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Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats
Life Sciences, Vol. 60, No. 25, pp. 2291-2302, 1957 Copyright 0 1997 Ekvier Science Inc. Printed in the USA. AI1 rights resewed 0024-3205197 $17.00 + .%I
ELSEVIER
PII SOO24-3205(97)00284-1
DIFFERENTIAL
EFFECTS OF LOW- AND HIGH-DOSE ESTROGEN ON VASCULAR RESPONSES IN FEMALE RATS
TREATMENTS
Chiara Bolego, Andrea Cignarella, Raffaella Ruzza, Claude Zaarour, Elio Messi*, Mariarosa Zanisi* and Lina Puglisi Institute of Pharmacological Sciences and *Institute of Endocrinology University of Milan, Via Balzaretti, 9 20133 Milan, ltaly (Received in hal
form March 17, 1997)
Summary In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-P-estradiol injections (5 pg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and No-Methyl-Larginine acetate, which was found to be greater in 17-&estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-P-estradiol-treated females released significantly more prostacyclin than those from oiltreated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 pg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens. Key Words:
aorta, ovariectomy, estrogen, nitric oxide, prostacych
Women in the reproductive age are known to have a lower risk for coronary heart disease (CHD) when compared to men (1,2). Postmenopausal women, due to the loss of ovarian function, are exposed to increased incidence of hypertension and CHD (3) but appear to be protected by estrogen replacement therapy (45). Conversely, it is well established that women using oral contraceptive preparations are subjected to Correspondence to: L. Puglisi Institute of Pharmacological Sciences University of MilanVia Balzaretti, 9. 20133, Milan, Italy Tel. 02/20488305 Fax. 02/29404961
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increased relative risk of CHD (6). These observations, therefore, clearly suggest an influence of estrogens on the vessel wall, although the molecular mechanisms are not yet fully understood. The generation of vasoactive mediators, like nitric oxide (NO) and prostacyclin (PGls), plays a pivotal role in maintaining the cardiovascular system in a state of constant active vasodilation (7) and numerous studies indicateded that alterations in the release of vasodilators synthesized by the vessel wall occur at an early stage in the atherosclerotic process, both in animals and humans (8,Q). Estrogens have been shown to affect the production of vasoactive mediators by the vessel wall. The basal release of NO from rabbit aortic rings was shown to be greater in female than in male rabbis, but ovariectomy eliminated the difference (10). In addition, estrogen treatment was reported to induce in vitro stimulation of rat smooth muscle and piglet aortic endothelial PGlp synthesis (11 ,12), modulation of contractile and relaxant responses in ex vivo vascular tissue isolated from both ovariectomized (OVX) rabbits and rats (13-l 5) and increase of endotheliumdependent relaxation in vivo (16,17). The present experiments were designed in order to test the hypothesis that different protocols of estrogen administration to female rats may induce opposite effects, in terms of NO- and PGls-mediated vascular responses. We therefore compared a low-dose, short-term estrogen treatment of OVX female rats, as a model of replacement therapy, with a long-term, high-dose treatment of intact females, as a model of contraceptive regimen. We evaluated the release of NO indirectly by precontracting aortic rings with norepinephrine and by observing the effects on tone changes induced by superoxide dismutase (SOD), an enzyme able to scavenge superoxide anions, thereby preventing NO inactivation, and No-Methyl-L-arginine acetate (L-NMA), an L-arginine analogue which competes for NO synthase and inhibits NO formation. In addition, we evaluated the vasomotor responses elicited by acetylcholine (ACh) which triggers NO release by endothelial cells, and sodium nitroprusside (SNP), an exogenous NO donor. Methods Animals : The experiments were performed on isolated aortas taken from adult male and female Sprague Dawley rats weighing 200-250 g. The female rats were bilaterally ovariectomized under pentobatbital anesthesia (30 mg/Kg intraperitoneally). After a six weeks’ period of recovery, the animals were divided into three groups: 1) intact males, 2) OVX females, 3) OVX estrogen-treated females. Groups 1) and 2) received peanut oil (0.1 ml) as solvent of the estrogen solution; estrogen treatment consisted of a daily subcutaneous injection of 5 ug/Kg of 17-P_estradiol (E2) during the two days preceding the experiments. Plasma estradiol levels, as measured by radioimmunoassay following this treatment, resulted to be in the high physiological range (18) approximating the proestrus level in the rat (19). In addition, a series of experiments were carried out on intact females treated with high doses (100 us/Kg) of ethinylestradiol (EE). The hormone was injected subcutaneously once a day for one month. /so/a&d aortic ring preparations: At sacrifice, the aorta was removed and gently cleaned of adjacent tissue. Special care was taken to avoid contact with the luminal surface in order to preserve the endothelium. Rings approximately 3 mm in width were then cut, suspended in lo-ml or 20-ml organ baths and perfused (rate: 60 ml h-1 ) with Krebs solution at 37°C continuously bubbled with a 95% 02 and 5% CO2 mixture. The Krebs solution had the following composition (mmol.): NaCl 118.9, KCI 4.66, KH2P04 1.18, MgS04 1.1, CaCh 2.52, NaHCOs 25 and glucose 5.55, pH 7.4. Tissues were equilibrated for 1 h under a resting tension of 1.5 g. In preliminary length-tension
