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Effects of acute and chronic estrogenic treatment on vasomotor responses of aortic rings from ovariectomized rats
Life Sciences, Vol. 57, No. 5, pp. 473-486, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved @x4-3205/9s $9.50 + .oo
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EFFECTS OF ACUTE AND CHRONIC ESTROGENIC TREATMENT ON VASOMOTOR RESPONSES OF AORTIC RINGS FROM OVARIECTOMUED RATS M.C. Paredes-Carbajal, Department
M.A. Jukez-Oropeza*,
C.M. Ortiz-Mendoza
and D. Mascher
of Physiology and *Department of Biochemistry, School of Medicine, II.N.A.M., P.O. Box 70-250, Mexico D.F. 04510, MEXICO (Received in final form May 8,
1995)
Summary The effects of either chronic or acute estrogenic treatment on the “in vitro” vasomotor responses to phenylephrine (1 Oe9-10.’ M) and to carbachol ( 1OS“‘10.’ M) of aortic rings excised from ovariectomized rats were analyzed Chronic estrogenic treatment consisted in a single subcutaneous dose of 1 umol estradiol 17-stearate. Effects of acute estrogenic treatment were evaluated by recording the responses of aortic rings excised from untreated ovariectomized rats both before and after the addition of 17l3estradiol to the superfUsing solutions. In order to identify the endothelium-dependent responses each experiment was performed simultaneously on pairs of rings from the same aorta, one with and the other without functional endothelium. The contractile responses to phenylephrine of endothelium-intact vessels were attenuated by chronic estrogenic treatment; this attenuation was mrther increased by preincubation of the vessels with indomethacin and was reverted by No-nitro-L-arginine methyl ester. Either chronic or acute estrogenic treatment enhanced the carbachol-induced endothelium dependent relaxation of phenylephrine-precontracted rings. The results may be explained by assuming that estrogens increase the basal release of both nitric oxide and a cyclooxygenase-dependent vasoconstricting prostanoid as well as the receptor-mediated release of nitric oxide from the endothelium of the rat aorta. Kq Wotds: endothelium, estrogens, nitric oxide, prostanoids, aortic rings Women in the reproductive age have a lower prevalence of atherosclerosis related diseases than age matched men (1,2). This difference decreases after both surgical and natural menopause and postmenopausal women are at a much higher risk of developing coronary artery disease than premenopausal women of similar age (3,4). On the other hand, estrogen replacement therapy lowers the incidence of atherosclerotic diseases in ovariectomized and postmenopausal women (5-9). The beneficial effects of estrogens are even greater in women with already existing atherosclerotic disease (IO). Based on these epidemiological observations a “protective” role of estrogens, preventing atherosclerotic vascular diseases has been postulated. Several mechanisms by which this protection may be achieved have been proposed (5, 11-l 5). In the last few years, it has become evident that Corresponding author: Ma. Cristina Paredes-Carbajal. Department of Physiology, School of Medicine, U.N.A.M., P.O. Box 70-250, Mexico D.F. 045 10, MEXICO. FAX (525) 623-224 I E-mail [email protected].
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endothelium-dependent control of vascular tone is impaired early in atherosclerosis (16, 17) and, therefore, a putative role of the endothelium in the protective effects of estrogens has been suggested (18, 19). The effects of estrogens on endothelium-dependent responses of isolated arteries have been analyzed in different experimental models (18, 20-24). Evidence provided by these studies supports the hypothesis that estrogens modulate the release (and/or the action) of vasoactive substances from the endothelium. The present study was designed to answer two main questions: Does chronic or acute exposure to estrogens modify the contractile response of the isolated rat aorta to the CL,-adrenergic agonist phenylephrine? Do estrogens mow the carbachol-induced endothelium-dependent relaxation of this vessel? The results showed that estrogens attenuate the contractile response to the vasoconstrictor and accentuate the response to the vasodilator. Methods Animals. The experiments were performed on isolated aortas excised from adult nonpregnant female Wistar rats (250-300 g). The animals were ovariectomized bilaterally under sodium pentobarbital (32 mg/k& i.p.) anesthesia. Prophylactic crystalline penicillin (400,000 U/kg i.m.) was administered after surgery and eight days were allowed for recovery. Thereafter, the animals were randomly allocated to two groups, untreated and treated. Rats from the untreated group were used lo-14 days aRer surgery. Animals from the treated group received subcutaneously a single dose of 1 pmol estradiol 17-stearate dissolved in 0.3 ml castor oil. Previous work (25, 26) showed that, three days after administering a single dose (0.1-l .O pmol) of estradiol ester derivatives to ovariectomized rats, serum levels of 17&estradiol were restored to values close to those measured during estrus in nonpregnant rats and remained at these levels for up to 30-60 days. Rats of this group were used 1 l-l 3 days after treatment. AU animals were kept in individual cages with free access to food and water and exposed to a 12-h light-dark cycle. In vitro measurements of vascular responses. Animals were killed by cervical dislocation and the thoracic aorta was immediately removed, cleaned of connective tissue and cut transversely into rings (2 mm long). Special care was taken to avoid damage to the endothelium. In every other ring, the endothelium was removed by gently rubbing the intimal surface. For each experiment a pair of rings from the central portion of the same aorta (one with intact endothelium, the other without fimctional endothelium) was used. Each of these rings was suspended horizontally in the same miniature organ chamber (volume 1 ml) between two stainless steel hooks. One of the hooks was fixed to the chamber wall while the other was attached to an isometric force transducer (Grass, FT 03). The vessels were continuously superfused (3 ml/min) with prewarmed (37 C) aerated (95% 0, and 5% CO,) modified Tyrode solution (composition in mM: NaCl, 137; KC], 2.7; MgCl,, 0.69; NaHCO,, 11.9; NaHPO,, 0.4; CaCl,, 1.8 and glucose, 10; pH was 7.4). The rings were initially stretched until resting tension reached 2 g and allowed to equilibrate for one hour; during this period the resting tension was continuously monitored (Grass, Model 79 Polygraph) and, if needed, readjusted to 2 g by tirther stretching. Before starting an actual experiment, responsiveness of each pair of rings to phenylephrine and carbachol was tested. This was achieved by switching the superfUsing Tyrode solution for IO min to one containing phenylephrine (10S5 M) and, thereafter, to one containing, in addition to phenylephrine, carbachol (lo-’ M). Carbachol-induced relaxation of the phenylephrine precontracted vessels was taken as evidence for the preservation of an intact endothelium whereas lack of relaxation confirmed the absence of a functional endothelium.
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Experimental protocol, Two series of experiments were performed in order to analyze, respectively, the effects of either chronic or acute estrogenic treatment on the concentration-response curves of aortic rings from ovariectomized rats to phenylephrine and to carbachol. In each of these series, the contractile response of pairs of aortic rings to cumulative increasing concentrations ( 10m9- 10W5 M) of the a,-adrenergic agonist phenylephrine (concentrationresponse curve to phenylephrine) was initially recorded and, once tension development in response to the highest phenylephrine concentration reached its peak value, superfusion was switched to solutions having, in addition to phenylephrine, succesively increasing concentrations (1 Oe9- 1Oe5M) of carbachol (concentration-response curve to carbachol). In the first series of experiments these two succesive concentration-response curves were determined, both in abscence and presence of the cyclooxygenase inhibitor indomethacin ( 1O-5M), in rings excised from rats of either the treated or the untreated group ( n = 6 for each group). In the second series of experiments concentration-response curves to phenylephrine and to carbachol were obtained, in the presence of indomethacin (I Om5 M), in vessels excised from untreated animals, both before and after (40 - 60 min) addition of 178-estradiol (10m9M) to the perfusing solutions (n = 7). In both series of experiments the concentration response curves to phenylephrine were also obtained in the presence of both indomethacin and the competitive inhibitor of nitric oxide synthase No-nitro-L-arginine methyl ester (L-NAME, 300 PM; n = 6 for the first series of experiments; n = 4 for the second series). Data analysis. The contractile responses induced by phenylephrine are expressed as tension increment in grams above the basal tension (imposed on the vessel throughout the experiment). Carbachol-induced relaxation is expressed as the percent reduction in tension relative to maximal tension developed in response to phenylephrine (10” M). All data are expressed as means f S.E. EC,, (-Log of the mean molar concentration of agonist producing 50% of the maximal response) was determined with the software package Graph Pad Prism 10855 (San Diego, CA. 92121 USA). Comparisons of means were made by Student’s t test for unpaired (chronic treatment) and paired (acute treatment) values. Chemicals. All chemicals and drugs, except estradiol 17-stearate, were purchased from Sigma Chemical Co. (St. Louis, MO. USA) Estradiol 17-stearate was a gift of J.C. Diaz-Zagoya, and prepared according to V&zquez-Alc&ttara et al. (2526). Indomethacin was dissolved, by sonication, in 4% sodium carbonate. 17R-estradiol was dissolved in dimethyl sulfoxide. L-phenylephrine hydrochloride, carbachol (carbamoylcholine chloride) and No-nitro-L-arginine methyl ester hydrochloride (L-NAME) were dissolved in distilled water.
