (3), and supported by physiological and electrophysiological studies establishing this hormone as a basic factor controlling uterine contractility (4). In previous papers we have shown the uterine relaxant effect of natural progestins and androgens, either 4-ene or reduced at position 5. This action is produced mostly by 51]-progestins, however 5a-reduced androgens such as androsterone and androstanediol are also effective (5,6). Although the relaxant effect is quite clear in other organs such as epididymis,jejunum, ileum and coronary artery (7-9), the mechanism of action of these compounds remains almost unknown. The aims of this paper were twofold: Firstly, to support the hypothesis of a cell calcium influx blockade by the A-4 and 5-reduced steroids using calcium ionophores (A-23187 and X-537A), and Secondly, to answer the question of whether or not steroid uterine relaxation is produced through neurotransmittes release, for example by noradrenaline and histamine. Methods Uterine rings were obtained ~om adult (180-220 ~ Sprague- Dawley rats in diestrus. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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Non-genomic Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
Uteri were dissected in a Tris buffer solution containing (mM): NaC1120, KC14.6, Tris 10, MgCl2 1.2, KH2PO4 1.2, CaC12 1.5 and glucose 11.4; pH was adjusted to 7.4. Tissues were placed in a bath chamber containing the above solution and bubbled continuously with oxygen. Bath temperature was maintained at 37°C. Calcium depolarizant solution (Ca-DS) was obtained by partial equi-molar sustitution of NaC1 by KC1 and composed of (mM):NaCl 84.4, KC140, Tris 10, MgC12 1.2, KH2PO4 1.2. CaC12 1.5 and glucose 11.4. High-potassium calcium-free solution (HK-CF) contained (mM): NaC1 84.5, KC1 40, Tris 10, MgC12 1.2, KH2PO4 1.2, EGTA 2 and glucose 11.4. The following steroids were used (Sigma Chemical Co., St. Louis, Mo.) A N D R O G E N S : 17a-hydroxy-4-androsten-3-one (testosterone) 5-androstan-3a,17B-diol (androstanediol), 3 a - - h y d r o x y 5 a - a n d r o st an- 17- one (androsterone), 17B-hydroxy-5B-androstan-3-one (5B-dihydrotestosterone, 5B-DHT). PROGESTINS: 4-pregnen-3,20-dione (progesterone), 3B-hydroxy-5B- pregnan-20-one (pregnanolone), 5B-pregnan-3,20-dione (pregnanedione), 3a-hydroxy-5B-pregnan-20-one (epipregnanolone), 5 a - p r e g n a n e - 3 , 2 0 - d i o n e ( a l l o p r e g n a n e d i o n e ) and 3B-hydroxy-5a-pregnan-20-one (allopregnanokme). Hormones were made up in and diluted to a final volume of 10/~l ofpropylene glycol. Uterine contractions were detected through a strain gage muscle transducer FT O3C coupled to a Grass polygraph model 7D. The tension employed was 1 g. Spontaneous contractions were recorded during 20 minutes with tris buffer solution. After this, tissues were depolarized with HK-CF solution during all the experiment. This solution yielded a transient tonic contraction which was abolished after 30 minutes CaCl2 addition (1 mM) induced a full tonic contraction which was maintained for 10 minutes and assumed as control. Subsequently, tissues were washed with HK-CF solution, after the trial was repeated but with steroids added 10 minutes before to a second CaC12 stimulus (1 mM, during 10 minutes). In total, the steroid was maintained on the bath during 20 minutes the CaCl2 response showed a lower amplitude of contraction shape regarding to the control. Curves of these effects were obtained and effective concentrations50 (EC50) were calculated. The steroid potency was calculated by the Litchfield and Wilcoxon method (10) and compared with verapamil using the formula ECs0/EC50 steroid/verapamil. Experiments with ionophores had the same design and tissues were incubated with the steroid ECs0. Thus, under these conditions, steroids were tested against any of the ionophores (20/~M for A-23187 free acid and 20/~M for X-537A Lasalocid A), 10 minutes after the second CaC12 addition and the response was observed during 10 minutes. In order to discard steroid-induced relaxation through neurotransmitter release, tonic contractions of uterine rings from rats in diestrus were provoked by the CaDS maintaned 30 minutes for that contraction, noradrenaline (5/~M) was added 10 minutes after of the tonic contraction, its relaxant effect was observed during 10 minutes after noradrenaline addition. However, propranolol (20 ~M) 10 minutes before the tonic contraction prevented the relaxant effect of noradrenaline. A similar experiment with 20 ~M of histamine to produce relaxation blocked by cimetidine, 20 ~M was carried out. After 10 minutes under such conditions, steroids were tested (EC50) and their effect was observed during 10 minutes. The effects were quantified by measuring the area under the curve of the contractions. Results For spontaneous contractions exchanging the Tris buffer solution for HK-CF produced a tonic contraction which reached its maximum and decreased slowly to the basal line after 30 minutes with the addition of 1 mM of calcium the tissue responded with a sustained tonic contraction. Pretreatment with steroids prevented the contraction induced by Ca 2+ in the del~olarized tissues produced by the HK-CF solution, as observed after the second addition of Caz+ (steroid calcium-antagonistic effect). The effect was observed as a concentration-response
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
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curve. However, regardless of the high relaxant potency of the steroids, ionophores were able of reverting at least transiently the uterine relaxation (Fig.l). In all cases a peak of contraction was observed with both ionophores in a concentration-response fashion.