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experiments, optimal resting tension was determined to be 1.5 g for both control and treated aortic rings. Force was recorded by means of a microdynamometer consisting of a strain gauge transducer, a high-gain amplifier and a galvanometer (Pchannel recorder “Gemini” 7070, Basile, Italy). Concentration-response curves: After 1 h equilibration, rings were contracted with 0.1 pmol. norepinephrine (NE) to induce submaximal contraction. Increasing doses of acetylcholine (ACh) were then added to the bath to assess the integrity of the endothelium. If no relaxation to ACh was detected, the preparation was not used for the experiment. To establish whether the agonist sensitivity to NE remained unchanged over the course of the experimental period, two consecutive curves were carried out at about 30 minute intervals. After precontracting the aortic rings submaximally with 0.1 pmol. NE, the drugs to be tested were added cumulatively. Contractile and relaxant responses were expressed as a percentage of the NE contraction. Eicosanoids measurement : PGlp released from aortic tissue was determined as its stable metabolite 6-keto-PGF1, by EIA (Cayman Chemical). Segments approximately 23 cm in length were cut from the same aorta used for the above experiments and equilibrated in Krebs solution on ice for 60 minutes. The incubation medium was removed and the tissues were incubated in Krebs solution at 37% under gentle shaking for 5 minutes. At the end of incubation, the medium was separated by filtration and aliquots of it were assayed. The tissue was carefully desiccated by means of a vacuum-desiccator and the dry weight determined. Values were expressed as pg 6-keto-PGFtJmg dry aorta. Drugs: The following drugs were used: acetylcholine chloride (ACh), norepinephrine hydrochloride (NE), No-Methyl-L-arginine acetate (L-NMA), superoxide dismutase (SOD), sodium nitroprusside (SNP), 17-6-estradiol (E2), ethinylestradiol (EE); all of them were obtained from Sigma. All the drugs were freshly dissolved in saline solution except hormones, which were solubilited in peanut oil. Statistical Analysis: All data are expressed as means f S.E.M. of 5-7 experiments and represent unpaired data. Statistical analysis was performed with a Student’s t-test. A probability value of PeO.05 was considered statistically significant. When more than two means were compared (i.e. responses of aortic rings obtained from male, untreated and estradiol-treated female rats) an analysis of variance with repeated measurements was performed. If a significant F value was found, Scheffe’s test for multiple comparison was used to identify differences among groups. Results Effects of low-dose E2 treatment on the basal release of NO In studies designed to assess the tone-related release of NO from NE-precontracted aortic rings, the responses to superoxide dismutase (SOD) and No-Methyl-L-arginine (L-NMA) acetate were evaluated in aortic rings obtained from males, OVX oil- and OVX E24reated females. Fig. 1 shows that SOD (from 0.1 to 100 Unit/ml) produced concentration-related relaxant responses, with a level of magnitude greater in treated versus untreated female preparations. Moreover, the magnitude of relaxant responses to SOD in aortic rings obtained from OVX females resembled that observed in aortic rings from males.
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Application of increasing concentrations of the NO synthesis inhibitor L-NMA (l-100 pmol.) elicited vasoconstriction in a concentration-dependent manner. A significant increase in the contractile response was detected in tissues from females treated with E2, at least at the highest dose, when compared to tissues obtained from either untreated females or males (Fig. 2). so-
T w-
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&f T
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Fig. 1
10
loo
SOD, U/ml
Concentration-response curves to SOD in aortic rings obtained from male female OVX oil- (0) and E2- treated (5pg/Kg once a day per two days) rats. Data are expressed as percentage of the response to norepinephrine shown as means f S.E.M., n=5 in each group. Statistical significance: ** P
l
1
10
( q ), (o )
and and
100
L-NM&-Log Iw
Fig. 2 Concentration-response curves to L-NMA in aortic rings obtained from male ( q ), female OVX oil- (0) and OVX E2-treated ( l ) (5pg/Kg once a day per two days) rats. Data are expressed as percentage of the response to norepinephrine. Values are the mean f S.E.M. of 7 separate experiments. No differences were observed between male and female OVX-oil treated animals. Statistical significance: * PC 0.05.
