Mechanisms of relaxation of rat aorta in response to progesterone and synthetic progestins1

Maturitas 28 (1997) 181 – 191

Mechanisms of relaxation of rat aorta in response to progesterone and synthetic progestins1 E. Glusa a,*, T. Gra¨ser b, S. Wagner a, M. Oettel b a

Centre for Vascular Biology and Medicine, Uni6ersity of Jena, Medical Faculty, Nordha¨user Straße 78, D-99089 Erfurt, Germany b Department of Medical Research, Jenapharm GmbH and Company KG, Jena, Germany

Abstract Objecti6e: To compare the acute effects of progesterone, chlormadinone acetate (CMA), norethisterone acetate (NETA) and dienogest (DNG) with those of 17b-estradiol (17b-E2) on the vascular reactivity of male rat thoracic aorta. Methods: Aortic rings with or without endothelium were placed in an organ bath for isometric tension recording. The integrity of the endothelium was assessed by the relaxant response of precontracted rings to acetylcholine (1 and 10 mM), which was diminished after mechanical removal of the endothelium. The concentrations of the steroid hormones were 0.01–10 mM. Results: In vessels precontracted with phenylephrine (1 mM), CaCl2 (3 mM) or KCl (30 mM), progesterone, CMA and NETA (10 mM each) an endothelium-independent relaxation of 20–35% resulted, with a maximum response after 20–30 min, while DNG had a negligible effect in all experiments. The same concentration of 17b-E2 was twice as potent as the progestins. Indomethacin, the cyclooxygenase inhibitor and glibenclamide, an inhibitor of the ATP-sensitive potassium channels, did not affect the relaxant responses. The antagonists of progesterone receptors J 867 (1 mM) as well as of estrogen receptors ICI 182780 (1mM) did not counteract the relaxation induced by progesterone and 17b-E2, respectively. Progesterone (10 mM) did not interfere with endothelium-dependent acetylcholine-induced relaxation of precontracted aortic rings. Pretreatment of the vessels with the hormones attenuated the maximal contractile response to phenylephrine. In the presence of verapamil (1 mM) or progesterone (10 mM) or 17b-E2 (1 and 10 mM) the concentration-response curves for calcium-induced contractions in K + -depolarized vessels were shifted to the right, with suppression of the maximum response. Conclusions: These studies suggest that in addition to 17b-E2 the progestins, progesterone, CMA and NETA caused a reduction of vascular tone, probably due to blockade of voltage-dependent and/or receptor-operated calcium channels. © 1997 Elsevier Science Ireland Ltd. Keywords: Vascular relaxation; Rat aorta; Progestins; Endothelium; Calcium antagonism; Progesterone; 17b-estradiol; Chlormadinone acetate; Norethisterone acetate; Dienogest

* Corresponding author. Tel.: +49 361 7411313; fax: + 49 361 7411160. 1 Presented at the 8th International Congress on the Menopause, Sydney, Australia, 3 – 7 November 1996. 0378-5122/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 5 1 2 2 ( 9 7 ) 0 0 0 5 7 - 1

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1. Introduction The incidence of cardiovascular diseases in women increases dramatically after the menopause [1,2]. Clinical data indicate that hormone replacement therapy (HRT) in postmenopausal women reduces the incidence and severity of coronary heart disease by approximately 50% [3–6]. Angiographic studies have demonstrated that coronary artery spasm is reduced by estrogen replacement therapy, most markedly in women with severe lesions [7 – 9]. However, it remains to be clarified whether the addition of progestins to estrogen replacement therapy attenuates the cardiovascular benefits [2,10]. The mechanisms by which HRT reduces the risk of coronary heart disease in women are, as yet, incompletely understood [11]. Modulation of risk factors such as carbohydrate and lipid metabolism, e.g. prevention of insulin resistance as well as decrease in low-density and increase in high-density lipoprotein cholesterol may be important [2]. There is accumulating evidence that estrogens may also exert beneficial effects by directly modulating the arterial tone [9,12,13] via the activation of estrogen receptors that have been found in the arterial wall [14 – 16]. In postmenopausal women, HRT reduces the angiotensin-converting enzyme activity by about 20% [17]. In vitro experiments on vascular tissues have revealed that estrogens may exert direct endothelium-independent [12,18 – 22] as well as endothelium-dependent effects [23,24]. However, compared with normally circulating plasma levels of the hormones, these in vitro effects require 1000 –10 000 times higher concentrations. The direct vasodilator effects of estrogens are rapid in onset and are unlikely to be caused by alterations in gene expression. Further studies have shown that estrogens activate the L-arginine/nitric oxide pathway in vascular endothelial cells [25,26], stimulate prostacyclin biosynthesis [27] and inhibit endothelin production in rat aortic smooth muscle cells [28]. Moreover, estradiol has antioxidative [29] as well as calcium-channel blocking properties [22,30 – 32] that may contribute to its protective vascular

