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RU 38486 inhibits intracellular calcium mobilization and PGI2 release from human myometrium: Mechanism of action
.7. SteroidBiochem. Molec. Biol. Vol. 59, No. 1, pp. 63-73, 1996 Copyright © 1996 Elsevier Science Ltd. All fights reserved Printed in Great Britain PII: S0960-0760(96)00091-X 0960-0760/96 $15.00 + 0.00
Pergamon
R U 38486 Inhibits Intracellular C a l c i u m M o b i l i z a t i o n and PGI2 Release f r o m H u m a n M y o m e t r i u m : M e c h a n i s m of Action C. Lobaccaro-Henri, B. D e s c o m p s and H. Thaler-Dao* Centre de Recherche INSERM, 70 Rue de Navacelles, 34090, Montpellier, France
We p r e v i o u s l y d e m o n s t r a t e d t h a t t he a n t i p r o g e s t o g e n RU 486, w h e n s u p e r f u s e d on m y o m e t r i a l strips, i n d u c e s a r a p i d d e c r e a s e in s p o n t a n e o u s u t e r i n e c o n t r a c t i l e f r e q u e n c y , an i n c r e a s e in a m p l i t u d e a n d d u r a t i o n o f c o n t r a c t i o n s , a n d a c o n c o m i t a n t d e c r e a s e in 6-keto PGFI~ release. In this study, we p r e s e n t f u r t h e r w o r k on t he role o f c a l c i u m t r a n s i e n t s a n d t he i n v o l v e m e n t o f t he P L C / P K C p a t h w a y in m e d i a t i n g RU 486 effects. We f o u n d no cl ear causal r e l a t i o n s h i p b e t w e e n t he s p o n t a n e o u s c o n t r a c t i l i t y c o n t r o l l e d b y e x t e r n a l Ca + ÷ c o n c e n t r a t i o n a n d 6-keto PGFI~ rel ease d e p e n d i n g m o s t l y on i n t r a c e l l u l a r C a + ÷ m o b i l i z a t i o n . We show t h a t RU 486 s t r e n g t h e n e d t he i n h i b i t o r y effect o f TMB s , a p o t e n t i n h i b i t o r o f i n t e r n a l c a l c i u m , on b o t h s p o n t a n e o u s c o n t r a c t i l i t y a n d 6-keto PGFI~, r eleas e a n d a n t a g o n i z e d t he s t i m u l a t o r y act i on o f t h a p s i g a r g i n , a t oxi n blocking t he e n d o p l a s m i c r e t i c u l u m c a l c i u m p u m p ( E R C a + + A T P a s e ) . T h e s e d a t a i ndi cat e t h a t RU 486 could a c t as a n i n h i b i t o r o f i n t r a c e l l u l a r C a + ÷ m o b i l i z a t i o n . A slight b u t significant d e c r e a s e o f the p r o s t a n o i d l i b e r a t i o n was o b s e r v e d in t he p r e s e n c e o f U73122, an i n h i b i t o r o f P L C , b u t n o t in t he p r e s e n c e o f n e o m y c i n , a n o t h e r P L C i n h i b i t o r y c o m p o u n d . P K C i nhi bi t ors, s t a u r o s p o r i n e a n d H7 did not signific a ntly affect s p o n t a n e o u s 6-keto P G F ~ release, showing t h a t PIPe hydrolysis a n d P K C p a t h w a y w e r e n o t involved in t he bas a l r e l e a s e o f t he p r o s t a c y c l i n m e t a b o l i t e . V asopressi n (AVP), an a g e n t k n o wn to i n d u c e c o n t r a c t i l i t y o f the n o n - p r e g n a n t h u m a n u t e r u s , m a r k e d l y i n c r e a s e d 6-keto PGFI~ release in a d o s e - d e p e n d e n t m a n n e r . S t i m u l a t i o n o f G T P - r e g u l a t e d p r o t e i n s (G p r o t e i n s ) by ALF4 was a c c o m p a n i e d by a rise in 6-keto P G F ~ l i b e r a t i o n a n d a high c o n t r a c t i l e activity. T h e effects o f b o t h v a s o p r e s s i n a n d ALF4 w e r e n o t significantly o p p o s e d by RU 486, i n d i c a t i n g t h a t o t h e r s o u r c e s o f Ca ÷ ÷ , n o t c o n t r o l l e d b y t he s t er oi d, w e re involved in the a g o n i s t - s t i m u l a t e d p r o s t a n o i d release. S t u d ies with s t r u c t u r a l l y r e l a t e d RU 486 analogues show ed t h a t t he s t e r o i d effects w ere n o t d e p e n d e n t on t h e i r a n t i h o r m o n a l activity, b u t r a t h e r on a specific 11t3 a r y l s u b s t i t u t i o n a n d a 17fl-hydroxy 1 3 t - m e t h y l c o n f i g u r a t i o n o f t he 4 , 9 - e s t r a d i e n - 3 - o n e m o l e c u l e . C o p y r i g h t © 1996 E l sevi er Sc ie n c e Ltd.
J. Steroid Biochem. Molec. Biol., Vol. 59, No. 1, pp. 63-73, 1996
INTRODUCTION
lish than in rodents, it is clear that this hormone regulates uterine contractions as shown by the abortifacient effect of Rid 486 [11]. One of the proposed mechanisms was prostaglandin synthesis control [12]. Prostacyclin (PGI2) is the major uterine prostaglandin [13] and is assumed to mediate relaxation [14]. In an earlier study, we used the antiprogestogen RU 486 to study indirectly the putative effect of progesterone on prostacyclin release and the relationship between spontaneous contractility and PGI2 release, in a model of superfused human uterine strips [1]. We reported that RU 486 was able to decrease PGI2 release (measured by levels of its stable
Steroids modulate uterine contractile activity through genomic [2, 3] and non-genomic pathways [4-9]. Progesterone was long ago proposed as one of the major inhibitory agents responsible for the pregnant uterine quiescence [101] and, although its involvement in human pregnancy has been more difficult to estab*Correspondence to H. Thaler-Dao, Institut Universitaire de Recherche, Clinique, LBBL, 75, Rue de la Cardonille, 34093 MontpeUier Cedex 5, France. Tel: +33 67 41 59 17; Fax: +33 67 54 27 31. Received 5 Oct. 1995; accepted 9 Apr. 1996. 63
64
C. Lobaccaro-Henri et al.
metabolite 6-keto PGFI~) and that this action occurred after about 30 min of superfusion and was both reversible and dose dependent. Concomitant with this inhibition, we observed a modification of uterine spontaneous contractility with lower contraction frequency but increased amplitude and duration of contractions, indicating a slower but stronger contractile force. We demonstrated that the conjugated effects, on both contractility and PCI2 release, were specific to R U 486 and not obtained with the progesterone analogue R5020, nor with the corticosteroid dexamethasone; both agonist-hormonal steroids could act on the spontaneous motility but had no effect on 6-keto PGFI~ release. Therefore, R U 486 short-term action on P G release did not relate to the antihormonal activity of the molecule and was probably nongenomic. T h e rapidity of onset of R U 486 effects and their rapid reversal were considered to indicate a membrane action. Because uterine contractility and prostaglandin synthesis (release) are highly dependent on Ca + + transients [15, 16], we investigated the role of external Ca + + concentration, intracellular Ca + + mobilization and the possible involvement of the P L C - P I P 2 pathway, both in the control of spontaneous contractions and 6-keto PGFI~ release and in the putative mechanisms of R U 486 inhibitory effect. We present here further data indicating that R U 486 potentiates the inhibitory action of TMB8, an antagonist of intracellular Ca ++ [17], on 6-keto PGFI~ release and antagonizes the increased PGIe metabolite production induced by thapsigargin, an inhibitor of the endoplasmic reticulum Ca ++ ATPase (ER Ca ++ ATPase) [18]. T h e PIPz/PKC pathway was not involved in RU 486 action on PGIe release.
MATERIALS AND METHODS
Chemicals The progesterone antagonist RU 486, (mifepristone) [ 1113-(4-dimethylaminophenyl- 1)- 17t3hydroxy-17~-(1-propinyl)-4,9(10)-oestradien-3-one] and R U analogues were gifts from Roussel-Uclaf (Romainville, France). The antiprogestogens, ZK98.734, [1113-(4-dimethylaminophenyl- 1)- 17t3hydroxy-17~-(3-hydroxyprop-l-enyl)-4,9(10)-oestradien-3-one], ZK98.299 [1113-(4-dimethylaminophenyl- 1)- 17~-hydroxy- 1713-3-hydroxypropyl)- 13~-methy1-4,9-gonadien-3-one] and ZK112993 [1113-(4dimethylaminophenyl- 1)- 17t3-hydroxy- 17~- ( i -propinyl)-4,9(10)-oestradien-3-one] were kindly provided by Schering A G (Berlin, Germany). T h e steroids were dissolved in ethanol at concentrations of 1 0 - 2 M to 1 0 - S M and stored at - 2 0 ° C . Before use they were diluted to the appropriate concentrations in tyrode medium (NaCI 137 mM, KC1 2.6 mM, CaClz 1.8 mM, MgClz 1.05 m M , N a H C O 3
l l . 9 m M , NaH2PO4 0 . 4 2 m M , glucose 5 . 5 5 m M , p H 7.4). All reagents were analytical grade and obtained from Merck (Darmstadt, Germany). T M B s (3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester), thapsigargin, [ArgS]-vasopressin, NaF, phorbol esters (PMA and PDBu), staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), A23187, neomycin and nifedipine were purchased from Sigma (St Louis, MO, U.S.A.). U 73122 was supplied from T e b u (Rocky Hill, NJ, U.S.A.). Preparation of strips and superfusion Uterine corpus specimens (medium part) were obtained from cyclic premenopausal women following hysterectomy for fibromatosis or prolapsus. Since we did not observe any consistent relationship between the hormonal status and R U 486 effects, we randomly used strips obtained from the follicular or luteal phase. Institutional approval to use h u m a n tissue was obtained. T h e samples were immediately transferred into an ice-cold tyrode solution and kept at 4°C. U p o n arrival at the laboratory, the connective tissue was carefully removed and the myometrium dissected in strips parallel to the muscle fibre bundles. Prepared strips were approximately 20 m m long, 5 m m wide and 5 m m thick. T h e y were tied at each end with a silk thread and suspended in a superfusion chamber as described by Zahradnik et al. [19]. T h e strips were continuously superfused with oxygenated tyrode solution at a flow rate of 1 ml/min at 37°C. T h e y were allowed to equilibrate for at least 2 h. Isotonic registration of contractions was performed by means of a B368 transducer connected to a two-channel amplifier (Hugo Sachs Elektronik, March Hegstetten, Germany). After the establishment of regular contractions and monitoring of the control period (30 min), the strips were superfused with the same tyrode buffer containing RU 486 or the compound to be tested, at the same flow rate and temperature. Fractions of the superfused strip effluents (15ml) were collected. T h e samples were immediately stored at - 2 0 ° C . T h e RIA was performed after extraction with Sep Pak C18 columns (Waters Associates, Milford, MA, U.S.A.). T h e columns were washed with about 1 0 m l ethanol 95% (RP Carlo Erba, Milan, Italy) and 10 ml water/0.1% E D T A (Merck Darmstadt, Germany). T h e samples were acidified to p H 4 with citric acid 1 M, labelled with 1500 cpm ( [6, 9, 10, 12, 13, 15, 16] (n)-3H)-6 keto PGFI~ (specific activity 4.8-7.4 TBq/mmole, Amersham, U.K.) to estimate recovery, and then applied to the column; after washing with 0.1% E D T A and 2 m l hexane (RP SDS, France), the sample was eluted with 1 ml absolute ethanol. T h e ethanol was evaporated to dryness with nitrogen and the extract dissolved in 1 ml gelatine buffer (phosphate buffer 1 0 m M p H 7 . 4 , NaC1 9g/1 and
Inhibition of 6-Keto PGF~
gelatine 1 g/l). The RIA was performed with 3H-6keto PGFI~ (Amersham, U.K.) and an antiserum purchased from Biosys, Compi6gne, France, which does not crossreact with the steroids. Crossreactivity towards the other eicosanoids was respectively: PGFt~ 13%, PGE2 and dihydro-15-keto P G F ~ 4%, PGF2~, PGD2 and PGD1 2.8% and others 0.01%. The intraand inter-assay coefficients of variation were 5% and 6%, respectively. The concentration of 6-keto P G ~ displacing 50% of the labelled ligand was 71 pg and the lowest amount detectable was 18 pg. Unbound and bound ligand were separated by absorption on to dextran-charcoal, followed by centrifugation and measurement of the supernatant radioactivity in a liquid scintillation coun~er (Packard 460 CD). EIA was based on 6-keto PGFl~-bound-acetylcholinesterase tracer and quantification of acetylcholinesterase activity with an acetyl-thio-choline substrate. After hydrolysis the thiocholine reacted with Ellman's reagent and the absorbance of the 5-thio-2-nitrobenzoic acid was read at 412 nm. The ILIA was performed directly on the superfusion medium without previous extraction. Kits were purchased from Cayman (Ann Arbor, MI, U.S.A.) and used following the manufacturer instructions. The 50% B/BO was 82.4pg/ml and the threshold of detection was 23.6pg/ml. Crossreactivity was respectively: 2,3dinor-6-keto P G F ~ 8.7%, PGF2~ 2.1%, PGE2
Release by RU 486
0.92%, PGFI~ <0.01%.
