Prostaglandins
44:443-455,
1992
EFFECT OF THE PROGESTERONE ANTAGONIST RU486 ON HUMAN MYOMETRIAL SPONTANEOUS CONTRACTILITY ANU PG12 RELEASE C. Lobaccaro-Henri, M. Saintot, P. Laffazgue+ H.P. Bahradnik**, B. Descomps and H. Thaler-Dao Unite 58 I.N.S.E.JZ.M., 60 rue de navacellee, 34090 Montpellier and ,T$e Matemite, C.H.R. Universite Universitats Frauenklinik, Freiburg, Montpellier I France. Fm Briagau, Gemany
Abstract We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI2 release with human myometrial strips superfused “in vitro”. A decrease of PGIz release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dosedependent and reversible. Modifications of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as lo-9M, dexamethasone (IO-*M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contracti!ity, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 0x0 pregnene structure.
Introduction In many animal species progesterone inhibits uterine contractility, especially during pregnancy (l), and parturition is initiated by progesterone withdrawal (2,3). There is no definitive evidence of any progesterone block in humans. However, the efficacy of orally administered micronised progesterone in preventing premature labor (41, and the success of antiprogesterone RU 486 in inducing parturition (5), is evidence in favour of such a role. The mechanism by which this blockade could function is generally unknown, but decrease of oxytocin and Copyright 0 1992 Butterworth-Heinemann
444
Prostaglandins
adrenergic receptor concentration (6,7) and inhibition of gap junction formation (8) have been suggested. Since prostaglandins (PGs) are very effective contractile agents which have been used for more than 20 years to induce labor and abortion (9-ll), and since high levels of PGs, are synthesised during labor (12,13), it could be speculated that among other effects, progesterone blocks prostaglandin synthesis. Evidence reported in the literature is contradictory. Progesterone has been found to exert a “priming” effect on oestradiol-stimulated endometrial PGF2, synthesis in ovariectomized animals (14). In humans, endometrial PGF2e levels are higher during the secretory phase than during the proliferative phase (15). On the other hand, very low PG levels have been found in the decidua when ovarian progesterone production is high (16), and progesterone reportedly inhibits PG synthesis in endometrial explants (17,18). Recent observations with human endometrial (19) and rat myometrial (20) explants show that progesterone inhibits PG synthesis and that antiprogesterone reverses this effect. In the present study we used a superfusion model of human myometrial strips and antiprogestin RU 486, to investigate whether spontaneous prostacyclin release (the major myometrial PG) is modified by this steroid, and whether the changes are correlated with modifications of the spontaneous contractile activity pattern. Our study was voluntarily restricted to the release of PGI2, this PG being the major cycle-oxygenase metabolite within the myometrium ; PGF2, and PGE;! production in the superfusion medium was below the limit of our RIA sensitivity.
Materials and Methods The micronised progesterone antagonist RU 486, mifepristone (178 hydroxy-118 (4-dimethylaminophenyl-l)-17a (I-propynyl)-4,9-oestradien-3one and progesterone agonist R5020, promegestone (17,21-dimethyl-19nor-pregn-4,9-diene-3,20-dione) were gifts from Roussel-Uclaf, Romainville, France. The steroids were dissolved in ethanol at concentrations of lo-2M to lo-5M and stored at -2O“C. Before use they were diluted to the appropriate concentrations in tyrode medium (NaCl 137 mM, KC1 2.6 mM, CaC12 1.8 mM, MgC12 1.05 mM, NaHC03 11.9 mM, NaH2P04 0.42 mM, glucose 5.55 mM, pH 7.4). Uterine corpus specimens (medium part) were obtained from cyclic premenopausal women following hysterectomy for fibromatosis or prolapsus. Institutional approval to use human tissue was obtained. The samples were immediately transferred into an ice cold tyrode solution and kept at 4°C. Upon arrival at the laboratory, the connective tissue was carefully removed and the myometrium dissected in strips parallel to the muscle fiber bundles. Prepared strips were approximately 20mm long, 5mm wide and 5mm thick. They were tied at each end with a silk thread and suspended in a superfusion chamber as described by Zahradnik et al. (21). The strips were continuously superfused with oxygenated tyrode solution at a flow rate of 1 ml/min at 37’C. Isotonic registration of strip contractions was performed by means of a 8368 transducer connected to a
Prostaglandins
445
two channel amplifier (Hugo Sachs Elektronik, March Hegstetten, Germany). The strips were allowed to equilibrate for at least 2 h. After the establishment of regular contractions and monitoring of the control period (one hour), the strips were superfused with the same tyrode buffer containing RU 486 or steroid hormones at the same flow rate and temperature. In dose-response experiments for a given steroid, the same strip was successively superfused at each concentration for a period of one hour. The final concentration of RLJ 486 varied from lo-9M to lo-6M. The contraction frequency, amplitude and duration were registered over the same period of one hour for each concentration of a given product. At the end of the experiment the strip was superfused with tyrode medium, to check the return to the initial parameters of contractility and to PGI2 initial release rates. This was considered as a validation of the tissue viability for the period of observation. Fractions of the superfused strip effluents (5 ml) were collected during monitoring of the contractile activity. In time course study (fig.1) 6 ketoPGFla concentration (the stable metabolite of PGI2) was assayed on 5 ml samples (5 min). In dose-response studies, it was measured on 15 ml samples, (3 samples for each concentration of a given steroid). The samples were immediately stored at -20°C. The RIA was performed after extraction with Sep Pak Cl8 columns (Waters Associates, Milford Mass.). The columns were washed with about 10 ml ethanol 95 % (RF Carlo Erba, Milan, Italy) and 10 ml water/O.1 % EDTA (Merck Darmstadt, West Germany). The samples were acidified to pH 4 with citric acid 1 M, labelled with 1500 cpm (5, 6, 8, 9, 11, 12, 14, 15) 3H-6 keto PGFla (specific activity 5661 GBq/mmole, NEN Boston, Ma, USA) to estimate recovery, and then applied to the column ; after washing with 0.1 % EDTA and 2 ml hexane (RP SDS France), the sample was eluted with 1 ml absolute ethanol. The ethanol was evaporated to dryness with nitrogen and the extract dissolved in 1 ml gelatine buffer (phosphate buffer 10 mM pH 7.4, NaCl 9 g/l and gelatine 1 g/l). The RIA was performed with an antiserum purchased from Biosys, Compiegne, France, which does not cross- react with the steroids. Cross reactivity towards the other eicosanoids was respectively : PGFla 13 %, PGE;! and dihydro-15 keto PGFla 4 %, PGFza, PGD2 and PGDl 2,8 % and others 0.01 %. The intra- and inter-assay coefficients of variation were 5 % and 6 % respectively. The concentration of PG12 displacing 50 % of the labelled ligand was 30 pg and the lowest amount detectable was 4 pg. The data were expressed as mean percentage f SEM of control tyrode superfusion values. The 100 % value of PGI2release, represents the release rate of 6 keto PGF1, measured for a lh period (3 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 recoder) and their mean duration (set) measured over a lh period after the stabilization period, were considered as 100 %. The same calculations during one hour superfusion, at each steroid
446
percentage of or stimulation. data were f SEM control tyrode values at experiments and by Student’s paired test concentration).