effects. Animal experiments have also revealed the inhibitory effect of estrogens on the development of atherosclerosis and intima proliferation [33– 36]. Although in the last few years numerous studies have demonstrated direct cardiovascular effects of estrogens, there are few reports on the actions of progesterone and synthetic progestins as constituents of HRT on the vascular system [37–40]. Progesterone receptors have been demonstrated in venous and arterial vessels [37,41], indicating that blood vessels might be a target for progesterone action. Therefore, the aim of the present study was to examine the acute effects of progesterone, chlormadinone acetate (CMA), norethisterone acetate (NETA) and dienogest (DNG) in comparison to 17b-estradiol (17b-E2) on the vascular reactivity of isolated aorta from male rats.

2. Materials and methods

2.1. Substances The following substances were used: progesterone, chlormadinone acetate (CMA), norethisterone acetate (NETA), 17b-estradiol (17b-E2), dienogest (DNG), J 867 17b-(4-hydroxyiminomethylphenyl) -17b-methoxy-17a-methoxymethyl-estra-4,9-dien-3-one; (Jenapharm, Jena, Germany), ICI 182780 (Zeneca, Plankstadt, Germany), acetylcholine hydrochloride (Dispersa, Winterthur, Switzerland), phenylephrine and indomethacin (Serva, Heidelberg, Germany), glibenclamide (Research Biochemicals International, Natick, MA, USA), verapamil (Falicard®, Fahlberg List, Magdeburg, Germany), EGTA (Fluka, Buchs, Switzerland); IBMX (3-isobutyl-1methylxanthine) and forskoline (Sigma, Deisenhofen, Germany). Composition of Krebs-Henseleit solution (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, glucose 11. Composition of ‘calcium-free’ Krebs-Henseleit solution (mM): NaCl 62.7, KCl 60, MgSO4 1.2, KH2PO4 1.2, Tris 50, EDTA 0.03, glucose 11.

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Stock solutions of the hormones (10 mM) were prepared by dissolving the substances in ethanol (96%). The stock solution of EGTA (10 mM) was composed of 350 ml NaOH (0.1 M) and 650 ml NaCl (0.9%). Indomethacin (3 mM) was dissolved in a mixture of Na2HPO4/NaH2PO4 (0.5 M each), then diluted with distilled water. The other substances were dissolved in physiological saline. The concentration for each substance is expressed as final concentration in the organ bath.

2.2. Experiments with isolated rat aorta Adult male Wistar rats (250 – 300 g), under light ether anaesthesia, were killed by cervical dislocation and bled. The descending aorta was quickly explanted free and placed in Krebs – Henseleit solution. The aorta was then carefully cleaned of adhering connective tissue and cut into rings of 2 – 3 mm in length. Each ring was attached between L-shaped platinum hooks, placed in a 10ml organ bath containing Krebs – Henseleit solution (pH 7.4, 37°C) and gassed continuously with a mixture of 95% O2 and 5% CO2. Each vascular ring was connected to an isometric force transducer (Hugo Sachs Elektronik, March-Hugstetten, Germany) and changes in tension were recorded continuously. A passive resting tension of 20 mN was maintained throughout the experiments. During an initial stabilization period of 60 min, the bathing medium was changed every 20 min and the tension repeatedly readjusted to 20 mN. The vessel rings were contracted at 30-min intervals, once with KCl (30 mM) and three times with phenylephrine (1 mM), until the effect was reproducible. Functional integrity of the endothelium was assessed by relaxation of phenylephrineprecontracted vessels in response to the addition of acetylcholine (1 and 10 mM). This relaxation was absent after mechanical removal of endothelium by gently rubbing the intimal surface with a roughened plastic stick. To test the vasodilator activity of the hormones, rings were contracted with phenylephrine (1 mM), KCl (30 mM) or CaCl2 (3 mM) and relaxed by cumulative addition of the hormones. The relaxation was expressed as a percentage of the tonic contraction. In other experiments, the vessels were preincubated with