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Effects of low-dose of E2 treatment on the vascular responses to ACh and sodium nitroprusside ACh (0.1-l 0 pmol.) produced a concentration-dependent, NO-mediated relaxation of endothelium intact aortic rings precontracted with 0.1 umol. NE. As shown in Fig. 3, no significant differences in the relaxant responses to ACh could be observed in aortic rings from male, OVX oil-treated and OVX E2-treated female rats. Fig.4 illustrates the responses elicited by sodium nitroprusside (SNP), an endotheliumindependent vasodilator, cumulatively added into the incubation medium (0.001-l Hmol.). The sensitivity of the vascular smooth muscle in precontracted preparations was not affected by E2 treatment. Furthermore, the endothelium-independent relaxation to SNP of aortic rings from male was similar to that of OVX female animals.
1
-
7
ACh, -Log fmoL/ Fig. 3 Concentration-response curves to Ach cumulatively added to aortic rings obtained from male (0 ), female OVX oil- (0) and OVX EP-treated (5 ug/Kg once a day per two days) (0). Data are expressed as percentage of the response to norepinephrine and shown as means + S.E.M., n=7 in each group. No differences were observed between the three groups.
Effect of E2 treatment on PGI2 release The release of PGl2, measured as its stable metabolite 6-ketoPGF,,, was evaluated after incubation of aortic rings at 37°C in Krebs solution for 5 minutes. The results are shown in Fig. 5: in OVX E2-treated rats, the levels of PGls, released into the incubation medium were significantly higher than those observed in the preparations obtained from untreated animals (195.8k17.4 vs. 132.2+-9.6 pg/mg dry tissue, p
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8
9
6
SNP, -Log Im0l.l
Fig. 4 Percent of relaxation to SNP of aortic rings obtained from male (0 ), female OVX E2-treated (0 ) (5pg/Kg once a day per two days), and female OVX oiltreated rats (0). Aortic rings were submaximally contracted with norepinephrine before obtaining cumulative response to SNP. Values are the mean f S.E.M. of 5 separate experiments. Responses were similar in all three groups. 250
** ............................ ............ . ........................... .....................
50
Fig. 5 6-keto-PGFlo released into the incubation medium by aortic rings obtained from female OVX oil-treated (white column) and OVX E2-treated (black column) (5 ug/Kg once a day per two days) rats. Each value is the mean fS.E.M. of 7 experiments. Statistical significance: **PcO.Ol. shown in Figs. 6 and 7, such a treatment resulted in a significant decrease in the responses to both SOD and L-NMA. Furthermore, the concentration-dependent relaxation to either ACh or SNP was affected by the treatment with EE at this high dose. Indeed, the endothelium-dependent (ACh) and the endothelium-independent (SNP) relaxations were lower in aortic rings from EE-treated compared to untreated animals (Figs 8 and 9).
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Ii1
;
10
loo
SOD,Ulnd
Fig. 6 Concentration-response curves to SOD in aortic rings obtained from intact females rats treated with high doses of EE ( l) (100 pg/Kg daily for one month) or oil (0). Values are expressed as percentage of the response to norepinephrine and are the mean + S.E.M. of 7 separate experiments. Statistical significance: ’ P< 0.05. so
40
i” jm 10
0:
II
Fig. 7 Concentration-response curves to L-NMA in aortic rings obtained from intact females treated with high doses (100 pg/Kg daily for one month) of EE (a) or oil (0). Data are expressed as percentage of the response to norepinephrine and are the mean f S.E.M. of 6 separate experiments. Statistical significance: P< 0.05. l
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5
ACh, -Logfmo~ Fig. 8 Concentration-response curves to ACh cumulatively added to aortic rings obtained from intact female treated with high doses (100 pg/Kg daily for one month) of EE ( l) or oil (0). Data are expressed as percentage of the response to norepinephrine and are the mean f S.E.M. of 6 separate experiments. Statistical significance:* P< 0.05.