Prostaglandins
as mean beginning of vs each
Results The group of patients studied ranged from 40 to 52 years of age. All the strips were obtained from myometrium of cyclic women in the pre or perimenopausal period. The levels of basal 6 keto PGFl, release measured after the stabilization period (and considered as the 100 % control values) ranged from 0.3 to 3.0 pg/min/mg wet weight in agreement with previously reported data (22). The contractile activity tracings showed regular peaks occurring with a mean frequency (& SEM) of 20.2 f 10.5 (n = 16). The mean amplitude (height of the peaks measured on the recorder in our monitoring conditions) was 13.7 + 6.2 cm, and the mean duration of the contractions was 49 + 16 sec. There was no correlation between age or hormonal status (for instance, treatment with progesterone before hysterectomy) and the contractile parameters. When the myometrium samples were superfused with tyrode medium containing increasing concentrations of RU 486, a dose-dependent inhibition of PG12 release was observed. The time course of 6 keto PGFla output inhibition is shown in Fig. la. Inhibition occurred with a short delay of about 20 min after each change of RU 486 concentration. The inhibition was roughly 50 % at 10-7M and 80 % at lo-6M. This was reversed after a brief washing period of 20 min. The recovery after washing was 80 % of the initial control value. The dose-response studies (Fig. lb) shows that at lo-7M the 6 keto PGFl, release was 52,5 f 10 % of the control and at lo-6M it was 20 + 7 (n = 10). The difference from control was significant at IO-8M (p c 0.001) and the inhibition was dose dependent. The simultaneous recordings of the mechanical activity show that the decrease of 6 keto PGFla release was concommittant with a modification of uterine motility : inhibition of contraction frequency, increase of the amplitude and duration of the peaks (Fig. 2a and 2b). The effects on frequency were dose dependent and already significant at 10-8M (p c O.OOl), those on amplitude and duration were significant at lo-9M (p < 0.001) but reached a plateau at 10m7M.
447
Prostaglandins
a
50
100
Time (min)
b
0
9 8 7 6 - log RU 486 (M)
0
Fig. 1 : Effect of increasing concentrations of RU 486 on 6 keto PGFr, release from superfused human myometrial strips a- Time course effect : One myometrial strip was successively superfused with increasing RU 486 concentrations, for periods of 20 min. ; then the tissue was washed with tyrode medium for another 20 min. 6 keto PGFI, concentrations were measured on 5 ml fractions (5 min). The control value (100 %) at t = 0 was, in this case, 3 pg * O.O8/min/mg wet weight. b- Dose response efiecf : Each concentration was successively superfused for one hour. The release rate of 6 keto PGFl. was measured in 4 samples of 15 ml medium collected during this time and the mean value was expressed as percentage of the mean release rate during tyrode superfusion without steroid. For more details see text in the Materials and Methods section. Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, n = 10.
448
Prostaglandins
NS
100
0
9 8 7 6 - log RU 486 (M :>
0 0 0
Amplitude Duration
b
9 8 6 - log RU 486 (M)
0
Fig. 2 : Dose-response effect of increasing concentrations of RU 486 on the spontaneous contractile activity of superfused myometrial strips. The conditions of the superfusion are the same as in the Fig. lb. The data are expressed as percentage of the same parameters measured during tyrode medium superfusion. For more details see text in the Materials and Methods section. a- Frequency. b Amplitude q and Duration q Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, n = 10.
449
Prostaglandins
The progesterone analogue R5020, at concentrations ranging from 10-g to lo-6M, did not significantly inhibit 6 keto PGFla release (not shown), but had significant effects on the contractility at very low concentrations (Fig. 3a and 3b) : the frequency dropped to 41+ 7 % of control at IO-9M and the duration of the contractions increased to 248 + 5 %. The effect on the amplitude was less striking. In contrast to RU 486 and progesterone we did not observe any dose-response effect.
100
80 ,”
@ 1”3 gs
60
‘“e
20
*** ***
a
40
n 0
9
8
7
6
0
-log R5020 (M) Cl Amplitude Duration
b
8 7 6 -log R5020 (M)
0
Fig. 3 : Effect of a progesterone analogue R 5020 on the spontaneous contractile activity of superfused human myometrial strips. The conditions and expression of data are the same as in Fig. 2. For more details see text in the Materials and Methods section. a- Frequency. b- Amplitude 17 and Duration q Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, **p < O,Ol, n = 4.
Prostaglandins
450
Similarly, dexamethasone had no effect on 6 keto PGFla release (results not shown) but inhibited significantly uterine frequency. The amplitude and duration of contractions were also significantly altered (Fig.4a and 4b). Testosterone had no effect on the 6 keto PGFla release nor on the contractility, even at 10&M ( not shown).
0
8
7
- log Dex (M) 0
Amplitude Duration
b
8 -
7
log Dex (M)
Fig. 4 : Effects dexamethasone on the spontaneous contractile activity of superfused human myometrial strips. The conditions and expression of data are the same as in Fig. 2. For more details see text in the Materials and Methods section. a- Frequency. b- Amplitude 0 and Duration q Student’s t test : pair tested, control vs. each steroid concentration. *** p < 0.001, n = 9.