I. Effects of chronic (“in viva”) treatment with estradiol 17-stearate 1. Concentration-response
curves to phenylephrine
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A. Aortic rings with functionally
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intact endothelium.
Aortic rings excised from rats that received estrogenic treatment developed less tension in response to each of the tested phenylephrine concentrations than the rings harvested from untreated animals (compare filled circles and open circles in Fig. 1A). Statistical analysis showed that the difference was significant (pcO.05) for the responses to IO-‘, 10m6and 10m5M phenylephrine. Tension developed in response to the highest phenylephrine concentration tested (maximal contractile response) amounted to 1.69 i 0.23 g for rings excised from treated rats and to 2.21 f 0.13 g for vessels from untreated rats. Table I summarizes the EC,, calculated for this series of experiments; it can be seen that estrogenic treatment shifted the EC,, significantly (p < 0.05) from 6.82 * 0.04 to 6.42 z+z 0.01,
TABLE
I
Effects of Chronic Treatment with Estradiol17-stearate to Phenylephrine (10~9-10-SM) of rat Aortic Rings. Group
With endothelium Max. tension EC,,
on the Concentration-Response
Curve
Without endothelium Max. tension EC,,
Yntreated (-)indom. (+)indom.
682*004 6.86 & 0.03
2.2110 13 2.33 i 0.17
7 60 * 0.015 754iO.I0$
2.22 f 0 24 2.20 * 0.32
6.42 f O.Ol* 1 69 * 0.23* 6.52 + 0.03* 1.11 * 0.22”A
6.92 -I-0.123 7.16*0.06$
2.36 f 0 23 1.99 f 0.14
7.11 io.09
7 21 f 0.04
2.45 I 0.1 1
Treated (-)indom. (t)indom. (+)indom. (+)L-NAME
1.91 10 10
EC,,. -log of mean molar concentration causing 50% of maximal response to phenylephrine (IO-‘M). Max. tension: Tension (in grams) developed in response to phenylephrine (1 V5M). (-)indom: without indomethacin. (+)indom: with indomethacin ( 10m5M).(+)mdom (+)L-NAME: with indomethacin ( 10erM) and L-NAME (300pM). Data are presented as means i SE; n=6 for all groups * Denotes that differences among treated and untreated groups are significant (p
Additionally, it was observed that in the aortas from rats that received the hormonal treatment, tension development during continued exposure to a given concentration of phenylephrine tended to be transient, i.e., it started to decline gradually soon after reaching its peak value and, in some experiments, decayed to its initial level after a few minutes despite the continuous presence of the
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and Vasomotor Responses
contractile agonist. These transient responses of the vessels from estrogen treated animals contrasted strongly with the well sustained contractile response observed in aortas from untreated rats. The attenuation of the contractile response to phenylephrine observed in the aortic rings from treated rats was markedly accentuated when exposure to phenylephrine was performed in the presence of indomethacin (compare filled triangles and filled circles in Fig. 1 A). Under this experimental condition the maximal contractile response to phenylephrine amounted to 1.11 + 0.22 g in the rings from treated rats and to 2.33 * 0.17 g in those from untreated animals. Indomethacin also accentuated the transient nature of the phenylephrine mediated contractile response of the rings excised from treated animals. In the presence of indomethacin, addition of NAME (300 pM) to the superfUsing solutions abolished the above described differences among the contractile responses to phenylephrine of the aortic rings obtained from either the treated or the untreated rats (not shown).
B
_j Phenylephrine
I
I
I
1
7
-9 -8 -7 -6 -5 Phenylephrine (log M)
(log Ml
Fig. 1 Eff6cts of chronic treatment with estradiol 17-stearate on the concentrationresponse curves to phenylephrine, of aortic rings with (A) or without (B) endothelium. Each panel illustrates the responses of aortic rings excised from either untreated (open symbols) or estrogen treated (filled symbols) ovariectomized rats. Curves were obtained either in the absence (circles) or the presence (triangles) of indomethacin. Data are presented as means f SE of six rats in each group. For clarity SE-bars were omitted in panel B.
B. Aortic rings without functional endothelium Each of the experiments was performed simultaneously on a pair of aortic rings: one having a fUnctiona endothelium, the other without a fUnctional endothelium. In each pair, the contractile
Estrogens and Vasomotor Responses
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response to a given concentration of the ring with endothelium.
of phenylephrine
Vol. 57, No. 5, 199.5
of the ring without endothelium exceeded that
The dose-response curve of the rings excised from treated rats did not differ significantly from that of rings from untreated animals (compare open and filled circles in Fig 1B) Indomethacin endothelium-denuded
did not significantly rings.
2. Concentration-response
modify the contractile
responses to phenylephrine
of
curve to carbachol
Aortic rings with intact endothelium relaxed in a dose dependent manner in response to increasing concentrations of carbachol. The dose-response curve of the rings excised from the estrogen treated rats was significantly (p
TABLE II Effects of Chronic Treatment with Estradiol Carbachol (10m9 - 10.’ M) in Endothelium Phenylephrine (10” M). Group
(-)indom. (+)indom.
EC,,
17-stearate on the Relaxation induced by Intact Aortic Rings Precontracted with
Maximum Percent Relaxation
6.03 h 0.09 6.05 f- 0.06
76.90 i 8.42 75 00 + 10.81
6.5 1 f 0.29* 6.46 f 0.28*
89.98 f 2.57 82.46 f 5.97
Treated (-)indom. (+)indom.
EC,,: -Log of mean molar concentration causing 50% of maximal relaxation induced by carbachol. Maximum percent relaxation: maximal percent decrease in tension compared with maxima1 tension induced by phenylephrine (IO-’ M). Values are presented as means f SE; n=7 for all groups. * Denotes that differences between treated and untreated groups are significant (p
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.-
P 2
60
:: E
40-
-
0 s
20-
0’,
, -9
I
-8 Carbachol
I
-7 (log
I
I
-6
-5
M)
Fig. 2 Effects of chronic treatment with estradiol 17-stearate on the concentrationresponse curves to carbachol of endothelium intact aortic rings. Vessels were excised either from untreated (open symbols) or treated (tilled symbols) ovariectomized rats Responses recorded in the absence (circles) or in the presence (triangles) of indomethacin are plotted. Rings were precontracted with phenylephrine (lo-’ M) Data are expressed as percent of maximal phenylephrine-induced tension and shown as means h SE of six rats in each group. (*) Denotes that relaxations of the estrogen treated groups are significantly different from those of the untreated groups (p
II. Effects of acute (“in vitro”) treatment with 170-estradiol In this series of experiments concentration-response curves to phenylephrine and to carbachol were performed, in the presence of indomethacin, on pairs of aortic rings excised from ovariectomized rats (without estrogenic treatment) both, before and after (40 to 60 min) addition of 17g-estradiol ( 10m9M) to the supetising solutions. With this protocol each pair of rings served as its own experimental control for evaluating hormonal effects.