9
6
7
6
SNP, -Log knol.l Fig. 9 Percent of relaxation of aortic rings obtained from intact female treated with high doses (100 pg/Kg daily for one month) of EE (0) or oil (0). Aortic rings were submaximally contracted with norepinephrine before obtaining cumulative response to SNP. Values are the mean f S.E.M. of 6 separate experiments. Statistical significance: P< 0.05. l
We also evaluated the effect of the treatment with EE on the release of PGl2 by aortic tissue. After 5 min incubation, the release of 6-keto-PGF1, (the stable product of PGls catabolism) underwent a statistically significant decrease in aortic rings obtained from EE-treated females compared to controls (247.4k38.7 vs. 414.4k60.5 pg/mg dry tissue, peO.01) (Fig.1 0).
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Fig. 10 6-keto-PGF,, released into the incubation medium by aortic rings obtained from intact female rats treated with high doses (100 pg/Kg daily for a month) of EE (black column) or oil (white column). Each value is the mean + S.E.M. of 7 experiments. Statistical significance: **PC 0.01.
Discussion Several epidemiological studies pointed out the protective role of estrogens against the occurence of CHD, as indicated by the low CHD rates in premenopausal women, the narrowing of the gender gap in CHD mortality after menopause, and the decreased incidence of CHD in women undergoing estrogen replacement therapy after menopause (2,5,20,21). On the other hand, the significant increase in the risk of thromboembolic disease in oral contraceptive users has been ascribed to the estrogenic component (4,22). It can be speculated, therefore, that estrogens can induce remarkably different effects on the biology of the vascular wall depending on the physiological status, the duration of the treatment and the level of circulating hormones. In view of this hypothesis, we measured the vascular responses mediated by NO, as well as the release of PGl2, in aortic rings obtained from female rats following low- or high-dose estrogen treatment. The basal release of NO was indirectly evaluated by utilyzing SOD, an enzyme able to scavenge superoxide anions, and L-NMA, an L-arginine analogue which competes for NO synthase and inhibits NO formation (23). E2 replacement administration to OVX female rats induced a greater relaxant response to SOD and a more marked contractile response to L-NMA than that observed in tissue of untreated OVX females or males. No differences among the three experimental groups, however, were detected when either the ACh-stimulated release of NO from endothelial cells or the responsiveness of smooth muscle cells to SNP, a stable NO donor (24), was evaluated. These findings are in agreement with those previously reported by other investigators (10,25), suggesting that the gender differences in NO basal release are related to an increased release of NO by endothelial cells and not to an increased sensitivity of vascular smooth muscle to the released NO. Indeed, the relaxant responses elicited by endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilators were not affected by sex hormone treatment. In contrast, previous studies reported that estrogen treatment
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enhances endothelium-dependent relaxation of precontracted vascular preparations. Gisclard et al. (26) observed this effect in femoral artery tissue, whereas ParedesCarbajal et al. (15) reported enhanced carbachol-induced relaxation of 8x viva phenilephrine-precontracted aortic rings following one day estrogen administration. Cheng et al., (27) explained the enhanced ACh-induced responses after a 1Cday treatment with 17-fl-estradiol with a depressed acetylcolinesterase activity. The discrepancy with our results could be explained by differences in the activity of acetylcholinesterase and in the responsiveness to released NO among vascular tissues, as well as by the regional variation in the ACh-stimulated release of NO along the aorta (26), the use of carbachol, which is resistant to acetylcholinesterase and the different protocols of estrogen administration. In contrast to the low-dose E2 treatment, however, prolonged EE administration resulted not only in a reduced basal release of NO, but also in an impairement of endothelial function and/or smooth muscle responsiveness to NO, as suggested by the decreased relaxation to ACh and SNP of aortic rings excised from EE-treated animals. Taken together, our results would suggest that estrogens affect the vessel wall functionality at two levels. A relatively rapid vasodilatory effect might account for the observed differences in the basal release of NO, whereas a later-onset effect might eventually lead to alterations in endothelial functions and smooth muscle responsiveness to NO. The latter component is likely to be mediated by estrogen receptor-dependent alterations in NO synthase gene expression (29,30). Moreover, the contrasting effects of short- and