VERAPAillL EC50
A(X-557A) 20 pM
ALLOPREiIHANILIH!
A(X-S37A) 20 ~M
PREiNANOLME
A - 25187 20 ~M
5 rain
ECso
HK-CF
Co2+
w
1 mM
~
'~ $' Ca 2+ PLllEENANOLONEI rnM EC50
A(X-537A) 20 JiM
FIG. 1 Depicts the relaxant effect of verapamil (EC50 0.2 pM), allopregnanolone (10 pM same ECs0 that progesterone) and pregnanolone (EC50 4.8/~M) on the tonic contraction induced by HK- CF solution plus CaC12 (1 mM) as well as the transiently contractile peak elicited by the calcium ionophores A-23187 and A(X-537A). Epipregnanolone, pregnanolone and pregnanedione were the most potent amongst the progestins and 5J3-dihydrotestosterone was the most potent androgen. Although most steroids were capable of inhibiting the contraction induced by calcium, allopregnanedione and allopregnanolone were almost ineffective, but we tested these compounds at same progesterone ECs0 because their theoretical values for EC50 are very high in the curves. Steroids were less potent than veparamil in a range from 20 to 275 fold when equimolar concentrations were compared (Table I). Values for the EC50 calculated for steroids, effect of ionophores and a comparison of potency between steroids and verapamil are presented in Table I.
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Non-genomic Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
TABLE I Relaxation of Depolarized Rat Myometrium by Steroids, Reversion by Ionophore A(X-537A) and Calcium-Antagonism Compared with Verapamil. COMPOUND
Percent of Uterine ECs0 (pM) Calcium-Antagonist Effect of Steroids
Percent of Contraction by A(X-537A)
Folds Less P o t e n t of Steroids vs. Verapamil a
25.45 +_ 11.99
275.0
ANDROGENS Testosterone
55.0
43.49 _+ 9.52 b
Androstanediol
14.5
51.15 _+ 6.53
19.97 _+ 5.40
72.5
Androsterone
16.0
58.90 _+ 20.99
40.55 + 17.79
80.0
7.5
39.16 +_ 3.63
22.40 _+ 6.72
37.5
Progesterone
10.0
46.76 _+ 2.98
20.80 _+ 7.46
50.0
5B-DHT
PA~DJT~T2h~ Pregnanolone
4.8
54.40 _+ 7.98
29.10 _+ 12.26
24.0
Pregnanedione
7.2
50.19 +_ 9.95
66.80 _+ 21.78
37.5
Epipregnanolone
4.0
51.27 _+ 3.25
37.16 _+ 11.35
20.0
Allopregnanolone c
10.0
6.63 + 3.27
8.47 _+ 2.96
50.0
Allopregnanedione c
10.0
6.60 +_ 2.08
11.55 +_ 3.37
50.0
Percent of uterine calcium-antagonist effect of steroids, were calculated assuming as control = 100%, the calcium response without steroid. a Steroid potency was compareted with verapamil using the formula ECs0 steroidfEC50 verapamil in M. Verapamil EC50 = 0.2/~M. b Values are mean SEM n=7. Changes were computed assuming 100% contractility in vehicle (propylene glycol) as control. c EC50 for allopregnanolone and allopregnanedione are like progesterone (their theoretical values are high).
Depolarized myometrium with the CaDS showed a slow sustained contraction. After 10 minutes a complete relaxation was observed with the addition of either noradrenaline or histamine, however the slope of histamine-induced relaxation was less pronounced than that produced by noradrenaline. The addition of noradrenaline or histamine when propranolol or cimetidine were present in the bath produced an slight increase of the plateau of the tonic contraction instead of the relaxation. Under those conditions which were maintained for 10 minutes the steroids produced their relaxant effect (Fig. 2). The values are mean in percent of steroid relaxant effect by measuring the area under the curve from 9 experiments (Table II).