65
0.8%
and
other
prostaglandins
Data analysis The data were expressed as mean percentage + SD of control tyrode superfusion values. The 100% value of PGI2 release represents the release rate of 6-keto PGF1, measured for a 30-min period (two samples of 15 ml) at the beginning of the experiment (after the stabilization period) and expressed as pg/min/mg wet weight. Similarly, the frequency, the mean amplitude of the peaks (cm at the setting of the recorder), and their mean duration(s) measured over 30 min after the stabilization period were considered as 100%. Analysis of variance (ANOVA) and Student's t-test for paired samples were used for statistical analysis.
RESULTS
Role of external calcium Effects of decreasing external Ca ++ concentration and of nifedipine on spontaneous contractility and 6-keto PGFI~ release. As expected, decreasing tyrode medium Ca ++ concentration from 1.8 to 0 m M inhibited spontaneous contractility (Fig. 1). However, this inhibition was not accompanied by a significant decrease in 6-keto PGFI~ release, although a great variability of response was observed from one sample to another. [] Frequer~y
200
[]
6 keto I ~ F l a
--v--
I
[
o
7-1 I
,.~
o
s•s
100 m •
s
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• s•s
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¢ •
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/ s , • % % • % •
•s s • s•s s¢
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,
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0,90
0,62
0~1
• • s • sS
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% • / / , / / ,
EDTA
Ca++ mM Fig. 1. Effect o f C a + + c o n c e n t r a t i o n in t h e s u p e r f u s i o n m e d i u m on t h e f r e q u e n c y o f c o n t r a c t i o n s a n d 6 - k e t o P G F t ~ r e l e a s e f r o m h u m a n m y o m e t r i a l strips. T y r o d e m e d i u m at e a c h C a ++ c o n c e n t r a t i o n w a s s u p e r f u s e d for 30 rain. 1 5 - m l f r a c t i o n s w e r e c o l l e c t e d for 6 - k e t o P G F t ~ m e a s u r e m e n t s . T h e d a t a w e r e e x p r e s s e d as a p e r c e n t a g e + S D o f t h e m e a n v a l u e s d e t e r m i n e d d u r i n g t h e s u p e r f u s i o n c o n t r o l p e r i o d in s t a n d a r d t y r o d e m e d i u m (1.80 m M C a + + ) . S t u d e n t ' s t-test: p a i r t e s t e d , e a c h C a ++ c o n c e n t r a t i o n vs c o n t r o l . * P < 0.05, * * * P < 0.001, n = 12.
C. Lobaccaro-Henri et al.
66
~0
5 min I I
+
Fig. 2. Effect o f TMBs (10~ M) s u p e r f u s e d a l o n e a n d in t h e p r e s e n c e o f R U 486 (10 -6 M) on h u m a n m y o m e t r i a l s p o n t a n e o u s c o n t r a c t i o n s . T y r o d e r e f e r e n c e m e d i u m w a s s u p e r f u s e d for a 30-min c o n t r o l p e r i o d , t h e n T M B s a l o n e ( u p p e r trace) or t o g e t h e r w i t h R U 486 ( l o w e r trace) w a s a p p l i e d for 30 m i n m o r e . T h e strips w e r e t h e n w a s h e d with tyrode m e d i u m for I h.
In the presence of 1 m M E D T A , a slight but significant decrease in 6-keto P G F I ~ release, from about 20%, could be observed (P < 0.05). Nifedipine, an inhibitor of Ca ++ channels, was used at the concentration of 10 -8 and 10 - 7 M. It inhibited the spontaneous contractility in a dosedependent m a n n e r as expected, but did not significantly affect 6-keto P G F I ~ release (127 _+ 24%, n=5). Relationship between Ca + + entry, spontaneous contractility and 6-keto PGF1~ release. Superfusion with the ionophore A 23187 did not significantly increase the spontaneous contractility but stimulated 6-keto P G F I ~ production: 1 4 8 _ 20% at a concentration of 1 x 10 - 6 M ( P < 0.05) and 1 8 5 _ 14% a t 5 . 1 0 - 6 M ( P < 0.01) (n = 4). R U 486 (10 -6 M) did not inhibit the effect of A23187 on prostaglandin release (155 ___55%). High concentrations of KC1 in the superfusion m e d i u m induced a long-lasting contraction and stimulated 6-keto P G F I = release: 1 5 4 + 2 5 % at 30 m M and 2 5 2 _ 90% at 60 m M ( P < 0.05) (n = 4). R U 486 had no significant inhibitory action on KC1 effects.
Role of internal calcium Effect of TMB8. T M B s , an intracellular calcium inhibitor, stopped spontaneous contractility as shown in Fig. 2 and simultaneously decreased PGI2 metabolite release. Both effects were reversed by washing for a 30-min period. In the presence of R U 486, T M B s action on the two parameters was stronger and of longer duration (Figs 3A and B). Effect of thapsigargin. Thapsigargin is a marine toxin inhibiting the ER Ca +÷ ATPase. It increases intracellular Ca ÷ ÷ concentration.
Superfusion in the presence of 1 0 - 7 M thapsigargin stimulated only weakly uterine strip contractility, but basal 6-keto P G F I ~ production was increased twofold (Fig. 4). T h e effect of thapsigargin was significantly inhibited by R U 486 at 1 0 - 6 M ( P < 0.01). P L C and PIP2 pathway. T w o inhibitors of P L C , U73122 and neomycin, were used to test the involvem e n t of P L C on prostanoid release. A slight but significant inhibition of 6-keto PGl~ release was obtained in presence of 5 . 1 0 - 6 M U73122 (70 + 19% of control, P < 0.05), but not in presence of 1 0 - 3 M neomycin (Table 1). R U 486 did not amplify the slight decrease observed with P L C inhibitors. Activators of the P K C ( P M A or the di-butyrylated analogue PDBu) and protein C kinase inhibitors (staurosporine and H7) had no significant effect on the 6-keto P G F I ~ production (Table 1). Vasopressin superfusion, an excitatory agonist, induced a dose-dependent increase in contractility, which was accompanied by a significant stimulation of 6-keto P G F I ~ output (259 + 90% of control at 1 0 - 8 M AVP, P < 0.01) (Fig. 5). High levels of prostanoids, significantly different from control, were still noticeable after a washing period of 3 0 m i n (213 + 50%, P < 0.001), indicating that intracellular mechanisms were activated by AVP and remained effective some time after washing out the agonist. Superfusion of AVP together with R U 486 did not significantly decrease 6-keto P G F I ~ release when c o m p a r e d to AVP alone. However, we observed that the return to basal levels after washing occurred faster (30 min) in the presence of R U 486 (Fig. 5). This could m e a n that R U 486 inhibition of internal Ca ++ release was u n m a s k e d when AVP was washed out, and that the steroid could counteract some of the agonist effects on intracellular Ca + ÷ levels.
Inhibition of 6-Keto PGF~. Release by RU 486
67
200" Tyrode Tymde % , . ~ s % . . % , . %,
o
TMB8 10-6 M
o
A
100 %¢%,.%s%s%~ ',*sss
o
,*,*ss,
~ s , * s /
, / / s , * , s , * s /
%%%%~
0 15
30
45
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75
90
Time (min)
200 Tyrode (TMB8 + RU) 10-6 M
Tyrode c o
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r
r
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r
,
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Time (min) Fig. 3. S y n e r g i c effect o f T M B s a n d R U 486 s u p e r f u s i o n at 10 4 M c o n c e n t r a t i o n , on 6-keto P G F I ~ r e l e a s e f r o m h u m a n m y o m e t r i a l strips. T y r o d e m e d i u m w a s s u p e r f u s e d for 30 m i n , t h e n T M B s a l o n e (A) o r t o g e t h e r w i t h R U 486 (B) w a s a p p l i e d for 30 m i n a n d t h e t i s s u e s w e r e finally w a s h e d w i t h t y r o d e m e d i u m for a n o t h e r 30 rain. T h e r e l e a s e rate w a s m e a s u r e d in t w o s a m p l e s o f l $ - m l f r a c t i o n s a n d t h e d a t a w e r e e x p r e s s e d as a percentage_ SD of the mean values measured during the tyrode medium superfusion control period. S t u d e n t ' s t-test: p a i r t e s t e d , T M B s vs c o n t r o l a n d T M B s + R U 486 vs c o n t r o l . * * P < 0.01, * * * P < 0.001, n = 8.
Activation of G proteins by ALF~a. ALI~4, a potent activator of G proteins, induced very powerful and long-lasting contractile activity (not shown) together with a strong stimulation of 6-keto PGF1, release
(Fig. 6). Both effects were not significantly opposed by R U 486. W h e n superfused in Ca + ÷ free medium ALF~4 stimulatory effect on the prostanoid production was inhibited by 80%.
C. Lobaccaro-Henri et al.
68
200
[] Thaps 10-7M [ ] Thaps+RU 10-6M •.
o r~
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.d
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++
o
r
.
r
100 s s ~ s s s
2
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Fig. 4. Effect of thapsigarglne superfused alone (10 -7 M) or together with RU 486 (10~SM) on 6-keto PGFI~ release from human myometrial strips. Tyrode medium was superfused for 30 min, then thapsigargine alone, or together with RU 486 was applied for 30 min and the tissues were finally washed with tyrode medium for another 30 min. The release rate was measured in two samples of 15-ml fractions and the data were expressed as a percentage _+SD of the mean values measured during the tyrode medium superfusion control period. Student's t-test: pair tested, thapsigargine vs control. ***P < 0.001. Thapsigargin vs T h a p s . + R U 486. + +P<0.01, n = 5. STUDY OF THE SPECIFICITY OF ACTION OF llfl-ARYL SUBSTITUTED COMPOUNDS A series o f p r o g e s t o g e n or corticosteroid antagonists structurally related to R U 4 8 6 were studied to determ i n e the influence o f several substitutions on 6-keto P G F I ~ release f r o m superfused uterine strips. As s h o w n in T a b l e 2, the 17ct p o s i t i o n o f the hydrophobic substituent was m o r e i m p o r t a n t to confer an inhibitory activity than its degree o f unsaturation or its length: Z K 9 8 7 3 4 (17ct-hydroxy-propenyl) was inhibitory, like R U 4 8 6 (17~-propynyl), but Z K 9 8 2 9 9 (17B-hydroxy-propyl) had no effect. H o w e v e r , the analogue Z K 9 8 2 9 9 was also e p i m e r i c at C - 1 3 and presents a very different m o l e c u l a r shape ( C / D ring cis junction) w h i c h c o u l d be d e t e r m i n a n t in the lack o f effect. O n the other hand, the 17ct-propynyl analogues, Z K 1 1 2 9 9 9 and R U 4 0 2 2 6 , carrying a different substituent o n the 11B aryl ring, w e r e b o t h able to inhibit 6-keto P G F I ~ p r o d u c t i o n (Table 3). T h i s s h o w e d that the r e p l a c e m e n t o f the d i m e t h y l a m i n o
group in position para o f the phenyl ring by a d i m e t h y l or m e t h o x y radical did n o t alter the capacity to inhibit 6-keto P G F ~ output. H o w e v e r , this capacity was lost w h e n the aryl ring was suppressed or w h e n it was replaced by a m o r e bulky bicyclic substituent.