A. Concentration-response
curves to phenylephrine
As in the previous series of experiments (chronic estrogenic treatment) the dose-response curve to phenylephrine of the aortic rings with disrupted endothelium was significantly (p < 0.05) shifted to the left (EC,: 7.45 f 0.08) compared with the corresponding curve of the rings with intact endothelium (EC,,: 7.06 * 0.09) (Fig. 3 and Table III). Neither in the rings with intact endothelium nor in those without functional endothelium did the addition of 17LLestradiol modifji significantly the concentration-response curve to phenylephrine
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3-
52
-
5 ._ 2 I-“1
-
O-
I
1
-9
I
1
-7 -8 Phenylephrine
I
1
-6 -5 (log M)
Fig. 3 Effects of acute treatment with 17Pestradiol on the concentration-response curves to phenylephrine of aortic rings with (circles) or without (squares) endothelium excised from ovariectomized rats and preincubated with indomethacin. Responses were recorded in the same pair of rings before (open symbols) and after (tilled symbols) addition of 17lSestradiol to the superfusing solution. Data are expressed as means of rings from seven rats in each group. SE-bars were omitted for clarity.
TABLE II1 Effect of Acute Treatment with 17Gestradiol (10-9M) on the Concentration-Response Curve to Phenylephrine (10-g-10-5M) of Aortic Rings excised from ovariectomized rats and Preincubated with Lndomethacin (10.‘M). Group
With Endothelium
Without Endothelium
n
EC,,
ECS,
7.06 * 0.09
7.45 i 0.08*
7
6.89 f 0.05
7.21 i O.lO*
7
7.09*0.11
7.33 f 0.13
4
Treated f+)NAME
EC,,: -Log * SE of mean molar concentration causing 50% of maximal response to phenylephrine (1 O-‘M). * Denotes that the difference between the EC,, of rings with or without endothelium is significant (~~0.05).
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B. Concentration-response
curve to carbachol
The dose-response curve to increasing cumulative concentrations of carbachol of the rings with intact endothelium and precontracted with tom5M phenylephrine, performed in the presence of 17f&estradioi, was significantly (p < 0.05) shifted to the left, compared with the corresponding curve obtained before the exposure to the hormone (compare filled and open triangles in Fig. 4) The calculated values for the respective EC,,, are listed in table IV
100
0
1
1,
, -9
-8 Carbochol
I
I
I
-7
-6
-5
(log
M)
Fig. 4 Effects of acute treatment with 17&estradiol( IO-’ M) on the concentration-response curves to carbachol of endothelium intact aortic rings excised from ovariectomized rats and preincubated with indomethacin (10-j M). Responses to carbachol were obtained in the same ring before (open triangles) and after (filled triangles) addition of 17kestradiol to the super-king solution. Rings were precontracted with phenylephrine (10-j M). Data are expressed as percent of maximal phenylephrineinduced tension and shown as means k SE of vessels Corn seven rats in each group. * Denotes that relaxation of estrogen treated rings is significantly greater (~‘0.05) than that of the untreated ones.
Estrogens and Vasomotor Responses
TABLE
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IV
Effects of Acute Treatment with 17kestradiol (10.’ M) on the Relaxation induced by Carbachol (10-g-10-5M),in Endothelium Intact Aortic Rings excised from ovariectomized rats, Precontracted with Phenylephrine (10”M) and Incubated with Indomethacin (10.‘M). Group
EC,,
Maximum percent relaxation
Yntreated
6.04 f 0.05
75.86 * 5.51
Treated
6.64
87.58 * 4.44*
l
0.14’
EC,,: -Log of mean molar concentration causing 50% of maximal relaxation induced by carbachol (lO-M). Maximum percent relaxation: mean percent decrease in tension compared with maximal tension induced by phenylephrine (10-W). Data are presented as means * SE; s7 for all groups. * Denotes that differences among values observed before and after estrogenic treatment are significant (pcO.05).
One of the approaches that has been extensively used to elucidate the mechanisms by which estrogens perform their protective role on the vascular system has been to explore their effects on the “in vitro” responsiveness of isolated vessels to vasoactive agents. The results of these studies are controversial. This controversy may be explained, partly, by differences in the experimental model. These include the use of different species and vessels, the evaluation of varying vasoactive agents, differences in the methods of “in vitro” measurement of vascular responsiveness and differing schemes of estrogenic treatment. In the present study the effects of estrogens on the responsiveness of the rat aorta to just one vasoconstrictor -phenylephrine- and one vasodilator -carbachol- were analyzed. Lphenylephrine, a selective a,- adrenergic agonist was selected assuming that it would activate smooth muscle contraction without simultaneously inducing receptor mediated release of vasoactive substances from the endothelium. This assumption is based on the presumptive absence of aradrenergic receptors in this structure (27). Carbachol was selected because it consistently produces endothelium-dependent, nitric oxide-mediated, relaxation in the isolated rat aorta without having any relaxing effect on vessels lacking a hmctional endothelium (28). In addition, there is no evidence of carbachol-induced release of vasoactive metabolites of arachidonic acid. Regarding the “in vitro” measurement of vascular responsiveness, the method used in this study differs from those used by other authors. Vessels were continuously super-fused in a small organ chamber in an attempt to avoid, on the one hand, uncontrolled alterations of the extracellular medium (by either accumulation or depletion of molecules) and, on the other hand, insure -when desired- its fast and homogeneous modification. In each experiment a pair of rings (with and without fimctional endothelium, respectively) obtained from the same aorta and placed in a common organ chamber was studied; this allowed identification of the endothelium dependent responses during each individual experimental procedure. As to the estrogenic treatment, two schemes were used: chronic “in viva” treatment and acute “in vitro” exposure. Chronic treatment consisted in subcutaneous administration of a single dose
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of estradiol 17-stearate. According to Vazquez-Alcantara et al. (25, 26) this procedure insures longlasting estrogenic activity as evidenced by vaginal cytology, uterotrophic action, inhibition of serum gonadotrophins and serum levels of 17D-estradiol. Acute exposure to estrogen was achieved by addition of 17R-estradiol to the sup&using solutions Rings with endothelium excised from estrogen treated rats developed less tension in response to phenylephrine than corresponding rings from ovariectomized rats. This difference was markedly enhanced in the presence of indomethacin, and was no longer observed after addition of L-NAME to the superfusiig solutions. On the other hand, no differences were observed among the contractile responses to phenylephrine of rings without functional endothelium excised from either treated or untreated rats. Indomethacin did not modify the phenylephrine induced contraction of endotheliumdenuded rings frL>rneither group of rats. Taken together, these results may be explained by assuming that chronic estrogenic treatment of ovariectomized rats increases the basal synthesis and release -by the endothelium of the aorta- of both nitric oxide (NO) and a cyclooxygenase-dependent vasoconstricting metabolite of arachidonic acid. The assumption that estrogens increase the basal release of NO agrees with the results of Hayashi et al. (23) who found that basal (but tone related) release of NO is greater in endotheliumintact aortic rings excised from female rabbits than in those from either ovariectomized or male rabbits. It is, also, in line with the recent report by Weiner et al. (29) showing that estradiol treatment (and pregnancy) increases -in the guinea pig- the activity of calcium-dependent nitric oxide synthase in the uterine artery and in several organs as well as the amount of rnRNAs for both, the endothelial and the neuronal isoforms of the constitutive nitric oxide synthase, in skeletal muscle. The latter finding strongly suggests that estrogens may increase basal release of nitric oxide by enzyme induction. If this were indeed the case, and since enzyme induction requires a relatively long time, it would explain the present finding that acute exposure to 17Pestradiol had no significant effect on the contractile responses to phenylephrine of aortic rings excised from ovariectomized untreated rats and preincubated with indomethacin. The proposal that