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
l,
~A
f
4, p
+
NA
1551
I, STEROID
5 ml'--n +
K+40 mM H
C
H
STEROID
FIG. 2 Example of the relaxant effect of steroids (EC50 of pregnanolone) under the blockade of noradrenaline (NA 5 ~M) and histamine (H 20 ~M) effects in the depolarized rat myometrium by propranolol (P 20 ~M) and cimetidine (C 20 ~M), respectively.
TABLE II Relaxant Effect of Steroids under Blocking Condition of Noradrenaline and Histamine Effects in the Depolarized Rat myometrium. Compound
ECs0 ~uM)
Percent of Steroids Relaxation Effect under Noradrenaline Blockade
Percent of Steroids Relaxation Effect under Histamine Blockade
AND2~XkF2~ Testosterone
55.0
83.45 _+ 7.55 a
81.45 _+4.43
Androstanediol
14.50
72.63 _+ 5.79
78.09 _+ 13.16
Androsterone
16.0
67.43 _+ 7.96
74.74 _+ 9.97
5B-DHT
7.50
60.51 _+ 15.84
59.23 _+ 12.45
Progesterone
10.00
29.12 _+ 19.55
46.59 _+ 15.80
Pregnanolone
4.80
67.07 _+ 4.52
65.82 _+ 11.08
Pregnanedione
7.20
44.74 _+ 11.19
69.62 _+ 7.56
Epipregnanolone
4.00
21.42 _+ 14.65
67.00 _+ 7.16
Allopregnanoloneb
10.0
11.29 _+2.86
5.48 _+ 10.87
Allopregnanedioneb
10.0
2.05 _+ 12.20
5.81 _+ 8.50
a Values are mean SEM, n 7-9. b Same EC5o that progesterone with low effect.
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Non~genomlc Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
Discussion Androgens and progestins reduced at position-5 seem to play a physiological role; it has been found that in different kinds of smooth muscle such as uterus, epididymis jejunum, ileum and coronary artery these compounds elicit important relaxation (5-9). Other steroids such as corticosteroids also inhibit myometrium and ileum activity (8,11). The mechanisms by which these hormones influence the uterine contractility has not been well understood. Potassium-depolarized rat myometrium was used as a model to acquire reliable data to clarify the relaxant action of 5-reduced androgens and progestins. Moreover, the likelhood of neurotransmitter participation in this effect was discarded. Relaxation produced by steroids was quite similar to that observed in spontaneus contractility. Thus, 5B-progestins and 3a-hydroxy-5a-androgens (androsterone and androstanediol) and 5B-DHT, are better than the 4-ene precursors progesterone and testosterone relaxing uterine muscle (5,6). Smooth muscle contraction is dependent upon a sudden rise of intracellular calcium. Entrance of that ion into the cell is achieved by at least two different kinds of channels: voltageactivated and hormone-receptor activated channels (12-14). Depolarization of the myometrium with high-potassium produces the opening of the voltage-activated calcium channels (15); this, yields a tonic contraction by promoting calcium influx. Therefore any substance capable of producing relaxation under these conditions, could work by blocking calcium channels, either the voltage-activated or the hormone-receptor activated. With regard to noradrenaline and histamine both substances produce inhibition of calcium influx through specific receptors in the rat uterus. Thus, it is known that activation of B2-adrenergic and H2-histaminergic receptors induce relaxation of the myometrium in this species (1,2). The present observations demonstrated that the relaxant effect of 5-reduced steroids was not mediated through these neurotransmitters whose actions were antagonized by propranolol and cimetidine respectively. Thus, on disregarding the influence of the neurotransmitters, androgens and progestins showed their relaxant ability, by a direct myogenic mechanism. A-23187 and X-537A