DISCUSSION T h e control o f PGI2 synthesis (and release) in s m o o t h m u s c l e and in vascular tissues has b e e n the subject o f extensive investigations (for review see reference [20]). C o n t r a c t i o n s i n d u c e d by agonists and prostanoid release are m e d i a t e d through c o m m o n Ca + + d e p e n d e n t m e c h a n i s m s . T h e increase in intracellular Ca + + can be achieved t w o ways: by the o p e n i n g o f Ca + + channels or by intracellular Ca + + m o b i l i z a t i o n f r o m e n d o p l a s m i c reticulum. U s u a l l y b o t h m e c h a n i s m s are involved: intracellular Ca + + increase f o l l o w i n g PIP2 hydrolysis and IP3 formation
Table 1. 6-keto PGFI~ releasefrom human uterine strips supe~used in the presence of various effectors of the PLC-PIP2 pathway PLC inhibitors
PKC activators
Kinase inhibitors
U 73122 5 X 10 -6 M
Neomycin
TPA 10 -6 M
PDBU 10 -6 M
Staurosporine
H7
10 -3 M
10 -6 M
10 -6 M
70___ 19 p < 0.05 n=5
93 __. 16 n s n=5
126 ___ 19 n s n=3
93 ___ 16 n s n=3
117___ 17 n s n=3
120___ 10 n s n=3
Data are expressed as mean percentage _+ SD of control tyrode superfusion values, ns: non-significant vs. control; n: number of experiments. For more details, see Materials and Methods.
Inhibition o f 6 - K e t o P G F ~
Release by R U 4 8 6
69
~AVP
400
[] AVP +RU 10-6M o + o
300
o ,¢¢i ee
200 ,%
o
,.
,~
ns
,~/'sI '¢¢1 , ¢ ~1
q~
.'¢~sI
100 , ' / ~q
• ~' s I
x x '//I
0 Basal
AVP 10-8M
AVP 10-7M
Washing
Fig. 5. Effect o f v a s o p r e s s i n superfused alone (10 -s M and 10-7 M), or together with RU 486 (10-6 M), on 6-keto PGFxffi release from h u m a n m y o m e t r i a l strip. Tyrode m e d i u m was superfused for 30 m i n , then vasopressin w a s applied for 30 m i n at each concentration, alone or together with RU 486 and the tissues were finally washed with tyrode m e d i u m for 30 rain. The release rate was m e a s u r e d in two samples o f 15-mi fractions and the data were e x p r e s s e d as a percentage _ S D o f the m e a n values m e a s u r e d during the tyrode m e d i u m superfusion control period. Student's t-test. Pair tested: vasopressine vs control and vasopressine + RU 486 vs control. * * * P < 0.001, * * P < 0.01, ns, non-significant. Washing after A V P superfuslon vs washing after A V P + RU 486 superfusion. +P < 0.05. N u m b e r o f experiments were six (in duplicate) for A V P and four (in duplicate) for A V P + RU 486. leads to the opening of Ca ++ channels and prostaglandin release. However, the relative importance of each mechanism depends on the cell type and the a g o n i s t u s e d . I n m a n y s m o o t h m u s c l e c e l l s s u c h as
trachea, urinary bladder or v e s s e l s , extracellular C a + + e n t r y s t i m u l a t e s P G I 2 s y n t h e s i s . I n t h e s e tissues, calcium channel blockers and removal of extracellular Ca ++ with chelators inhibits 6-keto PGFx~
500
[]
A1F4- 1 0 m M
[]
ALF4-+RU 486
40'0
TT
8 o
[,T., c~
300
*¢s */s ,s/
200
q~
/
%¢~¢,
100
r ~ S,
Basal
Test
Washing
Fig. 6. Effect o f AIF~4 (10 raM) superfused alone or together with RU 486 (10 -6 M) on 6-keto PGFI~ release from h u m a n m y o m e t r l a l strips. Tyrode m e d i u m was superfused for 30 rain, then ~ alone, or together with RU 486 was applied for 30 m i n and the tissues were finally washed with tyrode m e d i u m for another 30 rain. The release rate w a s m e a s u r e d in two samples o f 15-ml fractions and the data were expressed as a percentage +_ S D o f the m e a n values m e a s u r e d during the tyrode m e d i u m superfusion control period. Student's t-test: pair tested, AIF~4 vs control and AIF~4 + RU 486 vs control. * * * P < 0.001, * * P < 0.01, * P < 0.05, ns, non-significant. N u m b e r o f experiments were six (in duplicate) for ALI~4 and four (in duplicate) for ALF~4 + RU 486.
70
C. Lobaccaro-Henri et al.
Table 2. Inhibition
of 6-keto PGFI,
myometrial strips by RU 486 analogues d@erently
release from supefised
substituted at position
C13 and Cl7 of the steroid skeleton
Structure
Product
6-keto PGF1, release (% of control)
7t
RU 38486
48 + 12**’
8
RU 38486
48 + 12***
8
ZK 98734
65 f 17”
6
ZK 98734
65 f
6
ZK 98299
113+23 ns
6
ZK 98299
OH -- CH=CH-CH2OH
17**
OH (3I2+?H2-CH2OH
113f23ns
6
Data are expressed as mean percentage f SD of control tyrode superfusion values. Student’s t-test for paired samples. **PC 0.01, ***P < 0.001; ns: non-significant; n: number of experiments. release by 60%, indicating that calcium receptor-operated channels (ROC) are involved [2 l-241. The ionophore A23187 is able to stimulate PG synthesis and its effect is also inhibited by free calcium media and Ca+ + channel blockers [22, 23, 251. High K concentrations do not stimulate 6-keto PGFi, release in the vascular tissues [21, 221 nor in the guinea pig myometrium [25]. However, reports from the literature indicate that intracellular Ca+ + mobilization is an important pathway in vascular cells: in bovine vascular endothelial cells, 50% of the basal 6-keto PGFr,, production and 80-100% of the thrombin or A23187 stimulated production were inhibited in presence of the [Ca’ + ]i antagonist TMBs [26-281. In the human uterus, pacemaker cells initiate spontaneous contractility [ 161. This mechanical activity Table 3. Inhibition
of 6-keto PGF,,
release from supe&sed
depends on Ca+ + entry through voltage-operated channels (VOC). We showed that 6-keto PGFi, release was not dependent on spontaneous contractility because blocking contractions, by depriving the cells of external Ca+ + or by nifedipine, did not significantly decrease that release. A23 187, although showing only a very slight stimulatory action on the contractility, had a marked effect on 6-keto PGFi, release. A23187 activity was lost in the absence of Ca+ + in the superfusion medium. In contrast to other reports from the literature indicating that high KC1 concentrations did not influence PGIs release from vascular tissues [21, 22, 291 or from superfused guinea pig uterus [30], we observed in our experiments a significant stimulation of 6-keto PGFr, production when superfusing KC1 30 and 60 mM. These
myometrial
strips by RU 486 analogues
d$terently substituted at position
Cl1 of the steroid skeleton
Structure
Product
6-keto PGF1, release (% of control)
n
RU 38486
48 f 12 ***
8
RU38486
48 f 12***
8
RU40226
66 + 11’S
6
RU40226
66k 11**
6
ZK112993
68 f 12 ***
6
ZK112993
68 f 12***
6
(-)ec+-J-c,,
RU43780
lOlf23
11s
4
RU43780
101 + 23 ns
4
lc,,
RU40540
122f26
ns
6
RU40540
122 f 26 ns
6
Lc,,
RU42764
113f13
ns
5
RU42764
113 _+13 ns
5
tQ_C,,
Data are expressed as mean percentage SD of control tyrode superfusion values. Student’s t-test for paired samples. **I’< 0.01, ***PC 0.001; ns: non-significant; n: number of experiments.
Inhibition of 6-Keto PGFI~ Release by RU 486 last two results indicate that in h u m a n uterus, an increase of 6-keto P G F I ~ release above basal levels can be mediated through Ca ++ entry by V O C and independently of contractions. O u r data strongly suggest that increased intracellular Ca+ + concentration is the major event controlling basal P G synthesis. This is clearly shown by the inhibitory action of T M B s and the stimulating effect of thapsigargin. This drug was reported to increase the level of intracellular Ca ++ by inhibiting the E R Ca ++ ATPase. Interestingly R U 486 seemed to antagonize the toxin action and to potentiate the effect of T M B s . W h e t h e r the steroid inhibits the E R Ca + + p u m p or increases Ca ++ binding to the internal m e m b r a n e s remains to be established. Spontaneous 6-keto ]?GFI~ release did not depend on PIP2 hydrolysis as shown by the marginal effect of P L C inhibitors and the lack of effect of P K C effectors. Neither P K C stimulation by P M A or its dibutyrylated analogue nor protein kinase inhibitors such as staurosporine or H 7 modified 6-keto P G F ~ release, which is in contrast to the data obtained by other groups in different tissues [30-33]. Although these inhibitors are not strictly specific to P K C , their lack of effect rules out P K C phosphorylation. Interestingly, R U 486 did not increase the slight inhibitory effect of U73122, indicating that the E R Ca + ÷ A T P a s e which is inhibited by thapsigargin and which m a y be the potential target for R U 486 action did not regulate the same intracellular Ca ++ pool as IP3. Vasopressin, an excitatory agonist m o r e active than oxytocin on n o n - p r e g n a n t uterus and known to exert its activity through the PIP2 pathway [34], was used to further estimate the role of IP3 dependent Ca *+ mobilization in our model. We showed that this agonist stimulated b o t h uterine contractility and PGI2 metabolite production and that R U 486 did not significantly counteract its action. These results suggest that the steroid inhibition of internal C a * + mobilization was not sufficient to oppose vasopressin effects. It is known, for instance, that excitatory agonists trigger Ca ++ entry through R O C , thus inducing a sustained contractility and prostaglandin release [20]. Similarly, we did not observe a significant inhibition of A L F 4 stimulatory action by R U 486. This substance is a known activator of various G proteins. It induced a marked activation of contractions and P G release in our experiments. T h e fact that 80% of its effect was inhibited in the absence of external Ca + ÷ indicated a G-protein-activated Ca + * channel as the possible AlF44 target. T h e lack of significant inhibitory effect of R U 486 on agonist-induced and ALF4induced P G synthesis is in agreement with a sequestering action on a specific Ca ++ intraceUular store and no action, or a marginal one, on external Ca ÷ * entry. An inhibitory action of R U 486 on intracellular
71
Ca + + levels was recently reported in h u m a n spermatozoa [35]. R U 486 action on Ca ++ internal sequestration is related to its specific structure and not to its h o r m o nal potential as discussed in a previous p a p e r [1] and as indicated by the structure-activity relationship study with analogues. Our data showed that the 11Baryl substitution was of prime importance to the inhibitory action on 6-keto P G F I ~ release: replacing the aromatic cycle by an ethylic or ethylenic radicals suppressed the activity. Otherwise, different hydrophobic substituent (propenyl or propynyl) could be admitted at the 17ct position. However, a complete lack of effect accompanied the epimerization at position 17 and 13: the 17ct-hydroxy-17B-3-hydroxypropyl)-13ctmethyl c o m p o u n d , Z K 98299, was not active. We cannot determine, with the few Z K c o m p o u n d s tested, which modification (13~-methyl or 17i3-substituent) caused the inactivity. T h e s e data on structureactivity relationship confirm that R U 486 action on 6keto P G F t ~ release did not depend on its antiprogestational capacity because Z K 98299, which is also an antiprogestogen [36], was not inhibitory. T h e same conclusion is also supported by the report of de N e e f on the hormonal potential o f the 1 l~t-methylsteroids [37]. In s u m m a r y , our data indicate that R U 486 inhibitory action on 6-keto P G F I ~ release is mediated by its blocking action on intracellular Ca ++ mobilization and that this effect was not related to its antihormonal potential. Steroid h o r m o n e s can show non-genomic m e m b r a n e effects, mediated either by m e m b r a n e receptors or by action on intracellular c o m m u n i c a t i o n pathways [38, 39]. R U 486 could act on intracellular Ca ++ mobilization: it increased T M B 8 inhibitory action and antagonized thapsigargin effect. R U 486 target could be the E R Ca ++ A T P a s e p u m p , an E R Ca ++ -binding protein or a small G T P - d e p e n d e n t protein [40, 41]. T h e s e effects of R U 486 indicating a relaxant action seemed very surprising and paradoxical in regard to its well-known antiprogesterone p r o p erties. R U 486 administration to pregnant w o m e n is able to restore uterine contractility and induce abortion or labour [11, 41]. By lowering intracellular Ca ++ levels, R U 486 would potentially inhibit uterine motility. This action is similar to that of progesterone in rat uterus reported in the literature [6]. However, in these experiments progesterone seemed to act via calcium channels or GABAA receptors [9]. In the h u m a n uterus, progesterone increased the A T P - d e p e n d e n t Ca ++ binding to microsomial preparations [42]. T h e progesterone-like relaxant effect of R U 486 is very surprising, but was also established by Perusquia et al. in the rat uterus [43]. T h e paradoxical action of R U 486 on intracellular Ca ++ could explain the modifications of contractile pattern observed: the higher amplitude and duration of contractions, indicating a stronger contractile force, could
72 be due
C. Lobaccaro-Henri et aL to a d e c r e a s e in t h e r e l a x a n t p r o s t a g l a n d i n
PGI2, whereas the concomitant lower frequency could depend
on
intracellular
Ca ++
sequestering.