chronic estrogen treatment may increase, also, the synthesis and release of a cyclooxygenase dependent vasoconstricting prostanoid is in line with the findings of Miller and Vanhoutte, (30). These authors reported that the endothelium- and cyclooxygenase-dependent contractions to arachidonic acid of aortic rings excised from estrogen treated ovariectomized rabbits were greater than those of vessels from untreated animals, In addition, the authors observed, in the same vessel, an increased endothelium- and cyclooxygenase-dependent sensitivity to norepinephrine. Based on these results they concluded that “under the influence of estrogens, norepinephrine must stimulate the metabolism of arachidonic acid in the endothelial cells that in turn enhances the contraction of the vascular smooth muscle evoked by the adrenergic agonist.” This conclusion, taken together with the above discussed evidence obtained by Hayashi et al. (23) could mean that, as suggested by the present results, estrogens increase, also in the rabbit aorta, the synthesis and release of both NO and a cyclooxygenase-dependent vasoconstrictor. Estrogenic treatment, either chronic or acute, increased significantly the carbachol-induced endothelium-dependent relaxation of phenylephrine-precontracted aortic rings. Indomethacin had no effect on the carbachol induced relaxation of vessels excised from either treated or untreated animals. Since it is well documented that the activation of endothelial muscarinic receptors induces the synthesis and release of NO (28) these results indicate that estrogenic treatment enhances the receptor mediated release of NO from the endothelium of the rat aorta. Regarding the effects of chronic estrogenic treatment on receptor mediated NO release, the same conclusion has been reached previously in several studies. G&lard et al. (18) reported that femoral arteries from 17R-estradiol
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treated rabbits show an enhanced endothelium-dependent relaxation in response to low doses of acetylcholine. Weiner et al. (2 1, 22) observed that acetylcholine-induced NO-mediated relaxation of uterine and carotid arteries from the guinea pig is increased during pregnancy. Williams et al. (3 1) reported that “in situ” atherosclerotic coronary arteries of ovariectomized cynomolgus monkeys responded to intracoronary inmsed acetylcholine with “paradoxical” constriction and that chronic estrogenic treatment reverted the constriction to a moderate dilation. Keany et al. (24) observed that chronic estrogenic treatment of ovariectomized hypercholesterolemic miniature swine preserves the endothelium-dependent relaxation of coronary artery rings to bradykinin and substance P, whereas vessels from untreated animals exhibited impaired relaxations to these agonists. It should be mentioned, however, that there are also studies reporting that chronic estrogenic treatment has no effect on the receptor-mediated release of NO. Miller and Vanhoutte, (30) observed no difference among acetylcholine-induced relaxations of aortas excised from either estrogen treated or untreated ovariectomized rabbits. Miller and Vanhoutte, (20) reported that the receptor-mediated relaxations of coronary arteries from ovariectomized dogs in response to the endothelium-dependent vasodilators, acetylcholine, adenosine diphosphate, and bradykinin, were similar in vessels from either estrogen treated or untreated animals, they observed, however, increased endothelium-dependent relaxations in response to the a,-adrenergic agonist BHT-920 in vessels from estrogen treated animals. Hayashi et al. (23) found no significant difference in the relaxant response to acetylcholine in aortic rings from male, ovary intact female, or ovariectomized rabbits. Improved receptor-mediated endothelium-dependent relaxation after short-term estrogenic treatment has also been reported previously. Williams et al. (32) observed that “in situ” atherosclerotic coronary arteries of ovariectomized cynomolgus monkeys, which responded with constriction to intracoronary infusion of acetylcholine, responded to this agonist with vasodilation 20 min a.I?erintravenous injection of ethinyl estradiol. Reis et el. (33) observed, in coronary arteries of postmenopausal women, a decrease in basal vasomotor tone and an attenuation of abnormal vasomotor response to acetylcholine I5 minutes after intravenous administration of ethinyl estradiol. The mechanism by which estrogens increase the receptor-mediated release of NO is not clear. In most of the above mentioned studies it was observed that the A23 187-stimulated, endotheliumdependent, relaxation of the vessels was unaltered by estrogens ( 18, 20-24). This calcium ionophore stimulates NO synthesis and release through a receptor-independent increase in cytosolic calcium that, in turn activates the calcium-dependent endothelial NO synthase. Therefore, the absence of estrogen mediated effects on the relaxation induced by A23 187 suggests that the increased receptor mediated relaxation is not mediated by an enhanced activity of the NO synthase. The increased receptormediated relaxation observed after short-term exposure to estradiol argues, also, against an estrogenstimulated enzyme induction. Gisclard et al. (18) and Weiner et al. (22) proposed an alteration of muscarinic receptor activity as a possible mechanism to explain the increased relaxant response to acetylcholine observed after estrogenic treatment. Alternatively, the link between the receptor and the enzyme activation could be involved. In this regard it would be of interest to investigate if estrogenic treatment modifies phosphoinositide turnover and inositol I ,4,5_triphosphate mediated calcium release from intracellular stores in endothelial cells. Increased activity of the enzyme phosphatidilinositol 4,Sbiphosphate phospholipase C after short term addition of I7ILestradiol to MCF-7 human breast cancer cell cultures or cell homogenates was recently reported (34).
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Acknowledgments We want to thank Dr. H. Vidrio for his useful suggestions and Dr. A. Arias for assisting us in the analysis of the results. We thank, also, M. Sgnchez-Negrete for his technical assistance. This work was supported in part by grant IN-205493 from DGAPA-UNAM, Mexico. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 22. 23. 24. 25.
W.B. KANNEL, M.C. HJORTLAND, P M. McNAMARA and T. GORDON, Ann Intern. Med. 81447-452 (1976). L.C. YOUNG, L.H. KULLER, G. RUTAN and C. BUNKER, Am J. Epidemiol. 138 973983 (1993). G. A. COLDITZ, W.C. WILLET, M.J. STAMPFER, B. ROSNER, F.E. SPEIZER and C H.HENNEKENS,N. Eng. J.Med.3161105-lllO(1987). W.B. KANNEL and P.S. VOKONAS, Ann. Epidemiol. 2 S-14 (1992). E. BARRETT-CONNOR, T.L. BUSH, J. A. M. A. 265 1861-1867 (1991). M.J. STAMPFER and G.A. COLDITZ, Prev. Med. 20 47-63 (1991). A. GREEN and C. BAIN. Baillieres Clin. Endocrinol. Metab. 7 95-l 12 (1993) A.A. NABULSI, A.R. FOLSOM, A. WHITE, W PATSCH, G. HEISS, K.K. WU and M. SZKLO, N. Eng. J. Med. 328 1069-1075 (1993). L. ROSENBERG, J. PALMENR and S SHAPIRO, Am. J. Epidemiol. 137 54-63 (1993). J.M. SULLIVAN, R. V. ZWAAG, G.F. LEMP, J.P. HUGHES, V. MADDOCK, F.W. KROETZ and K.B. RAMANATHAN, Ann. Intern. Med. 108 358-363 (1988). E. BARRETT-CONNOR, D. WINGARD and M.H. CRIQIJI, J A. M. A. 2612095-2100 (1989). T. BOURNE, T.C. HILLARD, M.J WHITEHEAD, D. CROOK and S. CAMPBELL, Lancet 335 1470-1471 (1990). F.M. SACKS and B.W. WALSH, Ann. N. Y. Acad. Sci. 592 272-285 (1990). R.A. LOBO, Ann. N. Y. Acad. Sci. 592 286-294 (1990). K.F. GANGAR, S. WAS, M. WHITEHEAD, D. CROOK, H. MEIRE and S CAMPBELL, Lancet 338 839-842 (1991). P.L. LUDMER, A.P. SELWIN, T.L.SHOOK, R.R. WAYNE, G.H. MUDGE, R.W. ALEXANDER and P.GANZ, N. Engl. J. Med. 315 1046-105 1 (1986). U. FORSTERMAN, A. MUGGE, U. ALHEID, A. HAVERICH and J.C. FROLICH, Circ. Res. Q 185-190 (1988). V. GISCLARD, V.M. MILLER and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther. 244 1922 (1988). S.P. WILLIAMS, D.P. SHACKELFORD, S. G. IAMS and S. J.MUSTAFA, Eur. J. Pharmacol. 141205-207 (1988). V.M. MILLER and P.M. VANHOUTTE, Am. J Physiol. 261 R1022- R1027 (1991). C. WEINER, E. MARTINEZ, L.K. ZHU, A. GHODSI and D. CHESTNUT, Am. J. Obstet. Gynecol. 161 1599-1605 (1989). C. WEINER, L.K. ZHU, L. THOMSON, J. HERRING and D. CHESTNUT, Am. J. Physiol. 2hl H1275-H1283 (1991). T. HAYASHI, J.M. FUKUTO, L.J. IGNARRO and G. CHAUDHURI, Proc. Natl. Acad. Sci. U.S.A. 89 11259-11263 (1992). J.F. KEANY, G.T. SHWAERY, A. XU, R.J. NICOLOSI, J. LOSCALZO, T L. FOXALL and J.A. VITA, Circulation 89 225 l-2259 (1994). M.A. VAZQUEZ-ALCANTARA, M.A. JUAREZ-OROPEZA, R. MIRANDA-ZAMORA, J.C. DIAZ-ZAGOYA and J.GARZA-FLORES, J. steroid. Biochem. 21.599-602 (1985).