ionophores are divalent cation ionophores for calcium and to lesser extent for magnesium (16). They have a variety of effects i.e. promote influx of calcium across thecell membrane and/or efflux of this ion from intracellular reservoirs in to the cytoplasm and subsequently out of the cell across the membrane (17). The challenge of these ionophores against the steroid-induced uterine relaxation produced a peak of contraction. Ionophore reversal of the steroid effect, might be due to extracellular calcium entrance through calcium-gates produced by the ionophores. These artificial calcium-gates are not voltageactivated calcium channels, therefore they are not blocked by the steroids thus explaining the peak observed with both ionophores. However, these sustances were not able of reverse completely the relaxant effect of steroids, thus we observed a small recovery in the uterine relaxation. The relaxation observed immediately after the contraction peak might be due to the high toxicity of ionophores. The present data suggest a non°genomic effect of steroids apparently blocking the external Caz+ influx. This hypothesis is supported by the fact that these hormones showed an effect resembling that produced by verapamil, which is a typical calcium channel blocker. Although the steroids had lower potency than verapamil, they were potent enough to produce an important uterine relaxation. This subtle effect of the steroids hormones is still significant allowing us to postulate that these steroids are calcium modulators. Despite that all the steroids studied showed a calcium-antagonism they differed in
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
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potency. This difference seems to be related to molecular stereospecificity and physico-chemical interaction with the cell membrane, resulting in a effect which may be blockade of calcium channels rather than interaction with specific receptors. References 1. E. BULBRING, H. OHASHI and T. TOMITA, Smooth Muscle, E. Bulbring, A.F. Brading, A.W. Jones and T. Tomita Eds. p. 219- 227, Edward Arnold London (1981). 2. M.F. SHUBA, Smooth Muscle, E. Bulbring, A.F. Brading, A.W. Jones and T. Tomita Eds. p 377-383, Edward Arnold, London (1981). 3. A.I. CSAPO, Amer. J. Anat. 98 273-281 (1956). 4. J.M. MARSHALL and A.I. CSAPO, Endocrinology 68 1026- 1035,(1959). 5. C. KUBLI-GARFIAS, L. MEDRANO-CONDE, C. BEYER and A. BONDANI, Steroids 34 609-619 (1979). 6. C. KUBLI-GARFIAS, A. LOPEZ-FIESCO, M. PACHECO-CANO, H. PONCE-MONTER and A. BONDANI, Steroids 35 633-641 (1980). 7. C. KUBLI-GARFIAS, C. HOYO VADILLO and H. PONCE-MONTER, Proc.West. Pharmacol. Soc. 26 31-34 (1983). 8. C. KUBLI-GARFIAS, M. MEDINA-JIMENEZ, E. GARCIA-YANEZ, A.M. VAZQUEZ-ALVAREZ, M. PERUSQUIA, N. GOMEZ-GARCIA, J. ALMANZA, R. IBANEZ and R. RODRIGUEZ, Acta Physiol. et Pharmacol.Latinoam. 37(3) 357-364 (1987). 9. C. KUBLI-GARFIAS, J. Steroid Biochem. 26:332-335 (1987). 10. J.T. LITCHFIELD and F.A. WILCOXON, J. Pharmacol. Exp. Ther. 96 99-108 (1949). 11. M. PERUSQUIA, C. HOYO-VADILLO and C. KUBLI-GARFIAS, Arch. Invest. Mdd. (Mdx). 17 203-209 (1986). 12. T.B. BOLTON, Physiol. Rev. 59 606-718 (1979). 13. G. DROOGMANS, B. HIMPENS and R. CASTEELS, Experientia. 41 895- 900 (1985). 14. M.E. CARSTEN and J.D. MILLER, Am. J. Obstet. Gynecol 157 1303- 1315 (1987). 15. S. ICHIDA, M. MORIYAMA, K. YOSHIOKA and S. ARIYOSHI, Japanese J. of Pharmacol. 44 51-61 (1987). 16. B.C. PRESSMAN and N.T. De GUZMAN, Ann. N.Y. Acad. Sci. 227 380-391 (1974). 17. J.R. LYMANGROVE and E. KEKU, Life Sciences, 34 371-377 (1984).