These
surprising results, given the clinical use o f R U 486 in abortion
and
the induction
of labour,
may
explain
why administration of prostaglandins with the steroid i m p r o v e s the clinical o u t c o m e .
REFERENCES 1. Lobaccaro-Henri C., Saintot M., Laffargue F., Zahradnik H. P., Descomps B. and Thaler-Dao H.: Effect of the progesterone antagonist RU 486 on human myometrial spontaneous contractility and PGI2 release. Prostaglandins 44 (1992) 443455. 2. Fuchs A. R. and Fuchs F.: Endocrinology of human parturition: a review. Br. J. Obstet. Gynaecol. 91 (1984) 948-967. 3. Casey L. M. and MacDonald P., Endocrinology of pregnancy and parturition. In Uterine Function. Molecular and Cellular Aspects, ed. M. E. Carsten and J. D. Miller. Plenum Press, NY, 1989, pp. 501-517. 4. Batra S. and Bengtsson B.: Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. J. Physiol. 276 (1978) 329-342. 5. Batra S. and Sj6gren C.: Effect of oestradiol treatment on calcium uptake by the rat uterine smooth muscle. Life Sciences 32 (1983) 315-319. 6. Kubli-Garfias C., Medrano-Conde L., Beyer C. and Bondani A.: In vitro inhibition of rat uterine contractility induced by 5ct and 513 progestins. Steroids 34 (1979) 609-615. 7. Perusquia M., Garcia-Yanez E., Ibanez R. and Kubli-Garfias C.: Nongenomic mechanism of action of a - 4 and 5-reduced androgens and progestins on the contractility of the isolated rat myometrium. Life Sciences 47 (1990) 1547-1553. 8. Gutierrez M., Martinez V., Cantabrana B. and Hildago A.: Genomic and non-genomic effects of steroidal drugs on smooth muscle contraction in vitro. Life Sciences 55 (1994) 437-443. 9. Pumam C. D., Brann D. W., Kolbeck R. C. and Mahesh V. B.: Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems. Biol. Reprod. 45 (1991) 266-272. 10. Csapo A. L.: Progesterone block. Am. J. Anat. 98 (1956) 273291. 11. Baulieu E. E., RU 486--an antiprogestin steroid with contragestive activity in women. In The Antiprogestin Steroid R U 486 and Human Fertility Control, ed. E. E. Baulieu and S. J. Segal. Plenum Press, NY, 1985, pp. 1-25. 12. Kelly R. W. and Smith S. K.: Progesterone and antiprogestins. A comparison of their effects on prostaglandin production by human secretory phase endometrium and decidua. Prostagland. Leuk. Med. 29 (1987) 181-190. 13. Abel M. H. and Kelly R. W.: Differential production of prostaglandins within the human uterus. Prostaglandins 18 (1979) 821-828. 14. Omini C., Pasargikilian R., Folco G. C., Fano M. and Berti F.: Pharmacological activity of PGI2 and its metabolite 6-oxoPGFI~. Prostaglandins 15 (1978) 1045-1053. 15. Carsten M. E. and Miller J. D., Calcium control mechanism in the myometrial cell and the role of the phosphoinositide cycle. In Uterine Function. Molecular and Cellular Aspects, ed. M. E. Carsten and J. D. Miller. Plenum Press, NY, 1989, pp. 121155. 16. Wray S.: Uterine contraction and physiological mechanisms of modulation. Am. J. Physiol. 264 (1993) C1-C18. 17. Chiou C. Y. and Malagodi M. H.: Studies on the mechanism of action of a new Ca 2 + antagonist -(N,N-diethylamino)octyl 3,4,5-trimethoxy benzoate hydrochloride in smooth and skeletal muscles. Br. J. Pharmacol. 53 (1975) 279-285.
18. Thastrup O., Cullen P. J., Drobak B. K., Hanley M. R. and Dawson A. P.: Thapsigargin, a tumor promotor, discharges intracellular Ca 2 + stores by specific inhibition of the endoplasmic reticulum Ca 2 ÷ ATPase. Proc. Natn. Acad, Sci. U.S.A. 87 (1990) 2466-2470. 19. Zahradnik H. P., Schoening R. and Breckwoldt M., Prostaglandins correlation to human myometrial activity in vitro. In Prostacyclin in Pregnancy, ed. P. J. Lewis. Raven Press, NY, 1983, pp. 147-151. 20. Jeremy J. Y., Mikhailidis D. P. and Dandona P.: Excitatory receptor-prostanoid synthesis coupling in smooth muscle: mediation by calcium, protein kinase C and G proteins. Prostagland. Leuk. Essential Fatty Acids 34 (1988) 215-227. 21. Stewart D., Poumey E. and Fitchett D.: Norepinephrine stimulated vascular prostacyclin synthesis. Receptor dependent calcium channels controls prostaglandin synthesis. Can. J. Physiol. Pharmacol. 62 (1984) 1341-1347. 22. Golub M. S., Berger M. E. and Camazola A. E.: Adrenergic stimulation of prostacyclin production in the rat tail artery. II. The role of calcium. Prostagland. Leuk. Med. 20 (1985) 313324. 23. Van de Velde V. J. S., Van den Bossche R. M., Bult H. and Herman A. G.: Modulation of prostacyclin biosynthesis by calcium entry blockers and extracellular calcium. Biochem. Pharmacol. 35 (1986) 253-256. 24. Jeremy J. Y., Mikhailidis D. P. and Dandona P.: The effect of nifedipine, nimodipine and nisoldipine on agonist- and traumastimulated vascular prostacyclin synthesis in vitro. Arch. Pharmacol. 332 (1986) 70-73. 25. Poyser N. L.: Effect of using calcium-free Krebs solution on basal and A23187-stimulated prostaglandin output from day 15 guinea-pig uterus superfused in vitro. Prostagland. Leuk. Med. 13 (1984) 259-269. 26. Adams Brotherton A. F. and Hoak J. C.: Role of Ca ~ ÷ and cyclic AMP in the regulation of the production of prostacyclin by the vascular endothelium. Proc. Nam. Acad. Sci. U.S.A. 79 (1982) 495-499. 27. Hassid A. and Oudinet J. P.: Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells. Prostaglandins 32 (1986) 457-478. 28. Watanabe K., Lam G. and Jaffe E. A.: The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells. Prostagland. Leuk. Essential Fatty Acids 46 (1992) 211-214. 29. Boeynaems J. M. and Ramboer I.: Effects of changes in extraand intracellular K ÷ on the endothelial production of prostacyclin. Br. J. Pharmacol. 98 (1989) 966-972. 30. Poyser N. L.: Effects of various factors on prostaglandin synthesis by the guinea-pig uterus. J. Reprod. Fertil. 81 (1987) 269-276. 31. Jeremy ]. Y. and Dandona P.: The role of the diacylglycerolprotein kinase C system in mediating adrenoreceptor-prostacyclin synthesis coupling in the rat aorta. Eur. J. Pharmacol. 136 (1987) 311-316. 32. Merkel L. A., Rivera L. M., Colussi D. J. and Perrone M. H.: Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation. J. Pharmacol. Exp. Ther. 257 (1991) 134-140. 33. Erbrich A. C., Church D. J., Valotton M. B. and Lang U.: Regulation of prostacyclin production by [Ca 2+ ]i and protein kinase C in aortic smooth muscle cells. Am. ft. Physiol. 263 (1992) E800-E806. 34. Schrey M. P., Conford P. A., Read A. M. and Steer P. J.: A role for phosphoinositide hydrolysis in human uterine smooth muscle during parturition. Am. J. Obstet. Gynecol. 159 (1988) 964-970. 35. Serres C., Yang J. and Jouannet P.: RU 486 and calcium fluxes in human spermatozoa. Biochem. Biophys. Res. Commun. 204 (1994) 1009-1015. 36. Elger W., Beier K., Chwalisz K., F~ihnrich M., Hasan H., Henderson D., Neef G. and Rohde R.: Studies on the mechanisms of action of progesterone antagonists, ft. Steroid Biochem. 25 (1986) 835-845. 37. Neef G., Beier S., Elger W., Henderson D. and Wiechert R.: New steroids with antiprogestational and antiglucocorticoid activities. Steroids 44 (1984) 349-361.
I n h i b i t i o n of 6 - K e t o P G F 1 , Release b y R U 4 8 6 38. Wehling M.: Non genornic actions of steroid hormones. T E M 5 (1994) 347-353. 39. Brann W. D., Hendry L. B. and Mahesh V. B.: Emerging diversities in the mechanism of action of steroid hormones. J. Steroid Biochem. Molec. Biol. 52 (1995) 113-133. 40. Bian J., Ghosh T. K., Wang J. C. and Gill D. L.: Identification of intracellular calcium pools. Selective modification by thapsigargin. J. Biol. Chem. 266 (1991) 8801-8806.
73
41. Missiaen L., De Smedt H., Droogmans G., Himpens B. and Casteels R.: Calcium ion homeostasis in smooth muscle. Pharmacol. Ther. 56 (1992) 191-231. 42. Carsten M. E.: Calcium accumulation by human uterine microsomal preparations: effects of progesterone and oxytocin. Am. 37. Obstet. Gynecol. 133 (1979) 598-601. 43. Perusquia M. and Kubli-Garfias C.: Progesterone-like relaxant effect of RU 486 in the rat myometrium. Life Sciences 54 (1994) 1501-1506.