4%
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M.A. VAZQUEZ-ALCANTARA, M. MENJIVAR, G A. GARCIA, J.C. DIAZ-ZAGOYA and J. GARZA-FLORES, J. steroid. Biochem. 33 11 I I- 1118 (1989). T.M. COCKS and J.A. ANGUS, Nature 305 627-630 (1983). R.F. FURCHGOTT and J.V. ZAWADZKI, Nature 288 373-376 (1980). C.P. WEINER, I. LIZASOAIN, S.A. BAYLIS, R.G. KNOWLES, LG. CHARLES and S. MONCADA Proc. Natl. Acad. Sci. U. S. A. 915212-5216 (1994). V.M. MILLER and P.M. VANHOUTTE, Am. J. Physiol. 258 R1502-RI507 (1990). J.K. WILLIAMS, M.R. ADAMS and H.S. KLOPFENSTEIN, Circulation &l 1680-1687 (1990). J.K. WILLIAMS, M.R. ADAMS, D.M. HERRINGTON and T.B. CLARKSON, J. Am. Coil. Cardiol. 20 452-457 (1992). SE. REIS, S.T. GLOTH, R.S. BLUMENTHAL, J.R. RESAR, H.A. ZACUR, G. GERSTENBLITH and J.A. BRINKER, Circulation Ss 52-60 (1994). R. GRABER, C. SUMIDA, G. VALLETTE and E.A. NUREZ, Cell. Signal. 5 18 I- I86 (1993).
Pergamon
0024-3205(95)00281-2
EFFECTS OF ACUTE AND CHRONIC ESTROGENIC TREATMENT ON VASOMOTOR RESPONSES OF AORTIC RINGS FROM OVARIECTOMUED RATS M.C. Paredes-Carbajal, Department
M.A. Jukez-Oropeza*,
C.M. Ortiz-Mendoza
and D. Mascher
of Physiology and *Department of Biochemistry, School of Medicine, II.N.A.M., P.O. Box 70-250, Mexico D.F. 04510, MEXICO (Received in final form May 8,
1995)
Summary The effects of either chronic or acute estrogenic treatment on the “in vitro” vasomotor responses to phenylephrine (1 Oe9-10.’ M) and to carbachol ( 1OS“‘10.’ M) of aortic rings excised from ovariectomized rats were analyzed Chronic estrogenic treatment consisted in a single subcutaneous dose of 1 umol estradiol 17-stearate. Effects of acute estrogenic treatment were evaluated by recording the responses of aortic rings excised from untreated ovariectomized rats both before and after the addition of 17l3estradiol to the superfUsing solutions. In order to identify the endothelium-dependent responses each experiment was performed simultaneously on pairs of rings from the same aorta, one with and the other without functional endothelium. The contractile responses to phenylephrine of endothelium-intact vessels were attenuated by chronic estrogenic treatment; this attenuation was mrther increased by preincubation of the vessels with indomethacin and was reverted by No-nitro-L-arginine methyl ester. Either chronic or acute estrogenic treatment enhanced the carbachol-induced endothelium dependent relaxation of phenylephrine-precontracted rings. The results may be explained by assuming that estrogens increase the basal release of both nitric oxide and a cyclooxygenase-dependent vasoconstricting prostanoid as well as the receptor-mediated release of nitric oxide from the endothelium of the rat aorta. Kq Wotds: endothelium, estrogens, nitric oxide, prostanoids, aortic rings Women in the reproductive age have a lower prevalence of atherosclerosis related diseases than age matched men (1,2). This difference decreases after both surgical and natural menopause and postmenopausal women are at a much higher risk of developing coronary artery disease than premenopausal women of similar age (3,4). On the other hand, estrogen replacement therapy lowers the incidence of atherosclerotic diseases in ovariectomized and postmenopausal women (5-9). The beneficial effects of estrogens are even greater in women with already existing atherosclerotic disease (IO). Based on these epidemiological observations a “protective” role of estrogens, preventing atherosclerotic vascular diseases has been postulated. Several mechanisms by which this protection may be achieved have been proposed (5, 11-l 5). In the last few years, it has become evident that Corresponding author: Ma. Cristina Paredes-Carbajal. Department of Physiology, School of Medicine, U.N.A.M., P.O. Box 70-250, Mexico D.F. 045 10, MEXICO. FAX (525) 623-224 I E-mail [email protected].
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endothelium-dependent control of vascular tone is impaired early in atherosclerosis (16, 17) and, therefore, a putative role of the endothelium in the protective effects of estrogens has been suggested (18, 19). The effects of estrogens on endothelium-dependent responses of isolated arteries have been analyzed in different experimental models (18, 20-24). Evidence provided by these studies supports the hypothesis that estrogens modulate the release (and/or the action) of vasoactive substances from the endothelium. The present study was designed to answer two main questions: Does chronic or acute exposure to estrogens modify the contractile response of the isolated rat aorta to the CL,-adrenergic agonist phenylephrine? Do estrogens mow the carbachol-induced endothelium-dependent relaxation of this vessel? The results showed that estrogens attenuate the contractile response to the vasoconstrictor and accentuate the response to the vasodilator. Methods Animals. The experiments were performed on isolated aortas excised from adult nonpregnant female Wistar rats (250-300 g). The animals were ovariectomized bilaterally under sodium pentobarbital (32 mg/k& i.p.) anesthesia. Prophylactic crystalline penicillin (400,000 U/kg i.m.) was administered after surgery and eight days were allowed for recovery. Thereafter, the animals were randomly allocated to two groups, untreated and treated. Rats from the untreated group were used lo-14 days aRer surgery. Animals from the treated group received subcutaneously a single dose of 1 pmol estradiol 17-stearate dissolved in 0.3 ml castor oil. Previous work (25, 26) showed that, three days after administering a single dose (0.1-l .O pmol) of estradiol ester derivatives to ovariectomized rats, serum levels of 17&estradiol were restored to values close to those measured during estrus in nonpregnant rats and remained at these levels for up to 30-60 days. Rats of this group were used 1 l-l 3 days after treatment. AU animals were kept in individual cages with free access to food and water and exposed to a 12-h light-dark cycle. In vitro measurements of vascular responses. Animals were killed by cervical dislocation and the thoracic aorta was immediately removed, cleaned of connective tissue and cut transversely into rings (2 mm long). Special care was taken to avoid damage to the endothelium. In every other ring, the endothelium was removed by gently rubbing the intimal surface. For each experiment a pair of rings from the central portion of the same aorta (one with intact endothelium, the other without fimctional endothelium) was used. Each of these rings was suspended horizontally in the same miniature organ chamber (volume 1 ml) between two stainless steel hooks. One of the hooks was fixed to the chamber wall while the other was attached to an isometric force transducer (Grass, FT 03). The vessels were continuously superfused (3 ml/min) with prewarmed (37 C) aerated (95% 0, and 5% CO,) modified Tyrode solution (composition in mM: NaCl, 137; KC], 2.7; MgCl,, 0.69; NaHCO,, 11.9; NaHPO,, 0.4; CaCl,, 1.8 and glucose, 10; pH was 7.4). The rings were initially stretched until resting tension reached 2 g and allowed to equilibrate for one hour; during this period the resting tension was continuously monitored (Grass, Model 79 Polygraph) and, if needed, readjusted to 2 g by tirther stretching. Before starting an actual experiment, responsiveness of each pair of rings to phenylephrine and carbachol was tested. This was achieved by switching the superfUsing Tyrode solution for IO min to one containing phenylephrine (10S5 M) and, thereafter, to one containing, in addition to phenylephrine, carbachol (lo-’ M). Carbachol-induced relaxation of the phenylephrine precontracted vessels was taken as evidence for the preservation of an intact endothelium whereas lack of relaxation confirmed the absence of a functional endothelium.
