External calcium dependence of the uterine contraction induced by prostaglandins E2 and F2 alpha and its antagonism with natural progestins

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External calcium dependence of the uterine contraction induced by prostaglandins E2 and F2 alpha and its antagonism with natural progestins

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and C. Kubli-Garfias

Unidad de Inveetigacion Biomedica, IMSS. Apartado Poetal 70492, Cd. Univereitaria C.P. 04511, Mexico City, Mexico

ABSTRACT Prostaglandins (PGs) E2 and F20 are strong inducers of uterine contraction by promoting a Ca2+ increase into the cell through specific receptors coupled with calcium the progesterone 5(3-reduced channels. On the contrary, and progestins promote smooth muscle relaxation by blocking the ion influx, this was designed to calcium Thus, study PGsemphasize the importance of external calcium in the rat uterus contraction. Likewise, also studied was induced antagonism and the interaction between PGs and the progestins its 5a and SB-reduced (progesterone and derivatives) in the myometrium. Results showed that uterine calcium, contraction induced by PGs depends on external since verapamil or extracellular calcium depletion abollshed Regarding the PGs-progestins antagonism, it the PGs effect. observed that pregnanedione, pregnanolone and was were quite effective for counteracting of epipregnanolone progesterone was effective PGs-induced contraction. However, middle range, whereas Sa-reduced progestins ;zllo:regnanedione and allopregnanolone) almost were ineffective. It has been concluded that the participation of PGs and progestins in the modulation of uterine contraction might be achieved through the control of calcium influx by opening (PGs) or blocking (progestins) receptor-operated calcium channels.

INTRODUCTION Both prostaglandi ns ( PGs) E2 and PGF2 II inducers of uterine muscle contraction. This carried out in the myometrium by the interaction smooth muscle membrane specific receptors (l-2).

are potent response is of PGs with

It has been proposed that the PGs stimulatory effect on may vary according to differences in the uterine must 1 e well and ion concentration as (8). status (3-51, hormonal thei r PGs is mechanism widely accepted for However, the ability to increase the free-cytoplasmic Ca2+ concentration. and is ions implies the mobilization of Ca2+ task This achieved in three different ways: a) increase of Ca2+ inward channels (7,8); b) voltage-dependent through current channels enhancing of Ca2+ influx through receptor-operated





Prostaglandins (8);








On the other hand q the membrane action of several steroids, namely, 5-reduced androgens, progestins and some corticosteroids on the uterus and other produce types of smooth muscle a marked relaxation of spontaneous and induced contractions 14). Thus, these steroids produce, for (13, instance, a clear Inhibition of the contraction induced by Cal + in the experimental model of high potassium-Ca2+ free medium (151, and interestingly those contractile responses elicited by oxytocin (161, acetylcholine (17) and serotonin in the isolated rat uterus (18). The uterine-relaxant reported to be connected permeability, probably through the voltage-activated receptor-activated calcium

effect with the by blocking calcium channels

of steroids has been regulation of calcium the calcium influx channels (15) and the (16-18).

The present study was carried out to i nvesti gate the following: first, to what extent the mechanism of PGEz and PGFza to induce uterine contraction depends on extracellular Ca*+ influx; second, to challenge the PGs excitatory effect against the relaxant effect of some natural steroids, i.e., progesterone and 5-reduced progestins in the uterine muscle; third, to establish a structure-activity relationship of the steroids; and some insight about the fourth, to gain interaction among PGs and steroids in the myometrial cell.

METHODS Animal and Tissue Preparation: Wistar rats were estrogen-primed estradiol (100 ug/Kg) dissolved were killed by spinal dislocation.