Prostaglandins
451
Discussion PGI2 (measured as 6 keto PGFla) is the major PG released by the myometrium (23). The release of this metabolite, probably as a result of PG synthesis activation (24), was very high just after handling of the strips.The rate of production then declined rapidly to a basic level as previously observed by several other groups (25,26). Our experiments were carried out after a 2 h resting period, when PG release was stable and when spontaneous uterine contractions were regular. RU 486 inhibited dose-dependently the spontaneous release of prostacycline by the superfused myometrial strips. The onset of this effect was rapid (about 20 min), was maintained as long as antiprogestin was superfused and was rapidly reversed (20 min) by tyrode medium washing. In our model, RU 486 was the only steroid to show a simultaneous effect on 6 keto PGFlcx release and uterine contractility. All the steroids tested in this study, except testosterone, affected uterine contractility without affecting the prostanoid release. Therefore, the simultaneous effects of RU 486 on PGI2 output and uterine contractility (although concommittant) may be independant and mediated by different mechanisms. Data of the literature indicate that other steroids act on spontaneous uterine contractility : oestradiol (27) and C21 steroids such as 5a and 5p pregnane compounds (28, 29) Therefore RU 486 effect on this parameter could be due to its ~4 C21 steroid structure and targeted on a membrane component such as a calcium channel (28) rather than a steroid receptor. It is also known that cortisone (30) and several progesterone metabolites are potent antagonists of GABA receptors (31). Therefore it should be of interest to carry out further investigations on structure-activity relationship of antiprogestins and C21 steroids. By contrast, the RU 486 effect on 6 keto PGFlu release is very specific of this molecule since it is not mimicked by other steroids. However, the fact that neither the progesterone analog R5020, (a progesterone agonist which was used in preference to progesterone because it was not metabolized in situ), nor the glucocorticoid effector dexamethazone, had any decreasing effect on 6 keto PGFI, release indicate that RU 486 action is not mediated by the progestin or the glucocorticoid receptor. Moreover, the short time course and the rapid reversibility of the effect do not support a classical steroid effect. It seems from our data that RU 486 inhibitory effect on PGI2 release is not related to its antihormonal activity. The lack of effect of the other steroids on PGI2 release in our data, differs from recently published observations in isolated human amnion cells : in this report (32), dexamethasone treatment (IO-8M) for period as short as 20 min, inhibited basal arachidonic acid release from these cells. This effect was not blocked by protein or RNA synthesis inhibitors, and was mimicked by RU 486 and all steroids tested, androgens, corticosteroids and progesterone. However these results were obtained with dispersed cells of a different tissue and concern arachidonic acid release and not PG release. As mentioned above, a striking point in our results concerns the very short lag period of the steroid effects. Studies on the regulation of uterine
452
Prostaglandins
PG synthesis by steroids are usually carried out using “in vivo” administration of hormones. In previous studies experiments on the “in vitro” control of prostaglandin synthesis by progesterone and antiprogestins, an incubation period of 18 h was chosen for rat myometrial explants (ZO), and 2 or 3 days for human endometrial explants (19). In the present study, the short time course of steroid activity, the lack of specificity of steroids in modifying the spontaneous uterine contractile activity and the reversibility of the action suggest the involvement of a membrane “receptor” site, such as ion channels (31) second messenger transmitters (32), phospholipases (33) or even of cycle oxygenase (34). Other non genomic effects were reported in the literature for estrogens and antiestrogens (35). Further investigations need to be carried out to determine whether RU 486 affect prostacyline release by acting on a membrane phospholipase or directly on the prostaglandin synthase or by another mechanism. We report that RU 486 decreased uterine contraction frequency while increasing simultaneously the amplitude and duration of contractions. Such mechanical activity changes could be considered as leading to a higher efficiency of contractions similar to that observed in labour. However, extrapolation of the present data to the pharmacological activity of RU 486 on pregnant myometrium should be very careful. At least 2 limits should be considered : firstly our data refer to an “in vitro” model and secondly they derived from observations on spontaneous contractions of non pregnant myometrium. It is quite probable that the regulation of uterine pregnant contractility is very different from that of cycling and peri menopausal women. The decrease of PGI2 release, observed in the present experiment in presence of RU 486, and which is not mimicked by other steroids, could partially mediates the “in vivo” activity of RU 486. It was already reported in longer term studies that inhibition of prostaglandin synthesis was one of RU 486 antiprogestin effect (19,201.
Acknowledgements We wish to thank Dr. A. Ulmann from Roussel Uclaf for financial and scientific support.
References 1. Lye P.T., D.G. Porter. Demonstration that progesterone “blocks” uterine activity in the ewe by a direct action on the myometrium. 1. of Reprod. and Fertil. 57 : 87 1978 2. Taylor M.J., R. Webb, M.D. Mitchell, J.S. Robinson. Effect of progesterone withdrawal in sheep during late pregnancy. J. Endocrinol. 92 : 85 1982. 3. Thornburn G.D., J.R.G. Challis. Endocrine control of parturition. Physiol. Rev. 59 : 863 1979.