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Experimental protocol, Two series of experiments were performed in order to analyze, respectively, the effects of either chronic or acute estrogenic treatment on the concentration-response curves of aortic rings from ovariectomized rats to phenylephrine and to carbachol. In each of these series, the contractile response of pairs of aortic rings to cumulative increasing concentrations ( 10m9- 10W5 M) of the a,-adrenergic agonist phenylephrine (concentrationresponse curve to phenylephrine) was initially recorded and, once tension development in response to the highest phenylephrine concentration reached its peak value, superfusion was switched to solutions having, in addition to phenylephrine, succesively increasing concentrations (1 Oe9- 1Oe5M) of carbachol (concentration-response curve to carbachol). In the first series of experiments these two succesive concentration-response curves were determined, both in abscence and presence of the cyclooxygenase inhibitor indomethacin ( 1O-5M), in rings excised from rats of either the treated or the untreated group ( n = 6 for each group). In the second series of experiments concentration-response curves to phenylephrine and to carbachol were obtained, in the presence of indomethacin (I Om5 M), in vessels excised from untreated animals, both before and after (40 - 60 min) addition of 178-estradiol (10m9M) to the perfusing solutions (n = 7). In both series of experiments the concentration response curves to phenylephrine were also obtained in the presence of both indomethacin and the competitive inhibitor of nitric oxide synthase No-nitro-L-arginine methyl ester (L-NAME, 300 PM; n = 6 for the first series of experiments; n = 4 for the second series). Data analysis. The contractile responses induced by phenylephrine are expressed as tension increment in grams above the basal tension (imposed on the vessel throughout the experiment). Carbachol-induced relaxation is expressed as the percent reduction in tension relative to maximal tension developed in response to phenylephrine (10” M). All data are expressed as means f S.E. EC,, (-Log of the mean molar concentration of agonist producing 50% of the maximal response) was determined with the software package Graph Pad Prism 10855 (San Diego, CA. 92121 USA). Comparisons of means were made by Student’s t test for unpaired (chronic treatment) and paired (acute treatment) values. Chemicals. All chemicals and drugs, except estradiol 17-stearate, were purchased from Sigma Chemical Co. (St. Louis, MO. USA) Estradiol 17-stearate was a gift of J.C. Diaz-Zagoya, and prepared according to V&zquez-Alc&ttara et al. (2526). Indomethacin was dissolved, by sonication, in 4% sodium carbonate. 17R-estradiol was dissolved in dimethyl sulfoxide. L-phenylephrine hydrochloride, carbachol (carbamoylcholine chloride) and No-nitro-L-arginine methyl ester hydrochloride (L-NAME) were dissolved in distilled water.
I. Effects of chronic (“in viva”) treatment with estradiol 17-stearate 1. Concentration-response
curves to phenylephrine
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A. Aortic rings with functionally
Vol. 57, No. 5, 1995
intact endothelium.
Aortic rings excised from rats that received estrogenic treatment developed less tension in response to each of the tested phenylephrine concentrations than the rings harvested from untreated animals (compare filled circles and open circles in Fig. 1A). Statistical analysis showed that the difference was significant (pcO.05) for the responses to IO-‘, 10m6and 10m5M phenylephrine. Tension developed in response to the highest phenylephrine concentration tested (maximal contractile response) amounted to 1.69 i 0.23 g for rings excised from treated rats and to 2.21 f 0.13 g for vessels from untreated rats. Table I summarizes the EC,, calculated for this series of experiments; it can be seen that estrogenic treatment shifted the EC,, significantly (p < 0.05) from 6.82 * 0.04 to 6.42 z+z 0.01,
TABLE
I
Effects of Chronic Treatment with Estradiol17-stearate to Phenylephrine (10~9-10-SM) of rat Aortic Rings. Group
With endothelium Max. tension EC,,
on the Concentration-Response
Curve
Without endothelium Max. tension EC,,
Yntreated (-)indom. (+)indom.
682*004 6.86 & 0.03
2.2110 13 2.33 i 0.17
7 60 * 0.015 754iO.I0$
2.22 f 0 24 2.20 * 0.32
6.42 f O.Ol* 1 69 * 0.23* 6.52 + 0.03* 1.11 * 0.22”A
6.92 -I-0.123 7.16*0.06$
2.36 f 0 23 1.99 f 0.14
7.11 io.09
7 21 f 0.04
2.45 I 0.1 1
Treated (-)indom. (t)indom. (+)indom. (+)L-NAME
1.91 10 10
EC,,. -log of mean molar concentration causing 50% of maximal response to phenylephrine (IO-‘M). Max. tension: Tension (in grams) developed in response to phenylephrine (1 V5M). (-)indom: without indomethacin. (+)indom: with indomethacin ( 10m5M).(+)mdom (+)L-NAME: with indomethacin ( 10erM) and L-NAME (300pM). Data are presented as means i SE; n=6 for all groups * Denotes that differences among treated and untreated groups are significant (p
Additionally, it was observed that in the aortas from rats that received the hormonal treatment, tension development during continued exposure to a given concentration of phenylephrine tended to be transient, i.e., it started to decline gradually soon after reaching its peak value and, in some experiments, decayed to its initial level after a few minutes despite the continuous presence of the
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477
and Vasomotor Responses
contractile agonist. These transient responses of the vessels from estrogen treated animals contrasted strongly with the well sustained contractile response observed in aortas from untreated rats. The attenuation of the contractile response to phenylephrine observed in the aortic rings from treated rats was markedly accentuated when exposure to phenylephrine was performed in the presence of indomethacin (compare filled triangles and filled circles in Fig. 1 A). Under this experimental condition the maximal contractile response to phenylephrine amounted to 1.11 + 0.22 g in the rings from treated rats and to 2.33 * 0.17 g in those from untreated animals. Indomethacin also accentuated the transient nature of the phenylephrine mediated contractile response of the rings excised from treated animals. In the presence of indomethacin, addition of NAME (300 pM) to the superfUsing solutions abolished the above described differences among the contractile responses to phenylephrine of the aortic rings obtained from either the treated or the untreated rats (not shown).
B
_j Phenylephrine
I
I
I
1
7
-9 -8 -7 -6 -5 Phenylephrine (log M)
(log Ml
Fig. 1 Eff6cts of chronic treatment with estradiol 17-stearate on the concentrationresponse curves to phenylephrine, of aortic rings with (A) or without (B) endothelium. Each panel illustrates the responses of aortic rings excised from either untreated (open symbols) or estrogen treated (filled symbols) ovariectomized rats. Curves were obtained either in the absence (circles) or the presence (triangles) of indomethacin. Data are presented as means f SE of six rats in each group. For clarity SE-bars were omitted in panel B.