Pergamon
R U 38486 Inhibits Intracellular C a l c i u m M o b i l i z a t i o n and PGI2 Release f r o m H u m a n M y o m e t r i u m : M e c h a n i s m of Action C. Lobaccaro-Henri, B. D e s c o m p s and H. Thaler-Dao* Centre de Recherche INSERM, 70 Rue de Navacelles, 34090, Montpellier, France
We p r e v i o u s l y d e m o n s t r a t e d t h a t t he a n t i p r o g e s t o g e n RU 486, w h e n s u p e r f u s e d on m y o m e t r i a l strips, i n d u c e s a r a p i d d e c r e a s e in s p o n t a n e o u s u t e r i n e c o n t r a c t i l e f r e q u e n c y , an i n c r e a s e in a m p l i t u d e a n d d u r a t i o n o f c o n t r a c t i o n s , a n d a c o n c o m i t a n t d e c r e a s e in 6-keto PGFI~ release. In this study, we p r e s e n t f u r t h e r w o r k on t he role o f c a l c i u m t r a n s i e n t s a n d t he i n v o l v e m e n t o f t he P L C / P K C p a t h w a y in m e d i a t i n g RU 486 effects. We f o u n d no cl ear causal r e l a t i o n s h i p b e t w e e n t he s p o n t a n e o u s c o n t r a c t i l i t y c o n t r o l l e d b y e x t e r n a l Ca + ÷ c o n c e n t r a t i o n a n d 6-keto PGFI~ rel ease d e p e n d i n g m o s t l y on i n t r a c e l l u l a r C a + ÷ m o b i l i z a t i o n . We show t h a t RU 486 s t r e n g t h e n e d t he i n h i b i t o r y effect o f TMB s , a p o t e n t i n h i b i t o r o f i n t e r n a l c a l c i u m , on b o t h s p o n t a n e o u s c o n t r a c t i l i t y a n d 6-keto PGFI~, r eleas e a n d a n t a g o n i z e d t he s t i m u l a t o r y act i on o f t h a p s i g a r g i n , a t oxi n blocking t he e n d o p l a s m i c r e t i c u l u m c a l c i u m p u m p ( E R C a + + A T P a s e ) . T h e s e d a t a i ndi cat e t h a t RU 486 could a c t as a n i n h i b i t o r o f i n t r a c e l l u l a r C a + ÷ m o b i l i z a t i o n . A slight b u t significant d e c r e a s e o f the p r o s t a n o i d l i b e r a t i o n was o b s e r v e d in t he p r e s e n c e o f U73122, an i n h i b i t o r o f P L C , b u t n o t in t he p r e s e n c e o f n e o m y c i n , a n o t h e r P L C i n h i b i t o r y c o m p o u n d . P K C i nhi bi t ors, s t a u r o s p o r i n e a n d H7 did not signific a ntly affect s p o n t a n e o u s 6-keto P G F ~ release, showing t h a t PIPe hydrolysis a n d P K C p a t h w a y w e r e n o t involved in t he bas a l r e l e a s e o f t he p r o s t a c y c l i n m e t a b o l i t e . V asopressi n (AVP), an a g e n t k n o wn to i n d u c e c o n t r a c t i l i t y o f the n o n - p r e g n a n t h u m a n u t e r u s , m a r k e d l y i n c r e a s e d 6-keto PGFI~ release in a d o s e - d e p e n d e n t m a n n e r . S t i m u l a t i o n o f G T P - r e g u l a t e d p r o t e i n s (G p r o t e i n s ) by ALF4 was a c c o m p a n i e d by a rise in 6-keto P G F ~ l i b e r a t i o n a n d a high c o n t r a c t i l e activity. T h e effects o f b o t h v a s o p r e s s i n a n d ALF4 w e r e n o t significantly o p p o s e d by RU 486, i n d i c a t i n g t h a t o t h e r s o u r c e s o f Ca ÷ ÷ , n o t c o n t r o l l e d b y t he s t er oi d, w e re involved in the a g o n i s t - s t i m u l a t e d p r o s t a n o i d release. S t u d ies with s t r u c t u r a l l y r e l a t e d RU 486 analogues show ed t h a t t he s t e r o i d effects w ere n o t d e p e n d e n t on t h e i r a n t i h o r m o n a l activity, b u t r a t h e r on a specific 11t3 a r y l s u b s t i t u t i o n a n d a 17fl-hydroxy 1 3 t - m e t h y l c o n f i g u r a t i o n o f t he 4 , 9 - e s t r a d i e n - 3 - o n e m o l e c u l e . C o p y r i g h t © 1996 E l sevi er Sc ie n c e Ltd.
J. Steroid Biochem. Molec. Biol., Vol. 59, No. 1, pp. 63-73, 1996
INTRODUCTION
lish than in rodents, it is clear that this hormone regulates uterine contractions as shown by the abortifacient effect of Rid 486 [11]. One of the proposed mechanisms was prostaglandin synthesis control [12]. Prostacyclin (PGI2) is the major uterine prostaglandin [13] and is assumed to mediate relaxation [14]. In an earlier study, we used the antiprogestogen RU 486 to study indirectly the putative effect of progesterone on prostacyclin release and the relationship between spontaneous contractility and PGI2 release, in a model of superfused human uterine strips [1]. We reported that RU 486 was able to decrease PGI2 release (measured by levels of its stable
Steroids modulate uterine contractile activity through genomic [2, 3] and non-genomic pathways [4-9]. Progesterone was long ago proposed as one of the major inhibitory agents responsible for the pregnant uterine quiescence [101] and, although its involvement in human pregnancy has been more difficult to estab*Correspondence to H. Thaler-Dao, Institut Universitaire de Recherche, Clinique, LBBL, 75, Rue de la Cardonille, 34093 MontpeUier Cedex 5, France. Tel: +33 67 41 59 17; Fax: +33 67 54 27 31. Received 5 Oct. 1995; accepted 9 Apr. 1996. 63
64
C. Lobaccaro-Henri et al.
metabolite 6-keto PGFI~) and that this action occurred after about 30 min of superfusion and was both reversible and dose dependent. Concomitant with this inhibition, we observed a modification of uterine spontaneous contractility with lower contraction frequency but increased amplitude and duration of contractions, indicating a slower but stronger contractile force. We demonstrated that the conjugated effects, on both contractility and PCI2 release, were specific to R U 486 and not obtained with the progesterone analogue R5020, nor with the corticosteroid dexamethasone; both agonist-hormonal steroids could act on the spontaneous motility but had no effect on 6-keto PGFI~ release. Therefore, R U 486 short-term action on P G release did not relate to the antihormonal activity of the molecule and was probably nongenomic. T h e rapidity of onset of R U 486 effects and their rapid reversal were considered to indicate a membrane action. Because uterine contractility and prostaglandin synthesis (release) are highly dependent on Ca + + transients [15, 16], we investigated the role of external Ca + + concentration, intracellular Ca + + mobilization and the possible involvement of the P L C - P I P 2 pathway, both in the control of spontaneous contractions and 6-keto PGFI~ release and in the putative mechanisms of R U 486 inhibitory effect. We present here further data indicating that R U 486 potentiates the inhibitory action of TMB8, an antagonist of intracellular Ca ++ [17], on 6-keto PGFI~ release and antagonizes the increased PGIe metabolite production induced by thapsigargin, an inhibitor of the endoplasmic reticulum Ca ++ ATPase (ER Ca ++ ATPase) [18]. T h e PIPz/PKC pathway was not involved in RU 486 action on PGIe release.
MATERIALS AND METHODS
Chemicals The progesterone antagonist RU 486, (mifepristone) [ 1113-(4-dimethylaminophenyl- 1)- 17t3hydroxy-17~-(1-propinyl)-4,9(10)-oestradien-3-one] and R U analogues were gifts from Roussel-Uclaf (Romainville, France). The antiprogestogens, ZK98.734, [1113-(4-dimethylaminophenyl- 1)- 17t3hydroxy-17~-(3-hydroxyprop-l-enyl)-4,9(10)-oestradien-3-one], ZK98.299 [1113-(4-dimethylaminophenyl- 1)- 17~-hydroxy- 1713-3-hydroxypropyl)- 13~-methy1-4,9-gonadien-3-one] and ZK112993 [1113-(4dimethylaminophenyl- 1)- 17t3-hydroxy- 17~- ( i -propinyl)-4,9(10)-oestradien-3-one] were kindly provided by Schering A G (Berlin, Germany). T h e steroids were dissolved in ethanol at concentrations of 1 0 - 2 M to 1 0 - S M and stored at - 2 0 ° C . Before use they were diluted to the appropriate concentrations in tyrode medium (NaCI 137 mM, KC1 2.6 mM, CaClz 1.8 mM, MgClz 1.05 m M , N a H C O 3
l l . 9 m M , NaH2PO4 0 . 4 2 m M , glucose 5 . 5 5 m M , p H 7.4). All reagents were analytical grade and obtained from Merck (Darmstadt, Germany). T M B s (3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester), thapsigargin, [ArgS]-vasopressin, NaF, phorbol esters (PMA and PDBu), staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), A23187, neomycin and nifedipine were purchased from Sigma (St Louis, MO, U.S.A.). U 73122 was supplied from T e b u (Rocky Hill, NJ, U.S.A.). Preparation of strips and superfusion Uterine corpus specimens (medium part) were obtained from cyclic premenopausal women following hysterectomy for fibromatosis or prolapsus. Since we did not observe any consistent relationship between the hormonal status and R U 486 effects, we randomly used strips obtained from the follicular or luteal phase. Institutional approval to use h u m a n tissue was obtained. T h e samples were immediately transferred into an ice-cold tyrode solution and kept at 4°C. U p o n arrival at the laboratory, the connective tissue was carefully removed and the myometrium dissected in strips parallel to the muscle fibre bundles. Prepared strips were approximately 20 m m long, 5 m m wide and 5 m m thick. T h e y were tied at each end with a silk thread and suspended in a superfusion chamber as described by Zahradnik et al. [19]. T h e strips were continuously superfused with oxygenated tyrode solution at a flow rate of 1 ml/min at 37°C. T h e y were allowed to equilibrate for at least 2 h. Isotonic registration of contractions was performed by means of a B368 transducer connected to a two-channel amplifier (Hugo Sachs Elektronik, March Hegstetten, Germany). After the establishment of regular contractions and monitoring of the control period (30 min), the strips were superfused with the same tyrode buffer containing RU 486 or the compound to be tested, at the same flow rate and temperature. Fractions of the superfused strip effluents (15ml) were collected. T h e samples were immediately stored at - 2 0 ° C . T h e RIA was performed after extraction with Sep Pak C18 columns (Waters Associates, Milford, MA, U.S.A.). T h e columns were washed with about 1 0 m l ethanol 95% (RP Carlo Erba, Milan, Italy) and 10 ml water/0.1% E D T A (Merck Darmstadt, Germany). T h e samples were acidified to p H 4 with citric acid 1 M, labelled with 1500 cpm ( [6, 9, 10, 12, 13, 15, 16] (n)-3H)-6 keto PGFI~ (specific activity 4.8-7.4 TBq/mmole, Amersham, U.K.) to estimate recovery, and then applied to the column; after washing with 0.1% E D T A and 2 m l hexane (RP SDS, France), the sample was eluted with 1 ml absolute ethanol. T h e ethanol was evaporated to dryness with nitrogen and the extract dissolved in 1 ml gelatine buffer (phosphate buffer 1 0 m M p H 7 . 4 , NaC1 9g/1 and
Inhibition of 6-Keto PGF~
gelatine 1 g/l). The RIA was performed with 3H-6keto PGFI~ (Amersham, U.K.) and an antiserum purchased from Biosys, Compi6gne, France, which does not crossreact with the steroids. Crossreactivity towards the other eicosanoids was respectively: PGFt~ 13%, PGE2 and dihydro-15-keto P G F ~ 4%, PGF2~, PGD2 and PGD1 2.8% and others 0.01%. The intraand inter-assay coefficients of variation were 5% and 6%, respectively. The concentration of 6-keto P G ~ displacing 50% of the labelled ligand was 71 pg and the lowest amount detectable was 18 pg. Unbound and bound ligand were separated by absorption on to dextran-charcoal, followed by centrifugation and measurement of the supernatant radioactivity in a liquid scintillation coun~er (Packard 460 CD). EIA was based on 6-keto PGFl~-bound-acetylcholinesterase tracer and quantification of acetylcholinesterase activity with an acetyl-thio-choline substrate. After hydrolysis the thiocholine reacted with Ellman's reagent and the absorbance of the 5-thio-2-nitrobenzoic acid was read at 412 nm. The ILIA was performed directly on the superfusion medium without previous extraction. Kits were purchased from Cayman (Ann Arbor, MI, U.S.A.) and used following the manufacturer instructions. The 50% B/BO was 82.4pg/ml and the threshold of detection was 23.6pg/ml. Crossreactivity was respectively: 2,3dinor-6-keto P G F ~ 8.7%, PGF2~ 2.1%, PGE2
Release by RU 486
0.92%, PGFI~ <0.01%.