Pergamon Press
NON-GENOMIC MECHANISM OF ACTION OF A-4 AND 5-REDUCED ANDROGENS AND PROGESTINS ON THE CONTRACTILITYOF THE ISOLATED RAT MYOMETRIUM. Mercedes Perusqufa*, Elvia Garcia-Yafiez, Rafael Ib~fiez and Carlos Kubli-Garfias. Unidad de Investigaci6n Biom~dica del CMN, IMSS., *Unidad de Investigaci6n Cient~fica, Escuela Superior de Medicina, IPN., Savona 52, M~xico D.F. 14300, MEXICO. (Received in final form August 15, 1990) Summary
Effective concentrations50 of androgens, i.e. testosterone, androsterone, androstanediol, 5B-dihydrotestosterone and progestins: progesterone, pregnanolone, pregnanedione, epipregnanolone, allopregnanolone and allopregnanedione were assayed on the tonic contractions of the isolated rat myometrium induced by calcium in high-potassium calcium-free depolarizant solutions. Steroids showed their relaxant effect by fadding the sustained contraction induced by calcium in a depolarized state. Also, the addition of the calcium ionophores A-23187 and X-537A reversed the steroid relaxant effect by increasing sharply the tonic contraction. The possibility of steroid-induced relaxation through release of noradrenaline or histamine was discarded by blocking their specific receptors. From the results it is concluded that A-4 and 5-reduced androgens and progestins produce relaxation by a myogenic mechanism acting on the smooth muscle cell, most likely by directly blocking the calcium channels they causing modulation of: the contraction-relaxation cycle. Modulation of uterine contractility is achieved mainly by nervous and humoral influx. For rat myometrial relaxation, some neurotransmitters are quite important. Thus, noradrenaline through its B2-receptors produces uterine relaxation (1), while histamine has the same effect by activation of its H2-receptors (2). With regard to steroids, the progesterone blocking effect in the myometrium has been reported (3), and supported by physiological and electrophysiological studies establishing this hormone as a basic factor controlling uterine contractility (4). In previous papers we have shown the uterine relaxant effect of natural progestins and androgens, either 4-ene or reduced at position 5. This action is produced mostly by 51]-progestins, however 5a-reduced androgens such as androsterone and androstanediol are also effective (5,6). Although the relaxant effect is quite clear in other organs such as epididymis,jejunum, ileum and coronary artery (7-9), the mechanism of action of these compounds remains almost unknown. The aims of this paper were twofold: Firstly, to support the hypothesis of a cell calcium influx blockade by the A-4 and 5-reduced steroids using calcium ionophores (A-23187 and X-537A), and Secondly, to answer the question of whether or not steroid uterine relaxation is produced through neurotransmittes release, for example by noradrenaline and histamine. Methods Uterine rings were obtained ~om adult (180-220 ~ Sprague- Dawley rats in diestrus. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
1548
Non-genomic Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
Uteri were dissected in a Tris buffer solution containing (mM): NaC1120, KC14.6, Tris 10, MgCl2 1.2, KH2PO4 1.2, CaC12 1.5 and glucose 11.4; pH was adjusted to 7.4. Tissues were placed in a bath chamber containing the above solution and bubbled continuously with oxygen. Bath temperature was maintained at 37°C. Calcium depolarizant solution (Ca-DS) was obtained by partial equi-molar sustitution of NaC1 by KC1 and composed of (mM):NaCl 84.4, KC140, Tris 10, MgC12 1.2, KH2PO4 1.2. CaC12 1.5 and glucose 11.4. High-potassium calcium-free solution (HK-CF) contained (mM): NaC1 84.5, KC1 40, Tris 10, MgC12 1.2, KH2PO4 1.2, EGTA 2 and glucose 11.4. The following steroids were used (Sigma Chemical Co., St. Louis, Mo.) A N D R O G E N S : 17a-hydroxy-4-androsten-3-one (testosterone) 5-androstan-3a,17B-diol (androstanediol), 3 a - - h y d r o x y 5 a - a n d r o st an- 17- one (androsterone), 17B-hydroxy-5B-androstan-3-one (5B-dihydrotestosterone, 5B-DHT). PROGESTINS: 4-pregnen-3,20-dione (progesterone), 3B-hydroxy-5B- pregnan-20-one (pregnanolone), 5B-pregnan-3,20-dione (pregnanedione), 3a-hydroxy-5B-pregnan-20-one (epipregnanolone), 5 a - p r e g n a n e - 3 , 2 0 - d i o n e ( a l l o p r e g n a n e d i o n e ) and 3B-hydroxy-5a-pregnan-20-one (allopregnanokme). Hormones were made up in and diluted to a final volume of 10/~l ofpropylene glycol. Uterine contractions were detected through a strain gage muscle transducer FT O3C coupled to a Grass polygraph model 7D. The tension employed was 1 g. Spontaneous contractions were recorded during 20 minutes with tris buffer solution. After this, tissues were depolarized with HK-CF solution during all the experiment. This solution yielded a transient tonic contraction which was abolished after 30 minutes CaCl2 addition (1 mM) induced a full tonic contraction which was maintained for 10 minutes and assumed as control. Subsequently, tissues were washed with HK-CF solution, after the trial was repeated but with steroids added 10 minutes before to a second CaC12 