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the hormones for 30–90 min and contractions were then induced by KCl, phenylephrine or CaCl2. In addition, concentration-response curves were performed by cumulative addition of phenylephrine or CaCl2, the concentration in the organ bath being increased when steady state contraction to the previous concentration was achieved. The substances tested were added 30–90 min before the cumulative application of the agonists. To determine if the relaxant effects of hormones were dependent on the Ca2 + -concentration, aortic rings were incubated in Ca2 + -free, high K + (60 mM) depolarizing Krebs–Henseleit solution, according to the procedure described by Ebeigbe et al. [42]. Cumulative concentration-response curves were then obtained by gradually increasing the Ca2 + concentration in the bath (0.3–20 mM). The results were expressed as a percentage of the preceding contractile response induced by 3 mM CaCl2.

2.3. Determination of cAMP and cGMP Endothelium-denuded aortic rings (2–3 mm length) were preincubated in oxygenized Krebs– Henseleit solution (pH 7.3) at 37°C for 20 min and then with IBMX (100 mM) for 10 min. Thereafter, 17b-estradiol, progesterone or chlormadinone acetate (10 mM each) were added to two rings each and incubated for 15, 30 and 60 min. The reaction was stopped by freezing in liquid nitrogen. Parallel experiments were done with forskoline (0.5 and 5 mM) and with vehicle (ethanol 96%). The frozen samples were homogenized with 0.5 ml distilled water by means of a pinned disk disintegrator (for 1 min) and then treated with perchloric acid (final concentration 5%) at 4°C for 60 min. The homogenates were centrifuged at 10 000× g at 4°C for 5 min. The precipitated proteins were dissolved in NaOH (1 M) for protein determination, using bovine serum albumin as standard, according to the method of Lowry [43]. To 400 ml of the supernatant were added 100 ml EDTA (10 mM, pH 7.5) and 450 ml of a mixture of Freon/trioctylamine (1:1). After a short centrifugation (350×g, 4°C, 2 min) the

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Fig. 1. Relaxation of precontracted rat aortic rings with and without endothelium induced by progestins and 17b-estradiol (10 mM each). Contractions were elicited by phenylephrine (1 mM, gray column), KCl (30 mM, black column) or CaCl2 (3 mM, hatched column). Means 9 S.E.M., n= 4–6. *P B0.05, significant differences between relaxant effects on phenylephrine- and KCl-induced contractions. 17b-E2, 17b-estradiol; CMA, chlormadinone acetate, NETA, norethisterone acetate; DNG, dienogest.

aqueous upper phase was removed and lyophilized. The samples were then dissolved in buffer (pH 6.3) and used for radioimmunoassay (125I-RIA-Kit, Du Pont NEN®, Bad Homburg, Germany). The results were expressed as pmol cAMP/mg protein and pmol cGMP/mg protein, respectively.

level of probability of PB 0.05 was considered statistically significant.

3. Results

3.1. Relaxant effects of hormones on precontracted aortic rings

2.4. Statistical analysis The data were expressed as means9S.E.M. of n separate experiments. Comparisons between the different groups were performed using the Student’s t-test, modified according to Bonferroni. A

The relaxant effects of progestins in comparison to 17b-E2 were obtained on vessels with intact endothelium or on endothelium-denuded vessels precontracted with phenylephrine (1 mM), KCl (30 mM) or CaCl2 (3 mM). When the contractile

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Fig. 2. Relaxant effect of progesterone and 17b-estradiol (10 mM each) alone and in combination in endothelium-denuded aortic rings precontracted by (A) phenylephrine (1 mM) or (B) KCl (30 mM). Means9S.E.M., n =3 – 6.