long-term estrogen administration on the NO release pathway and PG12production in aortic tissue could be accounted for by the down regulation or desensitization of estrogen receptors following prolonged exposure to unphysiolqical levels of hormone in the vessel wall. Alternatively, changes in ACh receptor characteristics might occur. As far as the effects of estrogens on PGl2 release are concerned, in vitro (31,32) and in viva studies (33) produced highly inconsistent results, probably on the account of the variable experimental conditions used by different investigators. In our hands, low-dose E2 treatment induced a rise in PGl2generation in OVX female, in comparison with untreated controls (34) whereas EE-treated intact females produces less PG12than controls (35). Interestingly, the aortic release of PG12by untreated OVX female turned out to be significantly lower than that observed in untreated intact females (compare Figs. 5 and lo), thereby supporting the concept of a reduced availability of vasodilating agents in estrogen deficiency. Our results suggest that the production of PGl2, as well as that of NO, is stimulated by physiological levels of estrogens impaired by higher hormone concentrations, consistently with the general impairement of the relaxant vascular responses under the latter conditions. The possibility that E2-treatment also increases the synthesis and release of vasocontricting cyclooxygenase products (15,36,37), which could further modulate vascular responses, cannot be rouled out. In addition, an intriguing link between NO and the cyclooxygenase pathway has been recently described (36) and we cannot exclude that such an interaction is relevant also under our experimental conditions. In conclusion, the protective effects exerted by physiological levels of estrogen are related to the enhanced availability of vasodilating agents (NO, PG12) within the vessel wall. Such a protection is dimmed when plasma estrogen concentrations are either below (OVX animals) or above (long-term EE treatment) the physiological range, thereby suggesting the existance of regulatory mechanisms (possibly mediated by specific receptors) which appear to be tightly controlled by the level of circulating estrogens.
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References 1. 2. 3. 4. 5. 6.
;: 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19. 20.
21. 22. 23. 24.
25. 26. 27.
N.K. WENGER, L. SPEROFF and B. PACKARD, N. Engl. J. Med. m 247-256 (1993). M.J. STAMPFER and G.A. COLDITZ, Prev. Med. 2Q 47-63 (1991). T.L. BUSH and E. BARRET-CONNOR, Epidemiol. Rev. Z 89-104 (1985). P.D. STOLLEY, J.A. TONASCIA, M.S. TOCKMAN, P.E. SARTWELL, A.H. RUTLEDGE and M.P. JACOBS, Am. J. Epidem. 142 197-201 (1975). D. GRADY, S.M. RUBIN, D.B. PETITTI, C.S. FOX, B. ETTINGER, U.L. ERNSTER and S.R. CUMMINGS, Ann. Inter. Med. 1z 1016-l 037 (1992). J.B. LABAT, S.D. BERGMANN and J.H. ZAVORAL, Cardiovasc. Risk Fact. 4 5159 (1994). R. BOTTING and J.R. VANE, Arch. Mal. Coer. 82 11-4 (1989). S.W. WERNS, J.A. WALTON, H.H. HSAI, E.G. NABEL, M.L. SANZ and B. PITT, Circulation z9 287-291 (1989). H. SHIMOKAWA and P.M. VANHOUlTE, J. Clin. Invest. 64 900-914 (1989). T. HAYASHI, J.M. FUKUTO, L.J. IGNARRO and G. CHAUNDURI, Procl. Natl. Acad. Sci. USA 89 11259-l 1263 (1992). W. CHANG, J. NAKAO, H. ORIMO and S. MUROTA, Biochim. Biophys. Acta m 107-l 18 (1989). C. SEILLAN, C. ODY, F. RUSSO-MARIE and D. DUVAL, Prostaglandins 263-12 (1983). V. GISCLARD, N.A. FLAVAHAN and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther. 24p 466-470 (1987). V.M. MILLER and M. VANHOUTTE, Am. J. Physiol. 261 R1022-1027 (1993). M.C. PAREDES-CARBAJAL, M.A. JUAREZ-OROPEZA, CM. ORTIZ-MENDOZA and D. MASHER, Life Sci. =473-486 (1995). J.K. WILLIAMS, M.R. ADAMS and H.S. KLOPFENSTEIN, Circulation 81 16801687 (1990). P. COLLINS, G.M.C. ROSANO, P.M. SARREL, L. ULRICH, S. ADAMOPOULOS, C.M. BEALE, J.G. MCNEILL and P.A. POOLEWILSON, Circulation 92 24-30 (1995). R.E. WATSON, G.E. HOFFMAN and S.J. WIEGAND, Brain Res. m 157-163 (1986). J.K.H. LU, P.S. LAPOLT, T.E. NASS, D.W. MAlT and H.L. JUDD, Endocrinology 116 1953-i 959 (1985). E.D. EAKER and W.P. CASTELLI, Coronary heart disease in women. E. Eaker, B. Packard, N.K. Wenger, T.B. Clarkson and H.A. Tyroler (Eds), 122-132, Haymarket Doyma, New York (1987). S.A. SAAMAN and M.H. CRAWFORD, J. Am. Coil. Cardiol. al403-1410 (1995). W.H.W. INMAN, M.P. VESSEY, B. WESTERHOLM and A. ENGELUND, Brit. Med. J. 2 203-208 (1970). D.D. REES, R.M.J. PALMER and S. MONCADA, Proc. Natl. Acad. Sci. USA u 3375-3378 (1989). L.J. IGNARRO, H. LIPTON, J.C. EDWARDS, W.H. BARICOS, A.L. HYMAN, P.J. KADOWITZ and C.A. GRUETTER, J. Pharmacol. Exp. Ther. 2LB 739-749 (1981). T. HAYASHI, J.M. FUKUTO, L.J. IGNARRO and G. CHADHURI, J. Cardiovasc. Pharmacol. 26792-802 (1995). V. GISCLARD, V. MILLER and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther.244 19-22 (1988). D.Y. CHENG, C.J. FENG, P. KADOWITZ and C.A. GRUETTER, Life Sci. ti 187-191
(1994).