stimulus (1 mM, during 10 minutes). In total, the steroid was maintained on the bath during 20 minutes the CaCl2 response showed a lower amplitude of contraction shape regarding to the control. Curves of these effects were obtained and effective concentrations50 (EC50) were calculated. The steroid potency was calculated by the Litchfield and Wilcoxon method (10) and compared with verapamil using the formula ECs0/EC50 steroid/verapamil. Experiments with ionophores had the same design and tissues were incubated with the steroid ECs0. Thus, under these conditions, steroids were tested against any of the ionophores (20/~M for A-23187 free acid and 20/~M for X-537A Lasalocid A), 10 minutes after the second CaC12 addition and the response was observed during 10 minutes. In order to discard steroid-induced relaxation through neurotransmitter release, tonic contractions of uterine rings from rats in diestrus were provoked by the CaDS maintaned 30 minutes for that contraction, noradrenaline (5/~M) was added 10 minutes after of the tonic contraction, its relaxant effect was observed during 10 minutes after noradrenaline addition. However, propranolol (20 ~M) 10 minutes before the tonic contraction prevented the relaxant effect of noradrenaline. A similar experiment with 20 ~M of histamine to produce relaxation blocked by cimetidine, 20 ~M was carried out. After 10 minutes under such conditions, steroids were tested (EC50) and their effect was observed during 10 minutes. The effects were quantified by measuring the area under the curve of the contractions. Results For spontaneous contractions exchanging the Tris buffer solution for HK-CF produced a tonic contraction which reached its maximum and decreased slowly to the basal line after 30 minutes with the addition of 1 mM of calcium the tissue responded with a sustained tonic contraction. Pretreatment with steroids prevented the contraction induced by Ca 2+ in the del~olarized tissues produced by the HK-CF solution, as observed after the second addition of Caz+ (steroid calcium-antagonistic effect). The effect was observed as a concentration-response
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
1549
curve. However, regardless of the high relaxant potency of the steroids, ionophores were able of reverting at least transiently the uterine relaxation (Fig.l). In all cases a peak of contraction was observed with both ionophores in a concentration-response fashion.
VERAPAillL EC50
A(X-557A) 20 pM
ALLOPREiIHANILIH!
A(X-S37A) 20 ~M
PREiNANOLME
A - 25187 20 ~M
5 rain
ECso
HK-CF
Co2+
w
1 mM
~
'~ $' Ca 2+ PLllEENANOLONEI rnM EC50
A(X-537A) 20 JiM
FIG. 1 Depicts the relaxant effect of verapamil (EC50 0.2 pM), allopregnanolone (10 pM same ECs0 that progesterone) and pregnanolone (EC50 4.8/~M) on the tonic contraction induced by HK- CF solution plus CaC12 (1 mM) as well as the transiently contractile peak elicited by the calcium ionophores A-23187 and A(X-537A). Epipregnanolone, pregnanolone and pregnanedione were the most potent amongst the progestins and 5J3-dihydrotestosterone was the most potent androgen. Although most steroids were capable of inhibiting the contraction induced by calcium, allopregnanedione and allopregnanolone were almost ineffective, but we tested these compounds at same progesterone ECs0 because their theoretical values for EC50 are very high in the curves. Steroids were less potent than veparamil in a range from 20 to 275 fold when equimolar concentrations were compared (Table I). Values for the EC50 calculated for steroids, effect of ionophores and a comparison of potency between steroids and verapamil are presented in Table I.
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Non-genomic Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
TABLE I Relaxation of Depolarized Rat Myometrium by Steroids, Reversion by Ionophore A(X-537A) and Calcium-Antagonism Compared with Verapamil. COMPOUND
Percent of Uterine ECs0 (pM) Calcium-Antagonist Effect of Steroids
Percent of Contraction by A(X-537A)
Folds Less P o t e n t of Steroids vs. Verapamil a
25.45 +_ 11.99
275.0
ANDROGENS Testosterone
55.0
43.49 _+ 9.52 b
Androstanediol
14.5
51.15 _+ 6.53
19.97 _+ 5.40
72.5
Androsterone
16.0
58.90 _+ 20.99
40.55 + 17.79
80.0
7.5
39.16 +_ 3.63
22.40 _+ 6.72
37.5
Progesterone
10.0
46.76 _+ 2.98
20.80 _+ 7.46
50.0
5B-DHT
PA~DJT~T2h~ Pregnanolone
4.8
54.40 _+ 7.98
29.10 _+ 12.26
24.0
Pregnanedione
7.2
50.19 +_ 9.95
66.80 _+ 21.78
37.5
Epipregnanolone
4.0
51.27 _+ 3.25
37.16 _+ 11.35
20.0
Allopregnanolone c
10.0
6.63 + 3.27
8.47 _+ 2.96
50.0
Allopregnanedione c
10.0
6.60 +_ 2.08
11.55 +_ 3.37
50.0
Percent of uterine calcium-antagonist effect of steroids, were calculated assuming as control = 100%, the calcium response without steroid. a Steroid potency was compareted with verapamil using the formula ECs0 steroidfEC50 verapamil in M. Verapamil EC50 = 0.2/~M. b Values are mean SEM n=7. Changes were computed assuming 100% contractility in vehicle (propylene glycol) as control. c EC50 for allopregnanolone and allopregnanedione are like progesterone (their theoretical values are high).