response to the agonists had reached a constant level, the hormones were added cumulatively at concentrations from 0.01 to 10 mM. The relaxant effect of the hormones was already evident at 1 mM, but markedly pronounced at 10 mM. Due to the limited solubility of the hormones and expected unphysiological effects, higher hormone concentrations were not tested. The results are summarized in Fig. 1. There was no significant difference between intact and denuded vessels. In phenylephrine-precontracted vessels the relaxant effects of NETA and CMA were less pronounced than the response to progesterone, whereas in the KCl- and CaCl2-precontracted vessels this difference disappeared. With the exception of progesterone, the relaxant responses were significantly (PB 0.05) more pronounced when the KCl-induced contractions were studied. The rank order of potency for relaxation was: 17b-E2 \progesterone \CMA=NETA. Dienogest and the solvent ethanol did not induce any significant relaxation. The combination of progesterone and 17b-E2 did not show a potentiation of relaxation, but rather an additive effect was observed (Fig. 2).

The relaxation induced by progesterone (10 mM) and other hormones could be repeated after washouts at intervals of 30 min, suggesting that the acute action of the hormones was reversible. The vasorelaxing effect of progesterone was timedependent. After a delay of 2–3 min, the relaxation increased gradually and reached a sustained maximum after 20–30 min. The same effect was seen with 17b-E2 (Fig. 3). The endothelium-independency of the responses could be confirmed in these experiments. The acetylcholine-induced endothelium-dependent relaxation of phenylephrine-precontracted aortic rings was not significantly affected by pretreatment (60 min) with progesterone (10 mM) (Fig. 4). The same result was obtained with 17bE2 (10 mM, not shown).

3.2. Influence on the contractile response to phenylephrine Pretreatment of aortic rings with progestins or 17b-E2 (1 and 10 mM each) produced a concentration-dependent decrease in the contractile response to 1 mM phenylephrine (Fig. 5). Progesterone and CMA at 10 mM for 60 min

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Fig. 3. Time-dependency of the response to progesterone and 17b-estradiol in intact (dotted lines) and endothelium-denuded (solid lines) rat aortic rings precontracted by KCl (30 mM). Means 9 S.E.M., n= 3 – 5.

attenuated the phenylephrine-induced contraction by about 25 and 27% respectively, (P B 0.05), whereas 17b-E2 reduced it by about 46% (P B 0.05). The inhibitory effects of NETA and DNG amounted to 18 and B10%, respectively. Preincubation of the vessels with progestins or 17b-E2 for 60 min caused a greater inhibition than for 30

Fig. 4. Influence of progesterone (10 mM) on acetylcholine-induced relaxation of intact rat aortic rings precontracted with phenylephrine (1 mM). Control (solid line), after pretreatment for 60 min with progesterone (dotted line). Means 9 S.E.M., n=4.

min, whereas a prolongation of the pretreatment time until 90 min did not further increase the inhibitory effect.

3.3. Studies on the mode of action of hormones To inhibit vascular eicosanoid production by cyclooxygenase, vessels were treated with indomethacin (3 mM) for 20 min and then contracted with phenylephrine or KCl. Under these conditions, the vasodilator effects of progesterone or 17b-E2 were not impaired, indicating that eicosanoid metabolism was not involved in the relaxant actions of the steroids. To exclude interference of the hormones with ATP-sensitive potassium channels, the vessels were treated with glibenclamide (1 mM) for 20 min. This specific potassium channel inhibitor did not influence the relaxant responses induced by progesterone or 17b-E2, indicating the absence of a role for potassium channels in these responses. Moreover, the question arose whether the acute vascular effects of hormones can be counteracted by antagonists of progesterone (J 867) or estradiol (ICI 182780) receptors. After preincubation of the endothelium-denuded vessels with these antagonists (1 mM each) for 20 min, contractions were induced by KCl, followed by the hormone-mediated relaxations. Neither the progesterone- nor

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Fig. 5. Inhibition of the contractile effects of phenylephrine (1 mM) caused by pretreatment for 60 min with progestins and 17b-estradiol (hatched columns, 1 mM; gray columns, 10 mM). Means 9 S.E.M., n = 5. *PB 0.05 to the control (without hormones).