65
0.8%
and
other
prostaglandins
Data analysis The data were expressed as mean percentage + SD of control tyrode superfusion values. The 100% value of PGI2 release represents the release rate of 6-keto PGF1, measured for a 30-min period (two samples of 15 ml) at the beginning of the experiment (after the stabilization period) and expressed as pg/min/mg wet weight. Similarly, the frequency, the mean amplitude of the peaks (cm at the setting of the recorder), and their mean duration(s) measured over 30 min after the stabilization period were considered as 100%. Analysis of variance (ANOVA) and Student's t-test for paired samples were used for statistical analysis.
RESULTS
Role of external calcium Effects of decreasing external Ca ++ concentration and of nifedipine on spontaneous contractility and 6-keto PGFI~ release. As expected, decreasing tyrode medium Ca ++ concentration from 1.8 to 0 m M inhibited spontaneous contractility (Fig. 1). However, this inhibition was not accompanied by a significant decrease in 6-keto PGFI~ release, although a great variability of response was observed from one sample to another. [] Frequer~y
200
[]
6 keto I ~ F l a
--v--
I
[
o
7-1 I
,.~
o
s•s
100 m •
s
•
s
•• ••• S••
T• • /
s•s •
, s ,
• s•s
•
•
• s•s
¢ •
T s , % • % •
• •s • ' s .
S•s • , s .
/ s , • % % • % •
•s s • s•s s¢
• s i
s
1,80
s
,
1~25
0,90
0,62
0~1
• • s • sS
0,00
% • / / , / / ,
EDTA
Ca++ mM Fig. 1. Effect o f C a + + c o n c e n t r a t i o n in t h e s u p e r f u s i o n m e d i u m on t h e f r e q u e n c y o f c o n t r a c t i o n s a n d 6 - k e t o P G F t ~ r e l e a s e f r o m h u m a n m y o m e t r i a l strips. T y r o d e m e d i u m at e a c h C a ++ c o n c e n t r a t i o n w a s s u p e r f u s e d for 30 rain. 1 5 - m l f r a c t i o n s w e r e c o l l e c t e d for 6 - k e t o P G F t ~ m e a s u r e m e n t s . T h e d a t a w e r e e x p r e s s e d as a p e r c e n t a g e + S D o f t h e m e a n v a l u e s d e t e r m i n e d d u r i n g t h e s u p e r f u s i o n c o n t r o l p e r i o d in s t a n d a r d t y r o d e m e d i u m (1.80 m M C a + + ) . S t u d e n t ' s t-test: p a i r t e s t e d , e a c h C a ++ c o n c e n t r a t i o n vs c o n t r o l . * P < 0.05, * * * P < 0.001, n = 12.
C. Lobaccaro-Henri et al.
66
~0
5 min I I
+
Fig. 2. Effect o f TMBs (10~ M) s u p e r f u s e d a l o n e a n d in t h e p r e s e n c e o f R U 486 (10 -6 M) on h u m a n m y o m e t r i a l s p o n t a n e o u s c o n t r a c t i o n s . T y r o d e r e f e r e n c e m e d i u m w a s s u p e r f u s e d for a 30-min c o n t r o l p e r i o d , t h e n T M B s a l o n e ( u p p e r trace) or t o g e t h e r w i t h R U 486 ( l o w e r trace) w a s a p p l i e d for 30 m i n m o r e . T h e strips w e r e t h e n w a s h e d with tyrode m e d i u m for I h.
In the presence of 1 m M E D T A , a slight but significant decrease in 6-keto P G F I ~ release, from about 20%, could be observed (P < 0.05). Nifedipine, an inhibitor of Ca ++ channels, was used at the concentration of 10 -8 and 10 - 7 M. It inhibited the spontaneous contractility in a dosedependent m a n n e r as expected, but did not significantly affect 6-keto P G F I ~ release (127 _+ 24%, n=5). Relationship between Ca + + entry, spontaneous contractility and 6-keto PGF1~ release. Superfusion with the ionophore A 23187 did not significantly increase the spontaneous contractility but stimulated 6-keto P G F I ~ production: 1 4 8 _ 20% at a concentration of 1 x 10 - 6 M ( P < 0.05) and 1 8 5 _ 14% a t 5 . 1 0 - 6 M ( P < 0.01) (n = 4). R U 486 (10 -6 M) did not inhibit the effect of A23187 on prostaglandin release (155 ___55%). High concentrations of KC1 in the superfusion m e d i u m induced a long-lasting contraction and stimulated 6-keto P G F I = release: 1 5 4 + 2 5 % at 30 m M and 2 5 2 _ 90% at 60 m M ( P < 0.05) (n = 4). R U 486 had no significant inhibitory action on KC1 effects.
Role of internal calcium Effect of TMB8. T M B s , an intracellular calcium inhibitor, stopped spontaneous contractility as shown in Fig. 2 and simultaneously decreased PGI2 metabolite release. Both effects were reversed by washing for a 30-min period. In the presence of R U 486, T M B s action on the two parameters was stronger and of longer duration (Figs 3A and B). Effect of thapsigargin. Thapsigargin is a marine toxin inhibiting the ER Ca +÷ ATPase. It increases intracellular Ca ÷ ÷ concentration.
Superfusion in the presence of 1 0 - 7 M thapsigargin stimulated only weakly uterine strip contractility, but basal 6-keto P G F I ~ production was increased twofold (Fig. 4). T h e effect of thapsigargin was significantly inhibited by R U 486 at 1 0 - 6 M ( P < 0.01). P L C and PIP2 pathway. T w o inhibitors of P L C , U73122 and neomycin, were used to test the involvem e n t of P L C on prostanoid release. A slight but significant inhibition of 6-keto PGl~ release was obtained in presence of 5 . 1 0 - 6 M U73122 (70 + 19% of control, P < 0.05), but not in presence of 1 0 - 3 M neomycin (Table 1). R U 486 did not amplify the slight decrease observed with P L C inhibitors. Activators of the P K C ( P M A or the di-butyrylated analogue PDBu) and protein C kinase inhibitors (staurosporine and H7) had no significant effect on the 6-keto P G F I ~ production (Table 1). Vasopressin superfusion, an excitatory agonist, induced a dose-dependent increase in contractility, which was accompanied by a significant stimulation of 6-keto P G F I ~ output (259 + 90% of control at 1 0 - 8 M AVP, P < 0.01) (Fig. 5). High levels of prostanoids, significantly different from control, were still noticeable after a washing period of 3 0 m i n (213 + 50%, P < 0.001), indicating that intracellular mechanisms were activated by AVP and remained effective some time after washing out the agonist. Superfusion of AVP together with R U 486 did not significantly decrease 6-keto P G F I ~ release when c o m p a r e d to AVP alone. However, we observed that the return to basal levels after washing occurred faster (30 min) in the presence of R U 486 (Fig. 5). This could m e a n that R U 486 inhibition of internal Ca ++ release was u n m a s k e d when AVP was washed out, and that the steroid could counteract some of the agonist effects on intracellular Ca + ÷ levels.
Inhibition of 6-Keto PGF~. Release by RU 486
67
200" Tyrode Tymde % , . ~ s % . . % , . %,
o
TMB8 10-6 M
o
A
100 %¢%,.%s%s%~ ',*sss
o
,*,*ss,
~ s , * s /
, / / s , * , s , * s /
%%%%~
0 15
30
45
60
75
90
Time (min)
200 Tyrode (TMB8 + RU) 10-6 M
Tyrode c o
1.00
F
r
r
r
r
,
, s / I / , ~ss,*s,
B
¢ ~ ¢ ¢ ¢ , ¢ ¢ ¢ ¢ . *
%%%%%
ts,*~s
, % ~ % % ~
0 15
30
45
60
75
90
Time (min) Fig. 3. S y n e r g i c effect o f T M B s a n d R U 486 s u p e r f u s i o n at 10 4 M c o n c e n t r a t i o n , on 6-keto P G F I ~ r e l e a s e f r o m h u m a n m y o m e t r i a l strips. T y r o d e m e d i u m w a s s u p e r f u s e d for 30 m i n , t h e n T M B s a l o n e (A) o r t o g e t h e r w i t h R U 486 (B) w a s a p p l i e d for 30 m i n a n d t h e t i s s u e s w e r e finally w a s h e d w i t h t y r o d e m e d i u m for a n o t h e r 30 rain. T h e r e l e a s e rate w a s m e a s u r e d in t w o s a m p l e s o f l $ - m l f r a c t i o n s a n d t h e d a t a w e r e e x p r e s s e d as a percentage_ SD of the mean values measured during the tyrode medium superfusion control period. S t u d e n t ' s t-test: p a i r t e s t e d , T M B s vs c o n t r o l a n d T M B s + R U 486 vs c o n t r o l . * * P < 0.01, * * * P < 0.001, n = 8.
Activation of G proteins by ALF~a. ALI~4, a potent activator of G proteins, induced very powerful and long-lasting contractile activity (not shown) together with a strong stimulation of 6-keto PGF1, release
(Fig. 6). Both effects were not significantly opposed by R U 486. W h e n superfused in Ca + ÷ free medium ALF~4 stimulatory effect on the prostanoid production was inhibited by 80%.
C. Lobaccaro-Henri et al.
68
200
[] Thaps 10-7M [ ] Thaps+RU 10-6M •.
o r~
% ,,
.d
' , s s i
++
o
r
.
r
100 s s ~ s s s
2
~ss¢
q~
I"q
Basal
Test
Washing
Fig. 4. Effect of thapsigarglne superfused alone (10 -7 M) or together with RU 486 (10~SM) on 6-keto PGFI~ release from human myometrial strips. Tyrode medium was superfused for 30 min, then thapsigargine alone, or together with RU 486 was applied for 30 min and the tissues were finally washed with tyrode medium for another 30 min. The release rate was measured in two samples of 15-ml fractions and the data were expressed as a percentage _+SD of the mean values measured during the tyrode medium superfusion control period. Student's t-test: pair tested, thapsigargine vs control. ***P < 0.001. Thapsigargin vs T h a p s . + R U 486. + +P<0.01, n = 5. STUDY OF THE SPECIFICITY OF ACTION OF llfl-ARYL SUBSTITUTED COMPOUNDS A series o f p r o g e s t o g e n or corticosteroid antagonists structurally related to R U 4 8 6 were studied to determ i n e the influence o f several substitutions on 6-keto P G F I ~ release f r o m superfused uterine strips. As s h o w n in T a b l e 2, the 17ct p o s i t i o n o f the hydrophobic substituent was m o r e i m p o r t a n t to confer an inhibitory activity than its degree o f unsaturation or its length: Z K 9 8 7 3 4 (17ct-hydroxy-propenyl) was inhibitory, like R U 4 8 6 (17~-propynyl), but Z K 9 8 2 9 9 (17B-hydroxy-propyl) had no effect. H o w e v e r , the analogue Z K 9 8 2 9 9 was also e p i m e r i c at C - 1 3 and presents a very different m o l e c u l a r shape ( C / D ring cis junction) w h i c h c o u l d be d e t e r m i n a n t in the lack o f effect. O n the other hand, the 17ct-propynyl analogues, Z K 1 1 2 9 9 9 and R U 4 0 2 2 6 , carrying a different substituent o n the 11B aryl ring, w e r e b o t h able to inhibit 6-keto P G F I ~ p r o d u c t i o n (Table 3). T h i s s h o w e d that the r e p l a c e m e n t o f the d i m e t h y l a m i n o
group in position para o f the phenyl ring by a d i m e t h y l or m e t h o x y radical did n o t alter the capacity to inhibit 6-keto P G F ~ output. H o w e v e r , this capacity was lost w h e n the aryl ring was suppressed or w h e n it was replaced by a m o r e bulky bicyclic substituent.