B. Aortic rings without functional endothelium Each of the experiments was performed simultaneously on a pair of aortic rings: one having a fUnctiona endothelium, the other without a fUnctional endothelium. In each pair, the contractile
Estrogens and Vasomotor Responses
418
response to a given concentration of the ring with endothelium.
of phenylephrine
Vol. 57, No. 5, 199.5
of the ring without endothelium exceeded that
The dose-response curve of the rings excised from treated rats did not differ significantly from that of rings from untreated animals (compare open and filled circles in Fig 1B) Indomethacin endothelium-denuded
did not significantly rings.
2. Concentration-response
modify the contractile
responses to phenylephrine
of
curve to carbachol
Aortic rings with intact endothelium relaxed in a dose dependent manner in response to increasing concentrations of carbachol. The dose-response curve of the rings excised from the estrogen treated rats was significantly (p
TABLE II Effects of Chronic Treatment with Estradiol Carbachol (10m9 - 10.’ M) in Endothelium Phenylephrine (10” M). Group
(-)indom. (+)indom.
EC,,
17-stearate on the Relaxation induced by Intact Aortic Rings Precontracted with
Maximum Percent Relaxation
6.03 h 0.09 6.05 f- 0.06
76.90 i 8.42 75 00 + 10.81
6.5 1 f 0.29* 6.46 f 0.28*
89.98 f 2.57 82.46 f 5.97
Treated (-)indom. (+)indom.
EC,,: -Log of mean molar concentration causing 50% of maximal relaxation induced by carbachol. Maximum percent relaxation: maximal percent decrease in tension compared with maxima1 tension induced by phenylephrine (IO-’ M). Values are presented as means f SE; n=7 for all groups. * Denotes that differences between treated and untreated groups are significant (p
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.-
P 2
60
:: E
40-
-
0 s
20-
0’,
, -9
I
-8 Carbachol
I
-7 (log
I
I
-6
-5
M)
Fig. 2 Effects of chronic treatment with estradiol 17-stearate on the concentrationresponse curves to carbachol of endothelium intact aortic rings. Vessels were excised either from untreated (open symbols) or treated (tilled symbols) ovariectomized rats Responses recorded in the absence (circles) or in the presence (triangles) of indomethacin are plotted. Rings were precontracted with phenylephrine (lo-’ M) Data are expressed as percent of maximal phenylephrine-induced tension and shown as means h SE of six rats in each group. (*) Denotes that relaxations of the estrogen treated groups are significantly different from those of the untreated groups (p
II. Effects of acute (“in vitro”) treatment with 170-estradiol In this series of experiments concentration-response curves to phenylephrine and to carbachol were performed, in the presence of indomethacin, on pairs of aortic rings excised from ovariectomized rats (without estrogenic treatment) both, before and after (40 to 60 min) addition of 17g-estradiol ( 10m9M) to the supetising solutions. With this protocol each pair of rings served as its own experimental control for evaluating hormonal effects.
A. Concentration-response
curves to phenylephrine
As in the previous series of experiments (chronic estrogenic treatment) the dose-response curve to phenylephrine of the aortic rings with disrupted endothelium was significantly (p < 0.05) shifted to the left (EC,: 7.45 f 0.08) compared with the corresponding curve of the rings with intact endothelium (EC,,: 7.06 * 0.09) (Fig. 3 and Table III). Neither in the rings with intact endothelium nor in those without functional endothelium did the addition of 17LLestradiol modifji significantly the concentration-response curve to phenylephrine
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3-
52
-
5 ._ 2 I-“1
-
O-
I
1
-9
I
1
-7 -8 Phenylephrine
I
1
-6 -5 (log M)
Fig. 3 Effects of acute treatment with 17Pestradiol on the concentration-response curves to phenylephrine of aortic rings with (circles) or without (squares) endothelium excised from ovariectomized rats and preincubated with indomethacin. Responses were recorded in the same pair of rings before (open symbols) and after (tilled symbols) addition of 17lSestradiol to the superfusing solution. Data are expressed as means of rings from seven rats in each group. SE-bars were omitted for clarity.
TABLE II1 Effect of Acute Treatment with 17Gestradiol (10-9M) on the Concentration-Response Curve to Phenylephrine (10-g-10-5M) of Aortic Rings excised from ovariectomized rats and Preincubated with Lndomethacin (10.‘M). Group
With Endothelium
Without Endothelium
n
EC,,
ECS,
7.06 * 0.09
7.45 i 0.08*
7
6.89 f 0.05
7.21 i O.lO*
7
7.09*0.11
7.33 f 0.13
4
Treated f+)NAME
EC,,: -Log * SE of mean molar concentration causing 50% of maximal response to phenylephrine (1 O-‘M). * Denotes that the difference between the EC,, of rings with or without endothelium is significant (~~0.05).
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B. Concentration-response
curve to carbachol
The dose-response curve to increasing cumulative concentrations of carbachol of the rings with intact endothelium and precontracted with tom5M phenylephrine, performed in the presence of 17f&estradioi, was significantly (p < 0.05) shifted to the left, compared with the corresponding curve obtained before the exposure to the hormone (compare filled and open triangles in Fig. 4) The calculated values for the respective EC,,, are listed in table IV
100
0
1
1,
, -9
-8 Carbochol
I
I
I
-7
-6
-5
(log
M)
Fig. 4 Effects of acute treatment with 17&estradiol( IO-’ M) on the concentration-response curves to carbachol of endothelium intact aortic rings excised from ovariectomized rats and preincubated with indomethacin (10-j M). Responses to carbachol were obtained in the same ring before (open triangles) and after (filled triangles) addition of 17kestradiol to the super-king solution. Rings were precontracted with phenylephrine (10-j M). Data are expressed as percent of maximal phenylephrineinduced tension and shown as means k SE of vessels Corn seven rats in each group. * Denotes that relaxation of estrogen treated rings is significantly greater (~‘0.05) than that of the untreated ones.
Estrogens and Vasomotor Responses
TABLE
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IV
Effects of Acute Treatment with 17kestradiol (10.’ M) on the Relaxation induced by Carbachol (10-g-10-5M),in Endothelium Intact Aortic Rings excised from ovariectomized rats, Precontracted with Phenylephrine (10”M) and Incubated with Indomethacin (10.‘M). Group
EC,,
Maximum percent relaxation
Yntreated
6.04 f 0.05
75.86 * 5.51
Treated
6.64
87.58 * 4.44*
l
0.14’
EC,,: -Log of mean molar concentration causing 50% of maximal relaxation induced by carbachol (lO-M). Maximum percent relaxation: mean percent decrease in tension compared with maximal tension induced by phenylephrine (10-W). Data are presented as means * SE; s7 for all groups. * Denotes that differences among values observed before and after estrogenic treatment are significant (pcO.05).