Virgin 200-250 by S.C. injection in corn oil 24 h

g of before

female 17Sthey

Uterine horns were removed and placed in a Krebsconcentration Henselei t solution (K-HS) with the following of salts (mM): NaCl 119, KC1 4.6, MgC12 1 .2, KHzPO4 1.2, 1.5, and glucose 11.1. Bubbled with a NaHCOa 20, CaC12 mixture of 95% 02 and 5% CO2 , the pH was adjusted to 7.4. Uterine horns were separated from fat and surrounding connective tissue; each horn was divided transversally into the two layers of muscle were two uterine rings (1 cm long), lo-ml not separated, and each ring was placed in a organ containing K-HS at 37°C. Uterine rings were suspended bath in a 10 mN force. The mechanical contractions exerted by FT-03 force t i ssues were recorded isometrically with an transducer connected to a Grass 70 polygraph and were al lowed to equilibrate for approximately 1 h. After that, the force was readjusted to 10 mN. When the baseline force was stabilized, experiments were started. Contractions were evaluated in percentage measuring the area under the curve.



Drugs Used : Compounds were purchased from Sigma Chemical co. ) St Louis, MO. PGEz (1 .l uM) and PGFza (0.8 uM) were dissolved in ethanol (10%). This concentration of ethanol did not affect the uterine activity. Verapamil was dissolved distilled water in a final concentration of 1 uM. in The steroids (progesterone), tested were : 4-pregnen-3,20-dione 3B-hydroxy-5S-pregnan-20-one 5B-pregnane(pregnanolone), 3a-hydroxy-5S-pregnan-20-one 3,20-dione (pregnanedione), (epipregnanolone), 30-hydroxy-5a-pregnan-20-one (allopregnanolone) 5a-pregnane-3,20-dione and (allopregnanedione) all dissolved in propylene glycol at a concentration of 1.43 uM. This vehicle had no final effect on the PGs induced contractions. Calcium-Free and Verapami 1 Experiments: experiments These established the magnitude of calcium-antagonism of verapamil in our biological model. Thus, after the period of stabilization, tissues were depolarized with high potassiumcalcium free solution (HK-CF). This modified Krebs-Henseleit solution was obtained by equimolecular substitution of NaCl (84.5 mM) for KC1 (40 mM), without CaC12 and the addition of EGTA 2 mM. When the contraction reached the baseline, CaC12 (1 mM) was administered and the contractile response was recorded during a IO-min period. This contraction was assumed as control. After this, tissues were washed with HKCF provoking relaxation. When the baseline was reached again, tissues were loaded with verapamil 5 min before to a second contraction induced by the addition of 1 mM CaC12. Responses were allowed during a 10 min period and compared with the control. Prostaglandins vs. Verapami 1: For these experiments the were stabilized tissues under K-HS and the contraction induced by each PG was recorded for IO-min (control). Tissues were washed with the same solution, in order to return to the original position. After this, t i ssues were preincubated with verapami 1 5 min before with a second addition of PG at the same concentration as the control. Prostaglandins and Free-Ca induced 2+ Response: Contractions by PGE;! and PGF2a were recorded in K-HS for 10 min and these responses were assumed as 100% of contractile activity. The tissues were then washed. Thereafter, solution the was replaced by a calcium-free (CF) solution with 2 mM EGTA and tissues were incubated for 50 min in order to Ca2 + remove from membranes and the extracellular space. Subsequently, (in different experiments) each PG was added observing its response also during lo-min. After this, CaC12 (2 mM) was added and the effect was kept during a lo-min period, those responses were measured and compared with the control. PGs and incubated determined experiments;

Steroids Antagonism Experiments: T i ssues concentrations in K-HS and PGE2 and PGF2a concentration-response for particular the PG induced contraction was observed

were were during



IO-min control. After this period, tissues were washed. When the contraction was eliminated, tissues were incubated with progesterone at three different micromolar concentrations; only one concentration was tested for each uterine ring, 5 min before a second PG addition at the same concentration as the control. The response was observed for lo-min in the presence of progesterone, with a total time elapsed of 15 min. Concentration-response curves to progesterone effect to PGEz and PGFz (1 induced-contraction were plotted and the inhibitory concentrations 16, 50 and 84 (ICI of progesterone were obtained by interpolation, according to Litchfield and Wi lcoxon (20). In the same way, separated tissues were challenged with the progestins: pregnanolone, pregnanedione, epipregnanolone, allopregnanolone or allopregnanedione, against the excitatory effect of PGs. Progesti ns concentration was eouimolar to IClS of progesterone. Responses to progestins addition were compared with the IC16 of the precursor progesterone by the formula: progesti n inhibition (%) /progesterone inhibition (%I.