Prostaglandins
453
4. Erny R., A. Pigne, C. Prouvost, M. Gamerre, C. Malet, H. Serment, J. Barrat. The effects of oral administration of progesterone for premature labor. Am. J. Obstet. Gynecol. 154 : 525 1985. 5. Laffargue F., B. Boulot, L. Lafont, E. Badoc, J.M. Huet, B. Hedon, J.L. Viala. RU 486 and induction of labour in terminating pregnancies in the third cycle of the pregnancy : a preliminary clinical study. J. Gynecol. Obstet. Biol. Reprod. 17: 1095 1988. 6. Fuchs A.R., S. Periyasami, M.S. Soloff. Systemic and local regulation of oxytocin receptors in the rat uterus and their functional significance. Can. I. Biochem. Cell. Biol.. 61 : 615 1983. of myometrial 7. Roberts J.M., P.A Insel, A. Goldfien. Regulation adrenoreceptors and adrenergic response by sex steroids. Mol. Pharmacol. 20 : 52 1981. 8. Garfield R.E., M.S. Kannan, E.E. Daniel. Gap junction formation in myometrium : control by estrogens, progesterone and prostaglandins. Am. J. Physiol.. 238 : c 81 1980. 9. Embrey MI’., ed., The prostaglandins in the human reproduction. Churchill Livingston Edimburgh 1975, p 42. 10. Bygdeman M. The use of prostaglandins and their analogues for abortion. Clinics in Obstet *and Gynaecol. .U : 573 1984. 11. Cameron IT., D.T. Baird. Local prostaglandin administration for mid trimester abortion : a retrospective analysis. I. Obstet. Gynaecol.. 2 : 228 1987. 12. Dray F., R. Frydman. Primary prostaglandins in amniotic fluid in pregnancy and spontaneous labor. Am. J. Obstet. Gynecol. 126 : 13 1976. 13. Mitchell M.D., M.J.N.C. Keirse, A.B.M. Anderson, A.C. Turnbull. Evidence for a local control of prostaglandins within the pregnant human uterus. Br. J.. Obstet. Gynaecol. &I : 35 1977. 14 Castracane V.D., V.C. Jordan. The effect of estrogen and progesterone on uterine prostaglandin biosynthesis in the ovariectomized rat. Biol. Reprod. 13 : 587 1975. 15. Maathuis J.B., R.W. Kelly. Concentrations of PGF2, and E2 in the endometrium throughout the menstrual cycle after the administration of clomiphene or an oestrogen-progestagen pill and in early pregnancy. 1. Endocrinol. 77: 361 1978. 16. Abel M.H., S.K. Smith, D.T. Baird. Suppression of concentration of endometrial prostaglandins in early intrauterine and ectopic pregnancy in women. 1. Endocrinol. s5 : 379 1980. 17. Abel M.H., D.T. Baird. The effect of 17~ estradiol and progesterone in prostaglandin production by human endometrium maintained in organ culture. Endocrinol. 106 : 1599 1980. 18. Tsang B.K., T.C. Ooi. Prostaglandin secretion by human endometrium in vitro. Am. J. Obstet. Gynecol. 142 : 626 1982. 19. Kelly R.W., S.K. Smith. Progesterone and antiprogestins. A comparison of their effect on prostaglandin production by human secretory phase endometrium and decidua. Prostagl. Leuk. Med. 29 : 181 1987.
454
Prostaglandins
20. Jeremy J.Y., I’. Dandona. RU 486 antagonizes the inhibitory action of progesterone on prostacyclin and thromboxane A2 synthesis in cultured rat myometrial explants. Endocrinology 119 : 655 1986. 21. Zahradnik HF., R. Schoening, M. Breckwoldt. Prostaglandins correlation to human myometrial activity in vitro. In : Prostacyclin in Pregnancy. (P.J Lewis, S. Moncada, J. O’Grady, eds), Raven Press, N.Y. 1983, p 147. 22. Quaas L., J. Neulen, HF. Zahradnik. Prostaglandin (6 K PGFla, PGF2a) formation by human pre, peri and post menopausal myometrium in vitro. Acta Endocrinol. JlJ. Suppl. 274 : 43 1986. 23. Abel M.H., R.W. Kelly. Differential production of prostaglandins within the human uterus. Prostaglandins 18 : 821 1979. 24. Peek M.J., T.M. Norman, C. Morgan, IS. Fraser, R. Markham. Trauma induced human endometrial prostaglandin concentrations. Prostnglnndins 34 : 919 1987. 25. Mitchell M.D., P.F. Flint. Prostaglandin production by intra-uterine tissues from periparturient sheep : use of a superfusion technique. 1. Endocrinol. 76 : 111 1978. 26. Liggins G.C., G.A. Campos, C.M. Roberts. Production rates of prostaglandin F, 6-keto-PGF1, and thromboxane B2 by perifused human endometrium. Prostaglundins 19 : 461 1980. 27. Batra S. and 8. Bengtsson. Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. 1. Physiol. 276329 1978. 28 Kubli-Garfias C., L. Medrano-Conde, C. Beyer, A. Bondani. In vitro inhibition of rat uterine contractility induced by 5a and 5~ progestins. Steroids 34 : 609 1979. 29. Putnam C.D., D.W. Brann, R.C. Kolbeck and V.B. Mahesh Inhibition of uterine contractility by progesterone and progesterone metabolites : mediation by progesterone and gamma amino butyric acid A receptor system. Biol. Reprod. 45: 266 1991. 30. Walsh S.W., S. Coulters Increased placental progesterone may cause decreased placental prostacyclin production in preeclampsia Am. 1. Obstet. Gynecol. 161: 1586 1989. 31. Ong J., D.1.B Kerr ., H.R Capper, G.A.R. Johnston. Cortisone : a potent GABA A antagonist in the guinea-pig isolated ileum. J. Phurm. Phurmucol. 42 : 662 1990. 32. Putnam C.D., D.W. Brann, R.C. Kolbeck and V.B. Mahesh. Inhibition of uterine contractility by progesterone and progesterone metabolites : mediation by progesterone and gamma amino butyric acidA receptor systems Biol. Reprod. 45: 266 1991. 33. Potestio F.A., D.M. Olson. Arachidonic acid release from cultured human amnion cells : the effect of dexamethasone. J. Clin. Endocrinol. Metub. 70 : 647 1990. 34. Poli E., A. Merialdi, G. Coruzzi. Characterization of the spontaneous motor activity of the isolated human pregnant myometrium. Phurmucol. Res. 22 : 115 1990.
Prostaglandins
455
35. Jeremy J.Y., P. Dandona. The role of the diacylglycerol-protein
kinase C system in mediating adrenoreceptor-prostacyclin synthesis coupling in the rat aorta. Eur. J. Pharmacol. 136 : 311 1987. 36. Wilson T., G.C. Liggins, G.P. Aimer, E.J. Watkins. The effect of progesterone on the release of arachidonic acid from human endometrial cells stimulated by histamine. Prostaglandins. 31 : 343 1985. 37. Martyn Bailey J., A.N. Makheja, J. Pash, M. Verma. Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells. Biochem. Biophys. Res. Commun. 157 : 1159 1988. 38. Weiss D.J. and E. Gurpide. Non-genomic effects of estrogens and antiestrogens. 1. Steroid Biochem. 31 : 671 1988. Editor:
P.W. Ramwell
Received:
l-lo-92
Accepted:
7-7-92
44:443-455,
1992
EFFECT OF THE PROGESTERONE ANTAGONIST RU486 ON HUMAN MYOMETRIAL SPONTANEOUS CONTRACTILITY ANU PG12 RELEASE C. Lobaccaro-Henri, M. Saintot, P. Laffazgue+ H.P. Bahradnik**, B. Descomps and H. Thaler-Dao Unite 58 I.N.S.E.JZ.M., 60 rue de navacellee, 34090 Montpellier and ,T$e Matemite, C.H.R. Universite Universitats Frauenklinik, Freiburg, Montpellier I France. Fm Briagau, Gemany
Abstract We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI2 release with human myometrial strips superfused “in vitro”. A decrease of PGIz release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dosedependent and reversible. Modifications of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as lo-9M, dexamethasone (IO-*M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contracti!ity, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 0x0 pregnene structure.