One of the approaches that has been extensively used to elucidate the mechanisms by which estrogens perform their protective role on the vascular system has been to explore their effects on the “in vitro” responsiveness of isolated vessels to vasoactive agents. The results of these studies are controversial. This controversy may be explained, partly, by differences in the experimental model. These include the use of different species and vessels, the evaluation of varying vasoactive agents, differences in the methods of “in vitro” measurement of vascular responsiveness and differing schemes of estrogenic treatment. In the present study the effects of estrogens on the responsiveness of the rat aorta to just one vasoconstrictor -phenylephrine- and one vasodilator -carbachol- were analyzed. Lphenylephrine, a selective a,- adrenergic agonist was selected assuming that it would activate smooth muscle contraction without simultaneously inducing receptor mediated release of vasoactive substances from the endothelium. This assumption is based on the presumptive absence of aradrenergic receptors in this structure (27). Carbachol was selected because it consistently produces endothelium-dependent, nitric oxide-mediated, relaxation in the isolated rat aorta without having any relaxing effect on vessels lacking a hmctional endothelium (28). In addition, there is no evidence of carbachol-induced release of vasoactive metabolites of arachidonic acid. Regarding the “in vitro” measurement of vascular responsiveness, the method used in this study differs from those used by other authors. Vessels were continuously super-fused in a small organ chamber in an attempt to avoid, on the one hand, uncontrolled alterations of the extracellular medium (by either accumulation or depletion of molecules) and, on the other hand, insure -when desired- its fast and homogeneous modification. In each experiment a pair of rings (with and without fimctional endothelium, respectively) obtained from the same aorta and placed in a common organ chamber was studied; this allowed identification of the endothelium dependent responses during each individual experimental procedure. As to the estrogenic treatment, two schemes were used: chronic “in viva” treatment and acute “in vitro” exposure. Chronic treatment consisted in subcutaneous administration of a single dose
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of estradiol 17-stearate. According to Vazquez-Alcantara et al. (25, 26) this procedure insures longlasting estrogenic activity as evidenced by vaginal cytology, uterotrophic action, inhibition of serum gonadotrophins and serum levels of 17D-estradiol. Acute exposure to estrogen was achieved by addition of 17R-estradiol to the sup&using solutions Rings with endothelium excised from estrogen treated rats developed less tension in response to phenylephrine than corresponding rings from ovariectomized rats. This difference was markedly enhanced in the presence of indomethacin, and was no longer observed after addition of L-NAME to the superfusiig solutions. On the other hand, no differences were observed among the contractile responses to phenylephrine of rings without functional endothelium excised from either treated or untreated rats. Indomethacin did not modify the phenylephrine induced contraction of endotheliumdenuded rings frL>rneither group of rats. Taken together, these results may be explained by assuming that chronic estrogenic treatment of ovariectomized rats increases the basal synthesis and release -by the endothelium of the aorta- of both nitric oxide (NO) and a cyclooxygenase-dependent vasoconstricting metabolite of arachidonic acid. The assumption that estrogens increase the basal release of NO agrees with the results of Hayashi et al. (23) who found that basal (but tone related) release of NO is greater in endotheliumintact aortic rings excised from female rabbits than in those from either ovariectomized or male rabbits. It is, also, in line with the recent report by Weiner et al. (29) showing that estradiol treatment (and pregnancy) increases -in the guinea pig- the activity of calcium-dependent nitric oxide synthase in the uterine artery and in several organs as well as the amount of rnRNAs for both, the endothelial and the neuronal isoforms of the constitutive nitric oxide synthase, in skeletal muscle. The latter finding strongly suggests that estrogens may increase basal release of nitric oxide by enzyme induction. If this were indeed the case, and since enzyme induction requires a relatively long time, it would explain the present finding that acute exposure to 17Pestradiol had no significant effect on the contractile responses to phenylephrine of aortic rings excised from ovariectomized untreated rats and preincubated with indomethacin. The proposal that chronic estrogen treatment may increase, also, the synthesis and release of a cyclooxygenase dependent vasoconstricting prostanoid is in line with the findings of Miller and Vanhoutte, (30). These authors reported that the endothelium- and cyclooxygenase-dependent contractions to arachidonic acid of aortic rings excised from estrogen treated ovariectomized rabbits were greater than those of vessels from untreated animals, In addition, the authors observed, in the same vessel, an increased endothelium- and cyclooxygenase-dependent sensitivity to norepinephrine. Based on these results they concluded that “under the influence of estrogens, norepinephrine must stimulate the metabolism of arachidonic acid in the endothelial cells that in turn enhances the contraction of the vascular smooth muscle evoked by the adrenergic agonist.” This conclusion, taken together with the above discussed evidence obtained by Hayashi et al. (23) could mean that, as suggested by the present results, estrogens increase, also in the rabbit aorta, the synthesis and release of both NO and a cyclooxygenase-dependent vasoconstrictor. Estrogenic treatment, either chronic or acute, increased significantly the carbachol-induced endothelium-dependent relaxation of phenylephrine-precontracted aortic rings. Indomethacin had no effect on the carbachol induced relaxation of vessels excised from either treated or untreated animals. Since it is well documented that the activation of endothelial muscarinic receptors induces the synthesis and release of NO (28) these results indicate that estrogenic treatment enhances the receptor mediated release of NO from the endothelium of the rat aorta. Regarding the effects of chronic estrogenic treatment on receptor mediated NO release, the same conclusion has been reached previously in several studies. G&lard et al. (18) reported that femoral arteries from 17R-estradiol
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treated rabbits show an enhanced endothelium-dependent relaxation in response to low doses of acetylcholine. Weiner et al. (2 1, 22) observed that acetylcholine-induced NO-mediated relaxation of uterine and carotid arteries from the guinea pig is increased during pregnancy. Williams et al. (3 1) reported that “in situ” atherosclerotic coronary arteries of ovariectomized cynomolgus monkeys responded to intracoronary inmsed acetylcholine with “paradoxical” constriction and that chronic estrogenic treatment reverted the constriction to a moderate dilation. Keany et al. (24) observed that chronic estrogenic treatment of ovariectomized hypercholesterolemic miniature swine preserves the endothelium-dependent relaxation of coronary artery rings to bradykinin and substance P, whereas vessels from untreated animals exhibited impaired relaxations to these agonists. It should be mentioned, however, that there are also studies reporting that chronic estrogenic treatment has no effect on the receptor-mediated release of NO. Miller and Vanhoutte, (30) observed no difference among acetylcholine-induced relaxations of aortas excised from either estrogen treated or untreated ovariectomized rabbits. Miller and Vanhoutte, (20) reported that the receptor-mediated relaxations of coronary arteries from ovariectomized dogs in response to the endothelium-dependent vasodilators, acetylcholine, adenosine diphosphate, and bradykinin, were similar in vessels from either estrogen treated or untreated animals, they observed, however, increased endothelium-dependent relaxations in response to the a,-adrenergic agonist BHT-920 in vessels from estrogen treated animals. Hayashi et al. (23) found no significant difference in the relaxant response to acetylcholine in aortic rings from male, ovary intact female, or ovariectomized rabbits. Improved receptor-mediated endothelium-dependent relaxation after short-term estrogenic treatment has also been reported previously. Williams et al. (32) observed that “in situ” atherosclerotic coronary arteries of ovariectomized cynomolgus monkeys, which responded with constriction to intracoronary infusion of acetylcholine, responded to this agonist with vasodilation 20 min a.I?erintravenous injection of ethinyl estradiol. Reis et el. (33) observed, in coronary arteries of postmenopausal women, a decrease in basal vasomotor tone and an attenuation of abnormal vasomotor response to acetylcholine I5 minutes after intravenous administration of ethinyl estradiol. The mechanism by which estrogens increase the receptor-mediated release of NO is not clear. In most of the above mentioned studies it was observed that the A23 187-stimulated, endotheliumdependent, relaxation of the vessels was unaltered by estrogens ( 18, 20-24). This calcium ionophore stimulates NO synthesis and release through a receptor-independent increase in cytosolic calcium that, in turn activates the calcium-dependent endothelial NO synthase. Therefore, the absence of estrogen mediated effects on the relaxation induced by A23 187 suggests that the increased receptor mediated relaxation is not mediated by an enhanced activity of the NO synthase. The increased receptormediated relaxation observed after short-term exposure to estradiol argues, also, against an estrogenstimulated enzyme induction. Gisclard et al. (18) and Weiner et al. (22) proposed an alteration of muscarinic receptor activity as a possible mechanism to explain the increased relaxant response to acetylcholine observed after estrogenic treatment. Alternatively, the link between the receptor and the enzyme activation could be involved. In this regard it would be of interest to investigate if estrogenic treatment modifies phosphoinositide turnover and inositol I ,4,5_triphosphate mediated calcium release from intracellular stores in endothelial cells. Increased activity of the enzyme phosphatidilinositol 4,Sbiphosphate phospholipase C after short term addition of I7ILestradiol to MCF-7 human breast cancer cell cultures or cell homogenates was recently reported (34).
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Acknowledgments We want to thank Dr. H. Vidrio for his useful suggestions and Dr. A. Arias for assisting us in the analysis of the results. We thank, also, M. Sgnchez-Negrete for his technical assistance. This work was supported in part by grant IN-205493 from DGAPA-UNAM, Mexico. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 22. 23. 24. 25.
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