Depolarized myometrium with the CaDS showed a slow sustained contraction. After 10 minutes a complete relaxation was observed with the addition of either noradrenaline or histamine, however the slope of histamine-induced relaxation was less pronounced than that produced by noradrenaline. The addition of noradrenaline or histamine when propranolol or cimetidine were present in the bath produced an slight increase of the plateau of the tonic contraction instead of the relaxation. Under those conditions which were maintained for 10 minutes the steroids produced their relaxant effect (Fig. 2). The values are mean in percent of steroid relaxant effect by measuring the area under the curve from 9 experiments (Table II).
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
l,
~A
f
4, p
+
NA
1551
I, STEROID
5 ml'--n +
K+40 mM H
C
H
STEROID
FIG. 2 Example of the relaxant effect of steroids (EC50 of pregnanolone) under the blockade of noradrenaline (NA 5 ~M) and histamine (H 20 ~M) effects in the depolarized rat myometrium by propranolol (P 20 ~M) and cimetidine (C 20 ~M), respectively.
TABLE II Relaxant Effect of Steroids under Blocking Condition of Noradrenaline and Histamine Effects in the Depolarized Rat myometrium. Compound
ECs0 ~uM)
Percent of Steroids Relaxation Effect under Noradrenaline Blockade
Percent of Steroids Relaxation Effect under Histamine Blockade
AND2~XkF2~ Testosterone
55.0
83.45 _+ 7.55 a
81.45 _+4.43
Androstanediol
14.50
72.63 _+ 5.79
78.09 _+ 13.16
Androsterone
16.0
67.43 _+ 7.96
74.74 _+ 9.97
5B-DHT
7.50
60.51 _+ 15.84
59.23 _+ 12.45
Progesterone
10.00
29.12 _+ 19.55
46.59 _+ 15.80
Pregnanolone
4.80
67.07 _+ 4.52
65.82 _+ 11.08
Pregnanedione
7.20
44.74 _+ 11.19
69.62 _+ 7.56
Epipregnanolone
4.00
21.42 _+ 14.65
67.00 _+ 7.16
Allopregnanoloneb
10.0
11.29 _+2.86
5.48 _+ 10.87
Allopregnanedioneb
10.0
2.05 _+ 12.20
5.81 _+ 8.50
a Values are mean SEM, n 7-9. b Same EC5o that progesterone with low effect.
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Non~genomlc Mechanism of Action of Steroids
Vol. 47, No. 17, 1990
Discussion Androgens and progestins reduced at position-5 seem to play a physiological role; it has been found that in different kinds of smooth muscle such as uterus, epididymis jejunum, ileum and coronary artery these compounds elicit important relaxation (5-9). Other steroids such as corticosteroids also inhibit myometrium and ileum activity (8,11). The mechanisms by which these hormones influence the uterine contractility has not been well understood. Potassium-depolarized rat myometrium was used as a model to acquire reliable data to clarify the relaxant action of 5-reduced androgens and progestins. Moreover, the likelhood of neurotransmitter participation in this effect was discarded. Relaxation produced by steroids was quite similar to that observed in spontaneus contractility. Thus, 5B-progestins and 3a-hydroxy-5a-androgens (androsterone and androstanediol) and 5B-DHT, are better than the 4-ene precursors progesterone and testosterone relaxing uterine muscle (5,6). Smooth muscle contraction is dependent upon a sudden rise of intracellular calcium. Entrance of that ion into the cell is achieved by at least two different kinds of channels: voltageactivated and hormone-receptor activated channels (12-14). Depolarization of the myometrium with high-potassium produces the opening of the voltage-activated calcium channels (15); this, yields a tonic contraction by promoting calcium influx. Therefore any substance capable of producing relaxation under these conditions, could work by blocking calcium channels, either the voltage-activated or the hormone-receptor activated. With regard to noradrenaline and histamine both substances produce inhibition of calcium influx through specific receptors in the rat uterus. Thus, it is known that activation of B2-adrenergic and H2-histaminergic receptors induce relaxation of the myometrium in this species (1,2). The present observations