Introduction In many animal species progesterone inhibits uterine contractility, especially during pregnancy (l), and parturition is initiated by progesterone withdrawal (2,3). There is no definitive evidence of any progesterone block in humans. However, the efficacy of orally administered micronised progesterone in preventing premature labor (41, and the success of antiprogesterone RU 486 in inducing parturition (5), is evidence in favour of such a role. The mechanism by which this blockade could function is generally unknown, but decrease of oxytocin and Copyright 0 1992 Butterworth-Heinemann
444
Prostaglandins
adrenergic receptor concentration (6,7) and inhibition of gap junction formation (8) have been suggested. Since prostaglandins (PGs) are very effective contractile agents which have been used for more than 20 years to induce labor and abortion (9-ll), and since high levels of PGs, are synthesised during labor (12,13), it could be speculated that among other effects, progesterone blocks prostaglandin synthesis. Evidence reported in the literature is contradictory. Progesterone has been found to exert a “priming” effect on oestradiol-stimulated endometrial PGF2, synthesis in ovariectomized animals (14). In humans, endometrial PGF2e levels are higher during the secretory phase than during the proliferative phase (15). On the other hand, very low PG levels have been found in the decidua when ovarian progesterone production is high (16), and progesterone reportedly inhibits PG synthesis in endometrial explants (17,18). Recent observations with human endometrial (19) and rat myometrial (20) explants show that progesterone inhibits PG synthesis and that antiprogesterone reverses this effect. In the present study we used a superfusion model of human myometrial strips and antiprogestin RU 486, to investigate whether spontaneous prostacyclin release (the major myometrial PG) is modified by this steroid, and whether the changes are correlated with modifications of the spontaneous contractile activity pattern. Our study was voluntarily restricted to the release of PGI2, this PG being the major cycle-oxygenase metabolite within the myometrium ; PGF2, and PGE;! production in the superfusion medium was below the limit of our RIA sensitivity.
Materials and Methods The micronised progesterone antagonist RU 486, mifepristone (178 hydroxy-118 (4-dimethylaminophenyl-l)-17a (I-propynyl)-4,9-oestradien-3one and progesterone agonist R5020, promegestone (17,21-dimethyl-19nor-pregn-4,9-diene-3,20-dione) were gifts from Roussel-Uclaf, Romainville, France. The steroids were dissolved in ethanol at concentrations of lo-2M to lo-5M and stored at -2O“C. Before use they were diluted to the appropriate concentrations in tyrode medium (NaCl 137 mM, KC1 2.6 mM, CaC12 1.8 mM, MgC12 1.05 mM, NaHC03 11.9 mM, NaH2P04 0.42 mM, glucose 5.55 mM, pH 7.4). Uterine corpus specimens (medium part) were obtained from cyclic premenopausal women following hysterectomy for fibromatosis or prolapsus. Institutional approval to use human tissue was obtained. The samples were immediately transferred into an ice cold tyrode solution and kept at 4°C. Upon arrival at the laboratory, the connective tissue was carefully removed and the myometrium dissected in strips parallel to the muscle fiber bundles. Prepared strips were approximately 20mm long, 5mm wide and 5mm thick. They were tied at each end with a silk thread and suspended in a superfusion chamber as described by Zahradnik et al. (21). The strips were continuously superfused with oxygenated tyrode solution at a flow rate of 1 ml/min at 37’C. Isotonic registration of strip contractions was performed by means of a 8368 transducer connected to a
Prostaglandins
445
two channel amplifier (Hugo Sachs Elektronik, March Hegstetten, Germany). The strips were allowed to equilibrate for at least 2 h. After the establishment of regular contractions and monitoring of the control period (one hour), the strips were superfused with the same tyrode buffer containing RU 486 or steroid hormones at the same flow rate and temperature. In dose-response experiments for a given steroid, the same strip was successively superfused at each concentration for a period of one hour. The final concentration of RLJ 486 varied from lo-9M to lo-6M. The contraction frequency, amplitude and duration were registered over the same period of one hour for each concentration of a given product. At the end of the experiment the strip was superfused with tyrode medium, to check the return to the initial parameters of contractility and to PGI2 initial release rates. This was considered as a validation of the tissue viability for the period of observation. Fractions of the superfused strip effluents (5 ml) were collected during monitoring of the contractile activity. In time course study (fig.1) 6 ketoPGFla concentration (the stable metabolite of PGI2) was assayed on 5 ml samples (5 min). In dose-response studies, it was measured on 15 ml samples, (3 samples for each concentration of a given steroid). The samples were immediately stored at -20°C. The RIA was performed after extraction with Sep Pak Cl8 columns (Waters Associates, Milford Mass.). The columns were washed with about 10 ml ethanol 95 % (RF Carlo Erba, Milan, Italy) and 10 ml water/O.1 % EDTA (Merck Darmstadt, West Germany). The samples were acidified to pH 4 with citric acid 1 M, labelled with 1500 cpm (5, 6, 8, 9, 11, 12, 14, 15) 3H-6 keto PGFla (specific activity 5661 GBq/mmole, NEN Boston, Ma, USA) to estimate recovery, and then applied to the column ; after washing with 0.1 % EDTA and 2 ml hexane (RP SDS France), the sample was eluted with 1 ml absolute ethanol. The ethanol was evaporated to dryness with nitrogen and the extract dissolved in 1 ml gelatine buffer (phosphate buffer 10 mM pH 7.4, NaCl 9 g/l and gelatine 1 g/l). The RIA was performed with an antiserum purchased from Biosys, Compiegne, France, which does not cross- react with the steroids. Cross reactivity towards the other eicosanoids was respectively : PGFla 13 %, PGE;! and dihydro-15 keto PGFla 4 %, PGFza, PGD2 and PGDl 2,8 % and others 0.01 %. The intra- and inter-assay coefficients of variation were 5 % and 6 % respectively. The concentration of PG12 displacing 50 % of the labelled ligand was 30 pg and the lowest amount detectable was 4 pg. The data were expressed as mean percentage f SEM of control tyrode superfusion values. The 100 % value of PGI2release, represents the release rate of 6 keto PGF1, measured for a lh period (3 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 recoder) and their mean duration (set) measured over a lh period after the stabilization period, were considered as 100 %. The same calculations during one hour superfusion, at each steroid
446
percentage of or stimulation. data were f SEM control tyrode values at experiments and by Student’s paired test concentration).