the 17b-E2-induced relaxations were significantly inhibited by the used receptor antagonists. The relaxant effects of progestins and 17b-E2 were more pronounced when contractions were triggered by high K + depolarization. These findings encouraged us to investigate the influence of the hormones on Ca2 + -induced contractions in K + -depolarized aortic rings. After replacement of the normal Krebs–Henseleit solution by calciumfree solution containing EDTA and higher KCl (60 mM), a single contraction was induced by the addition of CaCl2 (3 mM). After washing and following re-equilibration, the hormones were added to the organ bath and 60 or 90 min later, cumulative concentration-response curves were obtained by addition of increasing concentrations of CaCl2 (from 0.1 to 20 mM). For comparison, the calcium channel blocker verapamil (0.3 mM) produced a marked antagonism, resulting in a pronounced rightward shift of the concentration response curve to CaCl2. A similar, but less pronounced effect, was seen with progesterone (up to 10 mM) (Fig. 6). The reponse to progesterone was not changed in the presence of the progesterone receptor antagonist J 867 (1 mM). NETA at 10 mM exhibited the same inhibition as progesterone (not shown). 17b-E2 (1 and 10 mM) also produced a concentration-dependent decrease of the contraction induced by Ca2 + and shifted the concentration-response curve to the right and

downward. The estrogen receptor antagonist ICI 182780 (1 mM) did not affect the action of 17b-E2 (Fig. 7). Several studies in vascular tissues have indicated that gonadal steroid hormones might modulate cAMP and cGMP levels in vascular smooth muscle [12,44]. After 15, 30 and 60 min incubation of endothelium-denuded aortic rings with progesterone, CMA or 17b-E2 (10 mM each), the tissues were quickly frozen and applied for determination of cyclic nucleotides by radioimmunoassays. The results at 15 min incubation time are given in Table 1. Independently of the incubation time, there was no increase in cAMP or cGMP. Forskoline (0.5 mM) was used as a positive control, causing a marked increase in cAMP levels.

4. Discussion Gonadal steroid hormones are capable of eliciting a variety of nongenomic effects in the vascular system. The effects on isolated vessels are dependent on the chemical structure of hormones, gender and species studied, as well as the type of vessels and localization. Therefore, great differences in the effects of hormones were observed. In our experiments using isolated aortas from male rats in addition to 17b-E2% progesterone and synthetic progestins, with the exception of

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Fig. 6. Inhibitory effect of progesterone (10 mM) in comparison to progesterone +J 876 (1 mM) and verapamil (0.3 mM) in aortic rings contracted by addition of CaCl2 (0.3–20 mM) to Ca2 + -free, high K + (60 mM) medium. Ordinate: Percentage of the preceding contraction obtained with 3 mM CaCl2. Means9 S.E.M., n=4. *PB0.05 to the control (without pretreatment).

dienogest, caused endothelium-independent longlasting vasodilator effects. The acetylcholine-induced endothelium-dependent transient relaxation was not enhanced in the presence of progesterone and 17b-E2. The acute relaxant response was reversible and the effect could be repeated after washouts. Besides the direct vasodilator effects, progestins also attenuated the contractile response to phenylephrine and calcium chloride. After treatment of dogs with estrogen and progesterone for several days, the in vitro relaxant effects on vessels induced by acetylcholine were lower in the combination than after treatment with estrogen alone [39]. Progesterone alone did not significantly affect the endothelium-dependent relaxation induced by acetylcholine, while estrogen alone somewhat enhanced the relaxation. In monkeys, treatment with equine estrogens for one month led to an endothelium-mediated dilatation of atherosclerotic coronary arteries, which was measured by coronary angiography [45]. However, the addition of medroxyprogesterone acetate diminished the vasodilating effect of estrogens [45]. In contrast to the present acute experiments on isolated vessels, there was a longer exposure of vessels to medroxyprogesterone in vivo, whereby interactions at the hormone receptor sites are possible. Moreover, progestins are known to dif-