2302
28. 29. 30. 31. 32. 33. 34 35. 36. 37. 38.
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Vol. 60, No. 25,1!%7
A.R. GREGG. L.P. THOMPSON. J.E. HERRIG and C.P. WEINER, J. Vast. Res. x 106-l 11 (l.995). C.P. WEINER. I. LIZASOAIN. S.A. BAYLIS. R.G. KNOWLES, LG. CHARLES and S. MONCADA, x5212-521 6 (1994). T. HAYASHI, K. YAMADA, T. ESAKI, M. KUZUYA, S. SATAKE, T. ISHIKAWA and A. IGUCHI, Biophys. Res. Commun.m 847-655 (1995). K. POMERANTZ, Y. MADDOX, F. MAGGI, E. RAMEY and P.W. RAMWELL, Life Sci. z1233-1236 (1980). M. DAVID, A. GRIESMACHER and M.M. MUELLER, Prostaglandins a431 -438 (1989). R.W. HULL, J.A. HASBARGEN, S. FALL and T.P. O’BARR, Chest. al 11611 19 (1991). T. MIKKOLA, P. TURUNEN, K. AVELA, A. ORPANA, L. VIINIKKA and 0. YLIKORKALA, J. Clin. Endocrinol. Metab. 8Q1832-1836 (1995). L.N. BERGE, J.B. HANSEN, B. SVENSSON, V. LYNGMO and Y. NORDO, Haemostasis 2Q 313-320 (1990). V.M. MILLER and P.M. VANHOUTTE, Am. J. Physiol. mR1502-1507 (1990). B. ZAMORANO, M.E. BRUZZONE and J.L. MARTINEZ, Gen. Pharmacol. 26 1613-1618 (1995). D. SALVEMINI, T.P; MISKO, J.L. MASFERRER, K. SEIBERT, M.G. CURRIE and P. NEEDLEMAN, Proc. Natl. Acad. Sci. USA Qe7240-7244 (1993).