DISCUSSION T h e control o f PGI2 synthesis (and release) in s m o o t h m u s c l e and in vascular tissues has b e e n the subject o f extensive investigations (for review see reference [20]). C o n t r a c t i o n s i n d u c e d by agonists and prostanoid release are m e d i a t e d through c o m m o n Ca + + d e p e n d e n t m e c h a n i s m s . T h e increase in intracellular Ca + + can be achieved t w o ways: by the o p e n i n g o f Ca + + channels or by intracellular Ca + + m o b i l i z a t i o n f r o m e n d o p l a s m i c reticulum. U s u a l l y b o t h m e c h a n i s m s are involved: intracellular Ca + + increase f o l l o w i n g PIP2 hydrolysis and IP3 formation
Table 1. 6-keto PGFI~ releasefrom human uterine strips supe~used in the presence of various effectors of the PLC-PIP2 pathway PLC inhibitors
PKC activators
Kinase inhibitors
U 73122 5 X 10 -6 M
Neomycin
TPA 10 -6 M
PDBU 10 -6 M
Staurosporine
H7
10 -3 M
10 -6 M
10 -6 M
70___ 19 p < 0.05 n=5
93 __. 16 n s n=5
126 ___ 19 n s n=3
93 ___ 16 n s n=3
117___ 17 n s n=3
120___ 10 n s n=3
Data are expressed as mean percentage _+ SD of control tyrode superfusion values, ns: non-significant vs. control; n: number of experiments. For more details, see Materials and Methods.
Inhibition o f 6 - K e t o P G F ~
Release by R U 4 8 6
69
~AVP
400
[] AVP +RU 10-6M o + o
300
o ,¢¢i ee
200 ,%
o
,.
,~
ns
,~/'sI '¢¢1 , ¢ ~1
q~
.'¢~sI
100 , ' / ~q
• ~' s I
x x '//I
0 Basal
AVP 10-8M
AVP 10-7M
Washing
Fig. 5. Effect o f v a s o p r e s s i n superfused alone (10 -s M and 10-7 M), or together with RU 486 (10-6 M), on 6-keto PGFxffi release from h u m a n m y o m e t r i a l strip. Tyrode m e d i u m was superfused for 30 m i n , then vasopressin w a s applied for 30 m i n at each concentration, alone or together with RU 486 and the tissues were finally washed with tyrode m e d i u m for 30 rain. The release rate was m e a s u r e d in two samples o f 15-mi fractions and the data were e x p r e s s e d as a percentage _ S D o f the m e a n values m e a s u r e d during the tyrode m e d i u m superfusion control period. Student's t-test. Pair tested: vasopressine vs control and vasopressine + RU 486 vs control. * * * P < 0.001, * * P < 0.01, ns, non-significant. Washing after A V P superfuslon vs washing after A V P + RU 486 superfusion. +P < 0.05. N u m b e r o f experiments were six (in duplicate) for A V P and four (in duplicate) for A V P + RU 486. leads to the opening of Ca ++ channels and prostaglandin release. However, the relative importance of each mechanism depends on the cell type and the a g o n i s t u s e d . I n m a n y s m o o t h m u s c l e c e l l s s u c h as
trachea, urinary bladder or v e s s e l s , extracellular C a + + e n t r y s t i m u l a t e s P G I 2 s y n t h e s i s . I n t h e s e tissues, calcium channel blockers and removal of extracellular Ca ++ with chelators inhibits 6-keto PGFx~
500
[]
A1F4- 1 0 m M
[]
ALF4-+RU 486
40'0
TT
8 o
[,T., c~
300
*¢s */s ,s/
200
q~
/
%¢~¢,
100
r ~ S,
Basal
Test
Washing
Fig. 6. Effect o f AIF~4 (10 raM) superfused alone or together with RU 486 (10 -6 M) on 6-keto PGFI~ release from h u m a n m y o m e t r l a l strips. Tyrode m e d i u m was superfused for 30 rain, then ~ alone, or together with RU 486 was applied for 30 m i n and the tissues were finally washed with tyrode m e d i u m for another 30 rain. The release rate w a s m e a s u r e d in two samples o f 15-ml fractions and the data were expressed as a percentage +_ S D o f the m e a n values m e a s u r e d during the tyrode m e d i u m superfusion control period. Student's t-test: pair tested, AIF~4 vs control and AIF~4 + RU 486 vs control. * * * P < 0.001, * * P < 0.01, * P < 0.05, ns, non-significant. N u m b e r o f experiments were six (in duplicate) for ALI~4 and four (in duplicate) for ALF~4 + RU 486.
70
C. Lobaccaro-Henri et al.
Table 2. Inhibition
of 6-keto PGFI,
myometrial strips by RU 486 analogues d@erently
release from supefised
substituted at position
C13 and Cl7 of the steroid skeleton
Structure
Product
6-keto PGF1, release (% of control)
7t
RU 38486
48 + 12**’
8
RU 38486
48 + 12***
8
ZK 98734
65 f 17”
6
ZK 98734
65 f
6
ZK 98299
113+23 ns
6
ZK 98299
OH -- CH=CH-CH2OH
17**
OH (3I2+?H2-CH2OH
113f23ns
6
Data are expressed as mean percentage f SD of control tyrode superfusion values. Student’s t-test for paired samples. **PC 0.01, ***P < 0.001; ns: non-significant; n: number of experiments. release by 60%, indicating that calcium receptor-operated channels (ROC) are involved [2 l-241. The ionophore A23187 is able to stimulate PG synthesis and its effect is also inhibited by free calcium media and Ca+ + channel blockers [22, 23, 251. High K concentrations do not stimulate 6-keto PGFi, release in the vascular tissues [21, 221 nor in the guinea pig myometrium [25]. However, reports from the literature indicate that intracellular Ca+ + mobilization is an important pathway in vascular cells: in bovine vascular endothelial cells, 50% of the basal 6-keto PGFr,, production and 80-100% of the thrombin or A23187 stimulated production were inhibited in presence of the [Ca’ + ]i antagonist TMBs [26-281. In the human uterus, pacemaker cells initiate spontaneous contractility [ 161. This mechanical activity Table 3. Inhibition
of 6-keto PGF,,
release from supe&sed
depends on Ca+ + entry through voltage-operated channels (VOC). We showed that 6-keto PGFi, release was not dependent on spontaneous contractility because blocking contractions, by depriving the cells of external Ca+ + or by nifedipine, did not significantly decrease that release. A23 187, although showing only a very slight stimulatory action on the contractility, had a marked effect on 6-keto PGFi, release. A23187 activity was lost in the absence of Ca+ + in the superfusion medium. In contrast to other reports from the literature indicating that high KC1 concentrations did not influence PGIs release from vascular tissues [21, 22, 291 or from superfused guinea pig uterus [30], we observed in our experiments a significant stimulation of 6-keto PGFr, production when superfusing KC1 30 and 60 mM. These
myometrial
strips by RU 486 analogues
d$terently substituted at position
Cl1 of the steroid skeleton
Structure
Product
6-keto PGF1, release (% of control)
n
RU 38486
48 f 12 ***
8
RU38486
48 f 12***
8
RU40226
66 + 11’S
6
RU40226
66k 11**
6
ZK112993
68 f 12 ***
6
ZK112993
68 f 12***
6
(-)ec+-J-c,,
RU43780
lOlf23
11s
4
RU43780
101 + 23 ns
4
lc,,
RU40540
122f26
ns
6
RU40540
122 f 26 ns
6
Lc,,
RU42764
113f13
ns
5
RU42764
113 _+13 ns
5
tQ_C,,
Data are expressed as mean percentage SD of control tyrode superfusion values. Student’s t-test for paired samples. **I’< 0.01, ***PC 0.001; ns: non-significant; n: number of experiments.
Inhibition of 6-Keto PGFI~ Release by RU 486 last two results indicate that in h u m a n uterus, an increase of 6-keto P G F I ~ release above basal levels can be mediated through Ca ++ entry by V O C and independently of contractions. O u r data strongly suggest that increased intracellular Ca+ + concentration is the major event controlling basal P G synthesis. This is clearly shown by the inhibitory action of T M B s and the stimulating effect of thapsigargin. This drug was reported to increase the level of intracellular Ca ++ by inhibiting the E R Ca ++ ATPase. Interestingly R U 486 seemed to antagonize the toxin action and to potentiate the effect of T M B s . W h e t h e r the steroid inhibits the E R Ca + + p u m p or increases Ca ++ binding to the internal m e m b r a n e s remains to be established. Spontaneous 6-keto ]?GFI~ release did not depend on PIP2 hydrolysis as shown by the marginal effect of P L C inhibitors and the lack of effect of P K C effectors. Neither P K C stimulation by P M A or its dibutyrylated analogue nor protein kinase inhibitors such as staurosporine or H 7 modified 6-keto P G F ~ release, which is in contrast to the data obtained by other groups in different tissues [30-33]. Although these inhibitors are not strictly specific to P K C , their lack of effect rules out P K C phosphorylation. Interestingly, R U 486 did not increase the slight inhibitory effect of U73122, indicating that the E R Ca + ÷ A T P a s e which is inhibited by thapsigargin and which m a y be the potential target for R U 486 action did not regulate the same intracellular Ca ++ pool as IP3. Vasopressin, an excitatory agonist m o r e active than oxytocin on n o n - p r e g n a n t uterus and known to exert its activity through the PIP2 pathway [34], was used to further estimate the role of IP3 dependent Ca *+ mobilization in our model. We showed that this agonist stimulated b o t h uterine contractility and PGI2 metabolite production and that R U 486 did not significantly counteract its action. These results suggest that the steroid inhibition of internal C a * + mobilization was not sufficient to oppose vasopressin effects. It is known, for instance, that excitatory agonists trigger Ca ++ entry through R O C , thus inducing a sustained contractility and prostaglandin release [20]. Similarly, we did not observe a significant inhibition of A L F 4 stimulatory action by R U 486. This substance is a known activator of various G proteins. It induced a marked activation of contractions and P G release in our experiments. T h e fact that 80% of its effect was inhibited in the absence of external Ca + ÷ indicated a G-protein-activated Ca + * channel as the possible AlF44 target. T h e lack of significant inhibitory effect of R U 486 on agonist-induced and ALF4induced P G synthesis is in agreement with a sequestering action on a specific Ca ++ intraceUular store and no action, or a marginal one, on external Ca ÷ * entry. An inhibitory action of R U 486 on intracellular
71
Ca + + levels was recently reported in h u m a n spermatozoa [35]. R U 486 action on Ca ++ internal sequestration is related to its specific structure and not to its h o r m o nal potential as discussed in a previous p a p e r [1] and as indicated by the structure-activity relationship study with analogues. Our data showed that the 11Baryl substitution was of prime importance to the inhibitory action on 6-keto P G F I ~ release: replacing the aromatic cycle by an ethylic or ethylenic radicals suppressed the activity. Otherwise, different hydrophobic substituent (propenyl or propynyl) could be admitted at the 17ct position. However, a complete lack of effect accompanied the epimerization at position 17 and 13: the 17ct-hydroxy-17B-3-hydroxypropyl)-13ctmethyl c o m p o u n d , Z K 98299, was not active. We cannot determine, with the few Z K c o m p o u n d s tested, which modification (13~-methyl or 17i3-substituent) caused the inactivity. T h e s e data on structureactivity relationship confirm that R U 486 action on 6keto P G F t ~ release did not depend on its antiprogestational capacity because Z K 98299, which is also an antiprogestogen [36], was not inhibitory. T h e same conclusion is also supported by the report of de N e e f on the hormonal potential o f the 1 l~t-methylsteroids [37]. In s u m m a r y , our data indicate that R U 486 inhibitory action on 6-keto P G F I ~ release is mediated by its blocking action on intracellular Ca ++ mobilization and that this effect was not related to its antihormonal potential. Steroid h o r m o n e s can show non-genomic m e m b r a n e effects, mediated either by m e m b r a n e receptors or by action on intracellular c o m m u n i c a t i o n pathways [38, 39]. R U 486 could act on intracellular Ca ++ mobilization: it increased T M B 8 inhibitory action and antagonized thapsigargin effect. R U 486 target could be the E R Ca ++ A T P a s e p u m p , an E R Ca ++ -binding protein or a small G T P - d e p e n d e n t protein [40, 41]. T h e s e effects of R U 486 indicating a relaxant action seemed very surprising and paradoxical in regard to its well-known antiprogesterone p r o p erties. R U 486 administration to pregnant w o m e n is able to restore uterine contractility and induce abortion or labour [11, 41]. By lowering intracellular Ca ++ levels, R U 486 would potentially inhibit uterine motility. This action is similar to that of progesterone in rat uterus reported in the literature [6]. However, in these experiments progesterone seemed to act via calcium channels or GABAA receptors [9]. In the h u m a n uterus, progesterone increased the A T P - d e p e n d e n t Ca ++ binding to microsomial preparations [42]. T h e progesterone-like relaxant effect of R U 486 is very surprising, but was also established by Perusquia et al. in the rat uterus [43]. T h e paradoxical action of R U 486 on intracellular Ca ++ could explain the modifications of contractile pattern observed: the higher amplitude and duration of contractions, indicating a stronger contractile force, could
72 be due
C. Lobaccaro-Henri et aL to a d e c r e a s e in t h e r e l a x a n t p r o s t a g l a n d i n
PGI2, whereas the concomitant lower frequency could depend
on
intracellular
Ca ++
sequestering.