Prostaglandins
as mean beginning of vs each
Results The group of patients studied ranged from 40 to 52 years of age. All the strips were obtained from myometrium of cyclic women in the pre or perimenopausal period. The levels of basal 6 keto PGFl, release measured after the stabilization period (and considered as the 100 % control values) ranged from 0.3 to 3.0 pg/min/mg wet weight in agreement with previously reported data (22). The contractile activity tracings showed regular peaks occurring with a mean frequency (& SEM) of 20.2 f 10.5 (n = 16). The mean amplitude (height of the peaks measured on the recorder in our monitoring conditions) was 13.7 + 6.2 cm, and the mean duration of the contractions was 49 + 16 sec. There was no correlation between age or hormonal status (for instance, treatment with progesterone before hysterectomy) and the contractile parameters. When the myometrium samples were superfused with tyrode medium containing increasing concentrations of RU 486, a dose-dependent inhibition of PG12 release was observed. The time course of 6 keto PGFla output inhibition is shown in Fig. la. Inhibition occurred with a short delay of about 20 min after each change of RU 486 concentration. The inhibition was roughly 50 % at 10-7M and 80 % at lo-6M. This was reversed after a brief washing period of 20 min. The recovery after washing was 80 % of the initial control value. The dose-response studies (Fig. lb) shows that at lo-7M the 6 keto PGFl, release was 52,5 f 10 % of the control and at lo-6M it was 20 + 7 (n = 10). The difference from control was significant at IO-8M (p c 0.001) and the inhibition was dose dependent. The simultaneous recordings of the mechanical activity show that the decrease of 6 keto PGFla release was concommittant with a modification of uterine motility : inhibition of contraction frequency, increase of the amplitude and duration of the peaks (Fig. 2a and 2b). The effects on frequency were dose dependent and already significant at 10-8M (p c O.OOl), those on amplitude and duration were significant at lo-9M (p < 0.001) but reached a plateau at 10m7M.
447
Prostaglandins
a
50
100
Time (min)
b
0
9 8 7 6 - log RU 486 (M)
0
Fig. 1 : Effect of increasing concentrations of RU 486 on 6 keto PGFr, release from superfused human myometrial strips a- Time course effect : One myometrial strip was successively superfused with increasing RU 486 concentrations, for periods of 20 min. ; then the tissue was washed with tyrode medium for another 20 min. 6 keto PGFI, concentrations were measured on 5 ml fractions (5 min). The control value (100 %) at t = 0 was, in this case, 3 pg * O.O8/min/mg wet weight. b- Dose response efiecf : Each concentration was successively superfused for one hour. The release rate of 6 keto PGFl. was measured in 4 samples of 15 ml medium collected during this time and the mean value was expressed as percentage of the mean release rate during tyrode superfusion without steroid. For more details see text in the Materials and Methods section. Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, n = 10.
448
Prostaglandins
NS
100
0
9 8 7 6 - log RU 486 (M :>
0 0 0
Amplitude Duration
b
9 8 6 - log RU 486 (M)
0
Fig. 2 : Dose-response effect of increasing concentrations of RU 486 on the spontaneous contractile activity of superfused myometrial strips. The conditions of the superfusion are the same as in the Fig. lb. The data are expressed as percentage of the same parameters measured during tyrode medium superfusion. For more details see text in the Materials and Methods section. a- Frequency. b Amplitude q and Duration q Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, n = 10.
449
Prostaglandins
The progesterone analogue R5020, at concentrations ranging from 10-g to lo-6M, did not significantly inhibit 6 keto PGFla release (not shown), but had significant effects on the contractility at very low concentrations (Fig. 3a and 3b) : the frequency dropped to 41+ 7 % of control at IO-9M and the duration of the contractions increased to 248 + 5 %. The effect on the amplitude was less striking. In contrast to RU 486 and progesterone we did not observe any dose-response effect.
100
80 ,”
@ 1”3 gs
60
‘“e
20
*** ***
a
40
n 0
9
8
7
6
0
-log R5020 (M) Cl Amplitude Duration
b
8 7 6 -log R5020 (M)
0
Fig. 3 : Effect of a progesterone analogue R 5020 on the spontaneous contractile activity of superfused human myometrial strips. The conditions and expression of data are the same as in Fig. 2. For more details see text in the Materials and Methods section. a- Frequency. b- Amplitude 17 and Duration q Student’s t test : pair tested, control vs. each steroid concentration. ***p < 0,001, **p < O,Ol, n = 4.
Prostaglandins
450
Similarly, dexamethasone had no effect on 6 keto PGFla release (results not shown) but inhibited significantly uterine frequency. The amplitude and duration of contractions were also significantly altered (Fig.4a and 4b). Testosterone had no effect on the 6 keto PGFla release nor on the contractility, even at 10&M ( not shown).
0
8
7
- log Dex (M) 0
Amplitude Duration
b
8 -
7
log Dex (M)
Fig. 4 : Effects dexamethasone on the spontaneous contractile activity of superfused human myometrial strips. The conditions and expression of data are the same as in Fig. 2. For more details see text in the Materials and Methods section. a- Frequency. b- Amplitude 0 and Duration q Student’s t test : pair tested, control vs. each steroid concentration. *** p < 0.001, n = 9.