ELSEVIER
PII SOO24-3205(97)00284-1
DIFFERENTIAL
EFFECTS OF LOW- AND HIGH-DOSE ESTROGEN ON VASCULAR RESPONSES IN FEMALE RATS
TREATMENTS
Chiara Bolego, Andrea Cignarella, Raffaella Ruzza, Claude Zaarour, Elio Messi*, Mariarosa Zanisi* and Lina Puglisi Institute of Pharmacological Sciences and *Institute of Endocrinology University of Milan, Via Balzaretti, 9 20133 Milan, ltaly (Received in hal
form March 17, 1997)
Summary In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-P-estradiol injections (5 pg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and No-Methyl-Larginine acetate, which was found to be greater in 17-&estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-P-estradiol-treated females released significantly more prostacyclin than those from oiltreated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 pg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens. Key Words:
aorta, ovariectomy, estrogen, nitric oxide, prostacych
Women in the reproductive age are known to have a lower risk for coronary heart disease (CHD) when compared to men (1,2). Postmenopausal women, due to the loss of ovarian function, are exposed to increased incidence of hypertension and CHD (3) but appear to be protected by estrogen replacement therapy (45). Conversely, it is well established that women using oral contraceptive preparations are subjected to Correspondence to: L. Puglisi Institute of Pharmacological Sciences University of MilanVia Balzaretti, 9. 20133, Milan, Italy Tel. 02/20488305 Fax. 02/29404961
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increased relative risk of CHD (6). These observations, therefore, clearly suggest an influence of estrogens on the vessel wall, although the molecular mechanisms are not yet fully understood. The generation of vasoactive mediators, like nitric oxide (NO) and prostacyclin (PGls), plays a pivotal role in maintaining the cardiovascular system in a state of constant active vasodilation (7) and numerous studies indicateded that alterations in the release of vasodilators synthesized by the vessel wall occur at an early stage in the atherosclerotic process, both in animals and humans (8,Q). Estrogens have been shown to affect the production of vasoactive mediators by the vessel wall. The basal release of NO from rabbit aortic rings was shown to be greater in female than in male rabbis, but ovariectomy eliminated the difference (10). In addition, estrogen treatment was reported to induce in vitro stimulation of rat smooth muscle and piglet aortic endothelial PGlp synthesis (11 ,12), modulation of contractile and relaxant responses in ex vivo vascular tissue isolated from both ovariectomized (OVX) rabbits and rats (13-l 5) and increase of endotheliumdependent relaxation in vivo (16,17). The present experiments were designed in order to test the hypothesis that different protocols of estrogen administration to female rats may induce opposite effects, in terms of NO- and PGls-mediated vascular responses. We therefore compared a low-dose, short-term estrogen treatment of OVX female rats, as a model of replacement therapy, with a long-term, high-dose treatment of intact females, as a model of contraceptive regimen. We evaluated the release of NO indirectly by precontracting aortic rings with norepinephrine and by observing the effects on tone changes induced by superoxide dismutase (SOD), an enzyme able to scavenge superoxide anions, thereby preventing NO inactivation, and No-Methyl-L-arginine acetate (L-NMA), an L-arginine analogue which competes for NO synthase and inhibits NO formation. In addition, we evaluated the vasomotor responses elicited by acetylcholine (ACh) which triggers NO release by endothelial cells, and sodium nitroprusside (SNP), an exogenous NO donor. Methods Animals : The experiments were performed on isolated aortas taken from adult male and female Sprague Dawley rats weighing 200-250 g. The female rats were bilaterally ovariectomized under pentobatbital anesthesia (30 mg/Kg intraperitoneally). After a six weeks’ period of recovery, the animals were divided into three groups: 1) intact males, 2) OVX females, 3) OVX estrogen-treated females. Groups 1) and 2) received peanut oil (0.1 ml) as solvent of the estrogen solution; estrogen treatment consisted of a daily subcutaneous injection of 5 ug/Kg of 17-P_estradiol (E2) during the two days preceding the experiments. Plasma estradiol levels, as measured by radioimmunoassay following this treatment, resulted to be in the high physiological range (18) approximating the proestrus level in the rat (19). In addition, a series of experiments were carried out on intact females treated with high doses (100 us/Kg) of ethinylestradiol (EE). The hormone was injected subcutaneously once a day for one month. /so/a&d aortic ring preparations: At sacrifice, the aorta was removed and gently cleaned of adjacent tissue. Special care was taken to avoid contact with the luminal surface in order to preserve the endothelium. Rings approximately 3 mm in width were then cut, suspended in lo-ml or 20-ml organ baths and perfused (rate: 60 ml h-1 ) with Krebs solution at 37°C continuously bubbled with a 95% 02 and 5% CO2 mixture. The Krebs solution had the following composition (mmol.): NaCl 118.9, KCI 4.66, KH2P04 1.18, MgS04 1.1, CaCh 2.52, NaHCOs 25 and glucose 5.55, pH 7.4. Tissues were equilibrated for 1 h under a resting tension of 1.5 g. In preliminary length-tension