These
surprising results, given the clinical use o f R U 486 in abortion
and
the induction
of labour,
may
explain
why administration of prostaglandins with the steroid i m p r o v e s the clinical o u t c o m e .
REFERENCES 1. Lobaccaro-Henri C., Saintot M., Laffargue F., Zahradnik H. P., Descomps B. and Thaler-Dao H.: Effect of the progesterone antagonist RU 486 on human myometrial spontaneous contractility and PGI2 release. Prostaglandins 44 (1992) 443455. 2. Fuchs A. R. and Fuchs F.: Endocrinology of human parturition: a review. Br. J. Obstet. Gynaecol. 91 (1984) 948-967. 3. Casey L. M. and MacDonald P., Endocrinology of pregnancy and parturition. In Uterine Function. Molecular and Cellular Aspects, ed. M. E. Carsten and J. D. Miller. Plenum Press, NY, 1989, pp. 501-517. 4. Batra S. and Bengtsson B.: Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. J. Physiol. 276 (1978) 329-342. 5. Batra S. and Sj6gren C.: Effect of oestradiol treatment on calcium uptake by the rat uterine smooth muscle. Life Sciences 32 (1983) 315-319. 6. Kubli-Garfias C., Medrano-Conde L., Beyer C. and Bondani A.: In vitro inhibition of rat uterine contractility induced by 5ct and 513 progestins. Steroids 34 (1979) 609-615. 7. Perusquia M., Garcia-Yanez E., Ibanez R. and Kubli-Garfias C.: Nongenomic mechanism of action of a - 4 and 5-reduced androgens and progestins on the contractility of the isolated rat myometrium. Life Sciences 47 (1990) 1547-1553. 8. Gutierrez M., Martinez V., Cantabrana B. and Hildago A.: Genomic and non-genomic effects of steroidal drugs on smooth muscle contraction in vitro. Life Sciences 55 (1994) 437-443. 9. Pumam C. D., Brann D. W., Kolbeck R. C. and Mahesh V. B.: Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems. Biol. Reprod. 45 (1991) 266-272. 10. Csapo A. L.: Progesterone block. Am. J. Anat. 98 (1956) 273291. 11. Baulieu E. E., RU 486--an antiprogestin steroid with contragestive activity in women. In The Antiprogestin Steroid R U 486 and Human Fertility Control, ed. E. E. Baulieu and S. J. Segal. Plenum Press, NY, 1985, pp. 1-25. 12. Kelly R. W. and Smith S. K.: Progesterone and antiprogestins. A comparison of their effects on prostaglandin production by human secretory phase endometrium and decidua. Prostagland. Leuk. Med. 29 (1987) 181-190. 13. Abel M. H. and Kelly R. W.: Differential production of prostaglandins within the human uterus. Prostaglandins 18 (1979) 821-828. 14. Omini C., Pasargikilian R., Folco G. C., Fano M. and Berti F.: Pharmacological activity of PGI2 and its metabolite 6-oxoPGFI~. Prostaglandins 15 (1978) 1045-1053. 15. Carsten M. E. and Miller J. D., Calcium control mechanism in the myometrial cell and the role of the phosphoinositide cycle. In Uterine Function. Molecular and Cellular Aspects, ed. M. E. Carsten and J. D. Miller. Plenum Press, NY, 1989, pp. 121155. 16. Wray S.: Uterine contraction and physiological mechanisms of modulation. Am. J. Physiol. 264 (1993) C1-C18. 17. Chiou C. Y. and Malagodi M. H.: Studies on the mechanism of action of a new Ca 2 + antagonist -(N,N-diethylamino)octyl 3,4,5-trimethoxy benzoate hydrochloride in smooth and skeletal muscles. Br. J. Pharmacol. 53 (1975) 279-285.
18. Thastrup O., Cullen P. J., Drobak B. K., Hanley M. R. and Dawson A. P.: Thapsigargin, a tumor promotor, discharges intracellular Ca 2 + stores by specific inhibition of the endoplasmic reticulum Ca 2 ÷ ATPase. Proc. Natn. Acad, Sci. U.S.A. 87 (1990) 2466-2470. 19. Zahradnik H. P., Schoening R. and Breckwoldt M., Prostaglandins correlation to human myometrial activity in vitro. In Prostacyclin in Pregnancy, ed. P. J. Lewis. Raven Press, NY, 1983, pp. 147-151. 20. Jeremy J. Y., Mikhailidis D. P. and Dandona P.: Excitatory receptor-prostanoid synthesis coupling in smooth muscle: mediation by calcium, protein kinase C and G proteins. Prostagland. Leuk. Essential Fatty Acids 34 (1988) 215-227. 21. Stewart D., Poumey E. and Fitchett D.: Norepinephrine stimulated vascular prostacyclin synthesis. Receptor dependent calcium channels controls prostaglandin synthesis. Can. J. Physiol. Pharmacol. 62 (1984) 1341-1347. 22. Golub M. S., Berger M. E. and Camazola A. E.: Adrenergic stimulation of prostacyclin production in the rat tail artery. II. The role of calcium. Prostagland. Leuk. Med. 20 (1985) 313324. 23. Van de Velde V. J. S., Van den Bossche R. M., Bult H. and Herman A. G.: Modulation of prostacyclin biosynthesis by calcium entry blockers and extracellular calcium. Biochem. Pharmacol. 35 (1986) 253-256. 24. Jeremy J. Y., Mikhailidis D. P. and Dandona P.: The effect of nifedipine, nimodipine and nisoldipine on agonist- and traumastimulated vascular prostacyclin synthesis in vitro. Arch. Pharmacol. 332 (1986) 70-73. 25. Poyser N. L.: Effect of using calcium-free Krebs solution on basal and A23187-stimulated prostaglandin output from day 15 guinea-pig uterus superfused in vitro. Prostagland. Leuk. Med. 13 (1984) 259-269. 26. Adams Brotherton A. F. and Hoak J. C.: Role of Ca ~ ÷ and cyclic AMP in the regulation of the production of prostacyclin by the vascular endothelium. Proc. Nam. Acad. Sci. U.S.A. 79 (1982) 495-499. 27. Hassid A. and Oudinet J. P.: Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells. Prostaglandins 32 (1986) 457-478. 28. Watanabe K., Lam G. and Jaffe E. A.: The correlation between rises in intracellular calcium and PGI2 production in cultured vascular endothelial cells. Prostagland. Leuk. Essential Fatty Acids 46 (1992) 211-214. 29. Boeynaems J. M. and Ramboer I.: Effects of changes in extraand intracellular K ÷ on the endothelial production of prostacyclin. Br. J. Pharmacol. 98 (1989) 966-972. 30. Poyser N. L.: Effects of various factors on prostaglandin synthesis by the guinea-pig uterus. J. Reprod. Fertil. 81 (1987) 269-276. 31. Jeremy ]. Y. and Dandona P.: The role of the diacylglycerolprotein kinase C system in mediating adrenoreceptor-prostacyclin synthesis coupling in the rat aorta. Eur. J. Pharmacol. 136 (1987) 311-316. 32. Merkel L. A., Rivera L. M., Colussi D. J. and Perrone M. H.: Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation. J. Pharmacol. Exp. Ther. 257 (1991) 134-140. 33. Erbrich A. C., Church D. J., Valotton M. B. and Lang U.: Regulation of prostacyclin production by [Ca 2+ ]i and protein kinase C in aortic smooth muscle cells. Am. ft. Physiol. 263 (1992) E800-E806. 34. Schrey M. P., Conford P. A., Read A. M. and Steer P. J.: A role for phosphoinositide hydrolysis in human uterine smooth muscle during parturition. Am. J. Obstet. Gynecol. 159 (1988) 964-970. 35. Serres C., Yang J. and Jouannet P.: RU 486 and calcium fluxes in human spermatozoa. Biochem. Biophys. Res. Commun. 204 (1994) 1009-1015. 36. Elger W., Beier K., Chwalisz K., F~ihnrich M., Hasan H., Henderson D., Neef G. and Rohde R.: Studies on the mechanisms of action of progesterone antagonists, ft. Steroid Biochem. 25 (1986) 835-845. 37. Neef G., Beier S., Elger W., Henderson D. and Wiechert R.: New steroids with antiprogestational and antiglucocorticoid activities. Steroids 44 (1984) 349-361.
I n h i b i t i o n of 6 - K e t o P G F 1 , Release b y R U 4 8 6 38. Wehling M.: Non genornic actions of steroid hormones. T E M 5 (1994) 347-353. 39. Brann W. D., Hendry L. B. and Mahesh V. B.: Emerging diversities in the mechanism of action of steroid hormones. J. Steroid Biochem. Molec. Biol. 52 (1995) 113-133. 40. Bian J., Ghosh T. K., Wang J. C. and Gill D. L.: Identification of intracellular calcium pools. Selective modification by thapsigargin. J. Biol. Chem. 266 (1991) 8801-8806.
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41. Missiaen L., De Smedt H., Droogmans G., Himpens B. and Casteels R.: Calcium ion homeostasis in smooth muscle. Pharmacol. Ther. 56 (1992) 191-231. 42. Carsten M. E.: Calcium accumulation by human uterine microsomal preparations: effects of progesterone and oxytocin. Am. 37. Obstet. Gynecol. 133 (1979) 598-601. 43. Perusquia M. and Kubli-Garfias C.: Progesterone-like relaxant effect of RU 486 in the rat myometrium. Life Sciences 54 (1994) 1501-1506.