Prostaglandins
451
Discussion PGI2 (measured as 6 keto PGFla) is the major PG released by the myometrium (23). The release of this metabolite, probably as a result of PG synthesis activation (24), was very high just after handling of the strips.The rate of production then declined rapidly to a basic level as previously observed by several other groups (25,26). Our experiments were carried out after a 2 h resting period, when PG release was stable and when spontaneous uterine contractions were regular. RU 486 inhibited dose-dependently the spontaneous release of prostacycline by the superfused myometrial strips. The onset of this effect was rapid (about 20 min), was maintained as long as antiprogestin was superfused and was rapidly reversed (20 min) by tyrode medium washing. In our model, RU 486 was the only steroid to show a simultaneous effect on 6 keto PGFlcx release and uterine contractility. All the steroids tested in this study, except testosterone, affected uterine contractility without affecting the prostanoid release. Therefore, the simultaneous effects of RU 486 on PGI2 output and uterine contractility (although concommittant) may be independant and mediated by different mechanisms. Data of the literature indicate that other steroids act on spontaneous uterine contractility : oestradiol (27) and C21 steroids such as 5a and 5p pregnane compounds (28, 29) Therefore RU 486 effect on this parameter could be due to its ~4 C21 steroid structure and targeted on a membrane component such as a calcium channel (28) rather than a steroid receptor. It is also known that cortisone (30) and several progesterone metabolites are potent antagonists of GABA receptors (31). Therefore it should be of interest to carry out further investigations on structure-activity relationship of antiprogestins and C21 steroids. By contrast, the RU 486 effect on 6 keto PGFlu release is very specific of this molecule since it is not mimicked by other steroids. However, the fact that neither the progesterone analog R5020, (a progesterone agonist which was used in preference to progesterone because it was not metabolized in situ), nor the glucocorticoid effector dexamethazone, had any decreasing effect on 6 keto PGFI, release indicate that RU 486 action is not mediated by the progestin or the glucocorticoid receptor. Moreover, the short time course and the rapid reversibility of the effect do not support a classical steroid effect. It seems from our data that RU 486 inhibitory effect on PGI2 release is not related to its antihormonal activity. The lack of effect of the other steroids on PGI2 release in our data, differs from recently published observations in isolated human amnion cells : in this report (32), dexamethasone treatment (IO-8M) for period as short as 20 min, inhibited basal arachidonic acid release from these cells. This effect was not blocked by protein or RNA synthesis inhibitors, and was mimicked by RU 486 and all steroids tested, androgens, corticosteroids and progesterone. However these results were obtained with dispersed cells of a different tissue and concern arachidonic acid release and not PG release. As mentioned above, a striking point in our results concerns the very short lag period of the steroid effects. Studies on the regulation of uterine
452
Prostaglandins
PG synthesis by steroids are usually carried out using “in vivo” administration of hormones. In previous studies experiments on the “in vitro” control of prostaglandin synthesis by progesterone and antiprogestins, an incubation period of 18 h was chosen for rat myometrial explants (ZO), and 2 or 3 days for human endometrial explants (19). In the present study, the short time course of steroid activity, the lack of specificity of steroids in modifying the spontaneous uterine contractile activity and the reversibility of the action suggest the involvement of a membrane “receptor” site, such as ion channels (31) second messenger transmitters (32), phospholipases (33) or even of cycle oxygenase (34). Other non genomic effects were reported in the literature for estrogens and antiestrogens (35). Further investigations need to be carried out to determine whether RU 486 affect prostacyline release by acting on a membrane phospholipase or directly on the prostaglandin synthase or by another mechanism. We report that RU 486 decreased uterine contraction frequency while increasing simultaneously the amplitude and duration of contractions. Such mechanical activity changes could be considered as leading to a higher efficiency of contractions similar to that observed in labour. However, extrapolation of the present data to the pharmacological activity of RU 486 on pregnant myometrium should be very careful. At least 2 limits should be considered : firstly our data refer to an “in vitro” model and secondly they derived from observations on spontaneous contractions of non pregnant myometrium. It is quite probable that the regulation of uterine pregnant contractility is very different from that of cycling and peri menopausal women. The decrease of PGI2 release, observed in the present experiment in presence of RU 486, and which is not mimicked by other steroids, could partially mediates the “in vivo” activity of RU 486. It was already reported in longer term studies that inhibition of prostaglandin synthesis was one of RU 486 antiprogestin effect (19,201.
Acknowledgements We wish to thank Dr. A. Ulmann from Roussel Uclaf for financial and scientific support.
References 1. Lye P.T., D.G. Porter. Demonstration that progesterone “blocks” uterine activity in the ewe by a direct action on the myometrium. 1. of Reprod. and Fertil. 57 : 87 1978 2. Taylor M.J., R. Webb, M.D. Mitchell, J.S. Robinson. Effect of progesterone withdrawal in sheep during late pregnancy. J. Endocrinol. 92 : 85 1982. 3. Thornburn G.D., J.R.G. Challis. Endocrine control of parturition. Physiol. Rev. 59 : 863 1979.