fer in their potency and probably in their vascular effects [13]. On the other hand, progesterone did not interfere with the inhibitory effect of 17b-E2 on growth factor-induced vascular smooth muscle proliferation [36]. In our study, peak vascular effects of progesterone and synthetic progestins occurred within 60–90 min. This is in contrast to the intracellular receptor-mediated genomic effects that require a longer period of time. Moreover, it has to be taken into account that only at concentrations considerably exceeding the physiological serum levels did progesterone and estradiol produced the vascular effects in vitro and in vivo such as could be confirmed in most studies [20,22,31,39,40,44]. Using an in vitro incubation technique Bell et al. [46] have shown that overnight exposure of porcine coronary arteries to 1 nM 17b-E2, a concentration close to physiological levels, enhanced the endothelium-dependent vasorelaxant responses to the calcium ionophore A-23187. For the acute reversible vascular effects the steroid receptor-mediated intracellular transcription processes do not seem to be important. Specific antagonists of estrogen or progesterone receptors did not antagonize the action of the steroid agonists on the vessels. In porcine coronary arteries, tamoxifen, an antagonist of the

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Fig. 7. Inhibitory effect of 17b-estradiol in comparison to 17b-estradiol+ ICI 182780 (1 mM) in aortic rings contracted by addition of CaCl2 (0.3 – 20 mM) to Ca2 + -free, high-K + (60 mM) medium. Ordinate: Percentage of the preceding contraction obtained with 3 mM CaCl2. Means9S.E.M., n=4. *PB0.05 to the control (without 17b-estradiol).

cytosolic estrogen receptor, did not change the action of estradiol [31]. The present data confirm previous studies on the action of progesterone on rabbit coronary arteries [39]. In those studies the progesterone-induced relaxation was also endothelium-independent, not inhibited by indomethacin or glibenclamide and the inhibition of calcium-induced contractions was quite similar to our results. In isolated rat aorta, Thomas et al. [20] have shown that the phenylephrine-induced contracTable 1 Influence of hormones and forskoline on cAMP and cGMP concentrations in endothelium-denuded rat aortic rings after 15 min incubation time Substances

Control (+ethanol) Progesterone 10 mM CMA 10 mM 17b-E2 10 mM Forskoline 0.5 mM Forskoline 0.5 mM+progesterone 10 mM

Concentration (pmol/mg protein) cAMP

cGMP

7.9 91.8 5.790.7 6.0 9 1.8 6.5 9 1.2 32.79 5.0 27.2 9 6.3

1.2 9 0.4 1.4 9 0.2 1.59 0.6 1.99 0.8 1.49 0.3 1.19 0.2

Values are expressed as means 9S.E.M.; n= 3.

tions were suppressed by 17b-E2 and progesterone to an extent similar to that observed in our studies. Furthermore, our results indicate that eicosanoids, changes in vascular cGMP and cAMP concentrations or release of endothelial nitric oxide, are unlikely to be involved in the mechanism of progesterone- as well as estrogen-induced relaxation in the rat aorta. The same seems to be true for activation of ATP-sensitive potassium channels, as has been shown by Jiang et al [39]. In porcine coronary arteries 17b-E2 did not influence the levels of cAMP and cGMP either [31], whereas other studies showed an augmentation [12,44]. The endothelium-independent mechanisms of progesterone-induced vasorelaxation may involve acute nongenomic alteration of cation channel function, as has been reported for estrogens. The latter inhibit or block voltage-operated Ca2 + channels in vascular smooth muscle [19,22,31,32]. Progesterone (1 mM) was found to suppress the Ca2 + -induced contractions of rat portal veins probably due to a K + channel opening mechanism [40]. Progesterone inhibited the contractile response induced by high K + depolarization and the Ca2 + -induced contractions in K + -depolarized aortic rings. These responses have been at-

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tributed to Ca2 + entry through voltage-operated channels. Moreover, progesterone also exerted an inhibitory effect on total contractile responses to phenylephrine, suggesting that it may also block Ca2 + in response to this agonist. Thus, these findings suggest that the vasodilator effects of progesterone, CMA and NETA as well as of 17b-E2 seem to be due to inhibition of Ca2 + uptake through voltage- and/or receptor-operated channels.

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