RESULTS the experiments In searching for the relationship between external calcium and PGs induced activity; it was observed that the contractile response elicited by the addition of 1 mM of CaClz in the calcium-free medium was prevented by verapamll in a range of 90.7% + 1.29 (Fig. 1A). These values were similar to those obtained in the excited verapamil-PGs antagonism experiments. Thus, tissues either with PGE2 or PGFza (Fig. lB), in K-HS responded with This effect was also prevented by an immediate contraction. verapami 1, 87.4% f 2.60 for PGEz and 76.6% + 1.80 for PGFza. Interestingly , the contraction elicited by the PGs was not observed when the tissues were incubated in a calcium-free solution containing 2mM EGTA. However, in these cond i ti ons the Caz+ addition at 2mM restored the contraction in 78.21% f 1.94 for PGEz and 74.26% 5 2.28 for PGFzc, (Fig. 1C).





Figure 1. Effects observed on myometrium contractility from estrogen-dominated rat. A) Tonic contraction induced by calcium and its preventlon by verapamil (Ver 1uM) in high potassi urn calcium free solution (HK-CF). B) Contractile responses elicited by PGEz (1.1 pM) and PGFza (0.8 pM) were prevented by verapamil (Ver 1pM) in a med i urn containing calcium. C) The induction of contraction by both PGs in a med i urn with calcium is not observed in a medium without calcium (Ca2+ 0 mM and EGTA 2 mM). However, those contractions are reestablished when calcium is added.

Figure 2 shows concentration-response progesterone on the two prostaglandins induced 1C50 and ICa4 of progesterone expressed The ICI 6, were derived from such curves.

curves for contraction. in table 1



2. Concentration response curves of progesterone Figure uterine contractions elicited by PGE2 at 1.1 uM the Each circles) and PGFza at 0.8 uM (closed circles). the mean and vertical bars are the f SEM represents experiments.

on (open point of 8

----_--_-----_---Table 1. progesterone contraction.

Inhibitory on the





concentrations of and PGF2 a -induced




26.33 24.91

40.34 45.00

54.35 80.00


-__-_--_--_--_---Experiments with progestins showed and 5-reduced progestins were outstanding contractile response induced by PGE2 and

progesterone that antagonists of the PGFza.

pregnanol one, i.e., Progest i ns 5Wreduced, than potent and epipregnanolone, were more pregnaned i one contraction inhibition of the to elicited progesterone compounds, Q-reduced induced by PGEz and PGFza . However, al most allopregnanedione and allopregnanolone were namely, ineffective (Fig. 3).



Figure 3. Typical recordings of the uterotoni c responses induced by 1.1 PM of PGEz (top) and 0.8 uM of PGFZCI ( bottom 1 a medium with calcium. Note the antagonistic effect of AFegnanolone (steroid 25 MM). Table 2 1 ists reduced progestins PGE2 and PGFzo .

Table PGEz-

2. and


the potencies antagonize

Percent of PGFe.i-induced


Progesterone Pregnanol one Pregnanedione Epi pregnanol one Allopregnanolone Allopregnanedione

inhibition of contraction.

% OF INHIBITION ON THE PGE2CONTRACTION 18.5 64.9 70.5 67.9 4.6 3.4

of progesterone and the contraction elicited

-+ + f f f: *

* Potency of each steroid % of progestin inhibition/% assuming value of 1.0 to All steroids were tested +SEM, n=8.