Prostaglandins
453
4. Erny R., A. Pigne, C. Prouvost, M. Gamerre, C. Malet, H. Serment, J. Barrat. The effects of oral administration of progesterone for premature labor. Am. J. Obstet. Gynecol. 154 : 525 1985. 5. Laffargue F., B. Boulot, L. Lafont, E. Badoc, J.M. Huet, B. Hedon, J.L. Viala. RU 486 and induction of labour in terminating pregnancies in the third cycle of the pregnancy : a preliminary clinical study. J. Gynecol. Obstet. Biol. Reprod. 17: 1095 1988. 6. Fuchs A.R., S. Periyasami, M.S. Soloff. Systemic and local regulation of oxytocin receptors in the rat uterus and their functional significance. Can. I. Biochem. Cell. Biol.. 61 : 615 1983. of myometrial 7. Roberts J.M., P.A Insel, A. Goldfien. Regulation adrenoreceptors and adrenergic response by sex steroids. Mol. Pharmacol. 20 : 52 1981. 8. Garfield R.E., M.S. Kannan, E.E. Daniel. Gap junction formation in myometrium : control by estrogens, progesterone and prostaglandins. Am. J. Physiol.. 238 : c 81 1980. 9. Embrey MI’., ed., The prostaglandins in the human reproduction. Churchill Livingston Edimburgh 1975, p 42. 10. Bygdeman M. The use of prostaglandins and their analogues for abortion. Clinics in Obstet *and Gynaecol. .U : 573 1984. 11. Cameron IT., D.T. Baird. Local prostaglandin administration for mid trimester abortion : a retrospective analysis. I. Obstet. Gynaecol.. 2 : 228 1987. 12. Dray F., R. Frydman. Primary prostaglandins in amniotic fluid in pregnancy and spontaneous labor. Am. J. Obstet. Gynecol. 126 : 13 1976. 13. Mitchell M.D., M.J.N.C. Keirse, A.B.M. Anderson, A.C. Turnbull. Evidence for a local control of prostaglandins within the pregnant human uterus. Br. J.. Obstet. Gynaecol. &I : 35 1977. 14 Castracane V.D., V.C. Jordan. The effect of estrogen and progesterone on uterine prostaglandin biosynthesis in the ovariectomized rat. Biol. Reprod. 13 : 587 1975. 15. Maathuis J.B., R.W. Kelly. Concentrations of PGF2, and E2 in the endometrium throughout the menstrual cycle after the administration of clomiphene or an oestrogen-progestagen pill and in early pregnancy. 1. Endocrinol. 77: 361 1978. 16. Abel M.H., S.K. Smith, D.T. Baird. Suppression of concentration of endometrial prostaglandins in early intrauterine and ectopic pregnancy in women. 1. Endocrinol. s5 : 379 1980. 17. Abel M.H., D.T. Baird. The effect of 17~ estradiol and progesterone in prostaglandin production by human endometrium maintained in organ culture. Endocrinol. 106 : 1599 1980. 18. Tsang B.K., T.C. Ooi. Prostaglandin secretion by human endometrium in vitro. Am. J. Obstet. Gynecol. 142 : 626 1982. 19. Kelly R.W., S.K. Smith. Progesterone and antiprogestins. A comparison of their effect on prostaglandin production by human secretory phase endometrium and decidua. Prostagl. Leuk. Med. 29 : 181 1987.
454
Prostaglandins
20. Jeremy J.Y., I’. Dandona. RU 486 antagonizes the inhibitory action of progesterone on prostacyclin and thromboxane A2 synthesis in cultured rat myometrial explants. Endocrinology 119 : 655 1986. 21. Zahradnik HF., R. Schoening, M. Breckwoldt. Prostaglandins correlation to human myometrial activity in vitro. In : Prostacyclin in Pregnancy. (P.J Lewis, S. Moncada, J. O’Grady, eds), Raven Press, N.Y. 1983, p 147. 22. Quaas L., J. Neulen, HF. Zahradnik. Prostaglandin (6 K PGFla, PGF2a) formation by human pre, peri and post menopausal myometrium in vitro. Acta Endocrinol. JlJ. Suppl. 274 : 43 1986. 23. Abel M.H., R.W. Kelly. Differential production of prostaglandins within the human uterus. Prostaglandins 18 : 821 1979. 24. Peek M.J., T.M. Norman, C. Morgan, IS. Fraser, R. Markham. Trauma induced human endometrial prostaglandin concentrations. Prostnglnndins 34 : 919 1987. 25. Mitchell M.D., P.F. Flint. Prostaglandin production by intra-uterine tissues from periparturient sheep : use of a superfusion technique. 1. Endocrinol. 76 : 111 1978. 26. Liggins G.C., G.A. Campos, C.M. Roberts. Production rates of prostaglandin F, 6-keto-PGF1, and thromboxane B2 by perifused human endometrium. Prostaglundins 19 : 461 1980. 27. Batra S. and 8. Bengtsson. Effects of diethylstilboestrol and ovarian steroids on the contractile responses and calcium movements in rat uterine smooth muscle. 1. Physiol. 276329 1978. 28 Kubli-Garfias C., L. Medrano-Conde, C. Beyer, A. Bondani. In vitro inhibition of rat uterine contractility induced by 5a and 5~ progestins. Steroids 34 : 609 1979. 29. Putnam C.D., D.W. Brann, R.C. Kolbeck and V.B. Mahesh Inhibition of uterine contractility by progesterone and progesterone metabolites : mediation by progesterone and gamma amino butyric acid A receptor system. Biol. Reprod. 45: 266 1991. 30. Walsh S.W., S. Coulters Increased placental progesterone may cause decreased placental prostacyclin production in preeclampsia Am. 1. Obstet. Gynecol. 161: 1586 1989. 31. Ong J., D.1.B Kerr ., H.R Capper, G.A.R. Johnston. Cortisone : a potent GABA A antagonist in the guinea-pig isolated ileum. J. Phurm. Phurmucol. 42 : 662 1990. 32. Putnam C.D., D.W. Brann, R.C. Kolbeck and V.B. Mahesh. Inhibition of uterine contractility by progesterone and progesterone metabolites : mediation by progesterone and gamma amino butyric acidA receptor systems Biol. Reprod. 45: 266 1991. 33. Potestio F.A., D.M. Olson. Arachidonic acid release from cultured human amnion cells : the effect of dexamethasone. J. Clin. Endocrinol. Metub. 70 : 647 1990. 34. Poli E., A. Merialdi, G. Coruzzi. Characterization of the spontaneous motor activity of the isolated human pregnant myometrium. Phurmucol. Res. 22 : 115 1990.
Prostaglandins
455
35. Jeremy J.Y., P. Dandona. The role of the diacylglycerol-protein
kinase C system in mediating adrenoreceptor-prostacyclin synthesis coupling in the rat aorta. Eur. J. Pharmacol. 136 : 311 1987. 36. Wilson T., G.C. Liggins, G.P. Aimer, E.J. Watkins. The effect of progesterone on the release of arachidonic acid from human endometrial cells stimulated by histamine. Prostaglandins. 31 : 343 1985. 37. Martyn Bailey J., A.N. Makheja, J. Pash, M. Verma. Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells. Biochem. Biophys. Res. Commun. 157 : 1159 1988. 38. Weiss D.J. and E. Gurpide. Non-genomic effects of estrogens and antiestrogens. 1. Steroid Biochem. 31 : 671 1988. Editor:
P.W. Ramwell
Received:
l-lo-92
Accepted:
7-7-92