demonstrated that the relaxant effect of 5-reduced steroids was not mediated through these neurotransmitters whose actions were antagonized by propranolol and cimetidine respectively. Thus, on disregarding the influence of the neurotransmitters, androgens and progestins showed their relaxant ability, by a direct myogenic mechanism. A-23187 and X-537A ionophores are divalent cation ionophores for calcium and to lesser extent for magnesium (16). They have a variety of effects i.e. promote influx of calcium across thecell membrane and/or efflux of this ion from intracellular reservoirs in to the cytoplasm and subsequently out of the cell across the membrane (17). The challenge of these ionophores against the steroid-induced uterine relaxation produced a peak of contraction. Ionophore reversal of the steroid effect, might be due to extracellular calcium entrance through calcium-gates produced by the ionophores. These artificial calcium-gates are not voltageactivated calcium channels, therefore they are not blocked by the steroids thus explaining the peak observed with both ionophores. However, these sustances were not able of reverse completely the relaxant effect of steroids, thus we observed a small recovery in the uterine relaxation. The relaxation observed immediately after the contraction peak might be due to the high toxicity of ionophores. The present data suggest a non°genomic effect of steroids apparently blocking the external Caz+ influx. This hypothesis is supported by the fact that these hormones showed an effect resembling that produced by verapamil, which is a typical calcium channel blocker. Although the steroids had lower potency than verapamil, they were potent enough to produce an important uterine relaxation. This subtle effect of the steroids hormones is still significant allowing us to postulate that these steroids are calcium modulators. Despite that all the steroids studied showed a calcium-antagonism they differed in
Vol. 47, No. 17, 1990
Non-genomic Mechanism of Action of Steroids
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potency. This difference seems to be related to molecular stereospecificity and physico-chemical interaction with the cell membrane, resulting in a effect which may be blockade of calcium channels rather than interaction with specific receptors. References 1. E. BULBRING, H. OHASHI and T. TOMITA, Smooth Muscle, E. Bulbring, A.F. Brading, A.W. Jones and T. Tomita Eds. p. 219- 227, Edward Arnold London (1981). 2. M.F. SHUBA, Smooth Muscle, E. Bulbring, A.F. Brading, A.W. Jones and T. Tomita Eds. p 377-383, Edward Arnold, London (1981). 3. A.I. CSAPO, Amer. J. Anat. 98 273-281 (1956). 4. J.M. MARSHALL and A.I. CSAPO, Endocrinology 68 1026- 1035,(1959). 5. C. KUBLI-GARFIAS, L. MEDRANO-CONDE, C. BEYER and A. BONDANI, Steroids 34 609-619 (1979). 6. C. KUBLI-GARFIAS, A. LOPEZ-FIESCO, M. PACHECO-CANO, H. PONCE-MONTER and A. BONDANI, Steroids 35 633-641 (1980). 7. C. KUBLI-GARFIAS, C. HOYO VADILLO and H. PONCE-MONTER, Proc.West. Pharmacol. Soc. 26 31-34 (1983). 8. C. KUBLI-GARFIAS, M. MEDINA-JIMENEZ, E. GARCIA-YANEZ, A.M. VAZQUEZ-ALVAREZ, M. PERUSQUIA, N. GOMEZ-GARCIA, J. ALMANZA, R. IBANEZ and R. RODRIGUEZ, Acta Physiol. et Pharmacol.Latinoam. 37(3) 357-364 (1987). 9. C. KUBLI-GARFIAS, J. Steroid Biochem. 26:332-335 (1987). 10. J.T. LITCHFIELD and F.A. WILCOXON, J. Pharmacol. Exp. Ther. 96 99-108 (1949). 11. M. PERUSQUIA, C. HOYO-VADILLO and C. KUBLI-GARFIAS, Arch. Invest. Mdd. (Mdx). 17 203-209 (1986). 12. T.B. BOLTON, Physiol. Rev. 59 606-718 (1979). 13. G. DROOGMANS, B. HIMPENS and R. CASTEELS, Experientia. 41 895- 900 (1985). 14. M.E. CARSTEN and J.D. MILLER, Am. J. Obstet. Gynecol 157 1303- 1315 (1987). 15. S. ICHIDA, M. MORIYAMA, K. YOSHIOKA and S. ARIYOSHI, Japanese J. of Pharmacol. 44 51-61 (1987). 16. B.C. PRESSMAN and N.T. De GUZMAN, Ann. N.Y. Acad. Sci. 227 380-391 (1974). 17. J.R. LYMANGROVE and E. KEKU, Life Sciences, 34 371-377 (1984).