3.7 2.7 3.0 3.0 1.1 0.5

* 1.0 3.4 3.7 3.6 0.2 0.1




% OF INHIBITION ON THE PGFz a CONTRACT1 ON 18.8 59.7 77.0 71.3 4.1 2.9

++ f + + -+


3.2 4.3 2.3 4.0 1.5 1.0

was calculated by the formula: of progesterone inhibition, progesterone. at ICIS of progesterone, means

* 1.0 3.1 4.0 3.7 0.2 0.1



DISCUSSION regulators Prostaglandins PGE2 and PGFza are important was the uterine contractility. In the present work, it of observed that the contractions induced by these compounds on antagonist calcium rat uterus can be prevented by the the verapamil and they are fully deleted when the tissues are in Ca* +-free a solution. The fact that contractions are restored when Ca2+ is added suggests that the effect of PGs depends on the influx of calcium ions into the myometrial from the extracel lular medium. cells Thus, an important point arising from this result is that PGs might act directly on the uterine smooth muscle membrane to produce contraction. This action takes place probably by interacting with their specific receptors identified previously on the myometrial membranes (1.2). Moreover, these receptors are probably involved in the receptor-operated Ca2 + channels activation, promoting the ionic influx, as has happened with other smooth muscle excitatory compounds (8). On the other hand, it has been reported that PGE2 and contraction of the uterus by PGF;! (L produce increasing the free-cytoplasmic Ca2+ concentration (22). The mobilization of the Ca2+ ions may be through three different mechanisms: increasing of Ca2+ current inward through vo 1tagea) dependent channels b) enhancing of Ca2 + influx (738); through receptor-operated channels (8); and/or c) release of Since intracellular stores are intracellular Ca2+ (9,10,11). sensitive to PGE2 and PGFna action, through most likely their specific receptors, it has that been suggested receptor-operated channels and agonist-sensitive Ca2+ stores can be integrated in a single mechanism (23). However, from Ca2 + the present results it appears that the main mode of calcium intracellular increase is through the external influx. Regarding the physiological correlation between PGs and regulation of uterine natural progestins on the is known that those steroids produce a contractility, it relaxant effect on spontaneous uterine contractions (13). it was noteworthy that progesterone and SR-progestins Thus, PGF2 0.showed a marked antagonistic effect on the PGEzand and Sa-progestins (allopregnanolone and induced contraction, almost ineffective. It was clear allopregnanedione) were are better than their precursor that 5i3-progest i ns Ring A reduction of progesterone in either 5a progesterone. Likewise, 5R position alters its biological properties. or estradiol, inhibitory action of similar effects of an activity estrone and progesterone on spontaneous myometrial to competition of steroids with the PGs for be due may selective binding sites on myometrial plasma membranes (19). The lowering of the frequency the contractions produced by produced by verapamil, likely by

and magnitude of uterine steroids resembles that blockade of the voltage-



operated Ca2+ channels, but to a lesser extent (15). This hypothesis might also explain the remarkable antagonism of these steroids to the contractions induced by oxytocin. Ach and 5-HT, suggesting that they are also capable of blocking the receptor-operated CaZ+ channels (16-18). steroid effect on the contraction induced by PGs The suggests an important antagonism between steroids and PGs on uterine contractility. the modulation of However, the possibility of a direct interaction of the progestins with the specific receptors to PGEz and PGFzo is discarded due to differences among PGs steroids. These the chemical and compounds probably do not bind at the same site and the decreased binding of PGs in the presence of steroids may be due to conformational changes, i.e., membranal stabilization in the myometrial plasma membranes induced by the steroids. Also, this non-genomic mechanism of steroids might modulate Ca*+ influx promoted by PGs by blocking their specific the Can+ channel as suggested oxytocin, receptor-operated for and 5-HT. Thus, it seems that SO-progesti ns acetylcholine important role in the play an regulation of calcium permeability in the cell antagonizing through a nonspecific mechanism of both receptorand voltage-operated channels.


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