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experiments, optimal resting tension was determined to be 1.5 g for both control and treated aortic rings. Force was recorded by means of a microdynamometer consisting of a strain gauge transducer, a high-gain amplifier and a galvanometer (Pchannel recorder “Gemini” 7070, Basile, Italy). Concentration-response curves: After 1 h equilibration, rings were contracted with 0.1 pmol. norepinephrine (NE) to induce submaximal contraction. Increasing doses of acetylcholine (ACh) were then added to the bath to assess the integrity of the endothelium. If no relaxation to ACh was detected, the preparation was not used for the experiment. To establish whether the agonist sensitivity to NE remained unchanged over the course of the experimental period, two consecutive curves were carried out at about 30 minute intervals. After precontracting the aortic rings submaximally with 0.1 pmol. NE, the drugs to be tested were added cumulatively. Contractile and relaxant responses were expressed as a percentage of the NE contraction. Eicosanoids measurement : PGlp released from aortic tissue was determined as its stable metabolite 6-keto-PGF1, by EIA (Cayman Chemical). Segments approximately 23 cm in length were cut from the same aorta used for the above experiments and equilibrated in Krebs solution on ice for 60 minutes. The incubation medium was removed and the tissues were incubated in Krebs solution at 37% under gentle shaking for 5 minutes. At the end of incubation, the medium was separated by filtration and aliquots of it were assayed. The tissue was carefully desiccated by means of a vacuum-desiccator and the dry weight determined. Values were expressed as pg 6-keto-PGFtJmg dry aorta. Drugs: The following drugs were used: acetylcholine chloride (ACh), norepinephrine hydrochloride (NE), No-Methyl-L-arginine acetate (L-NMA), superoxide dismutase (SOD), sodium nitroprusside (SNP), 17-6-estradiol (E2), ethinylestradiol (EE); all of them were obtained from Sigma. All the drugs were freshly dissolved in saline solution except hormones, which were solubilited in peanut oil. Statistical Analysis: All data are expressed as means f S.E.M. of 5-7 experiments and represent unpaired data. Statistical analysis was performed with a Student’s t-test. A probability value of PeO.05 was considered statistically significant. When more than two means were compared (i.e. responses of aortic rings obtained from male, untreated and estradiol-treated female rats) an analysis of variance with repeated measurements was performed. If a significant F value was found, Scheffe’s test for multiple comparison was used to identify differences among groups. Results Effects of low-dose E2 treatment on the basal release of NO In studies designed to assess the tone-related release of NO from NE-precontracted aortic rings, the responses to superoxide dismutase (SOD) and No-Methyl-L-arginine (L-NMA) acetate were evaluated in aortic rings obtained from males, OVX oil- and OVX E24reated females. Fig. 1 shows that SOD (from 0.1 to 100 Unit/ml) produced concentration-related relaxant responses, with a level of magnitude greater in treated versus untreated female preparations. Moreover, the magnitude of relaxant responses to SOD in aortic rings obtained from OVX females resembled that observed in aortic rings from males.
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Application of increasing concentrations of the NO synthesis inhibitor L-NMA (l-100 pmol.) elicited vasoconstriction in a concentration-dependent manner. A significant increase in the contractile response was detected in tissues from females treated with E2, at least at the highest dose, when compared to tissues obtained from either untreated females or males (Fig. 2). so-
T w-
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5 ; ;i
20-
d
/
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&f T
lo-
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a1
1
Fig. 1
10
loo
SOD, U/ml
Concentration-response curves to SOD in aortic rings obtained from male female OVX oil- (0) and E2- treated (5pg/Kg once a day per two days) rats. Data are expressed as percentage of the response to norepinephrine shown as means f S.E.M., n=5 in each group. Statistical significance: ** P
l
1
10
( q ), (o )
and and
100
L-NM&-Log Iw
Fig. 2 Concentration-response curves to L-NMA in aortic rings obtained from male ( q ), female OVX oil- (0) and OVX E2-treated ( l ) (5pg/Kg once a day per two days) rats. Data are expressed as percentage of the response to norepinephrine. Values are the mean f S.E.M. of 7 separate experiments. No differences were observed between male and female OVX-oil treated animals. Statistical significance: * PC 0.05.
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Effects of low-dose of E2 treatment on the vascular responses to ACh and sodium nitroprusside ACh (0.1-l 0 pmol.) produced a concentration-dependent, NO-mediated relaxation of endothelium intact aortic rings precontracted with 0.1 umol. NE. As shown in Fig. 3, no significant differences in the relaxant responses to ACh could be observed in aortic rings from male, OVX oil-treated and OVX E2-treated female rats. Fig.4 illustrates the responses elicited by sodium nitroprusside (SNP), an endotheliumindependent vasodilator, cumulatively added into the incubation medium (0.001-l Hmol.). The sensitivity of the vascular smooth muscle in precontracted preparations was not affected by E2 treatment. Furthermore, the endothelium-independent relaxation to SNP of aortic rings from male was similar to that of OVX female animals.
1
-
7
ACh, -Log fmoL/ Fig. 3 Concentration-response curves to Ach cumulatively added to aortic rings obtained from male (0 ), female OVX oil- (0) and OVX EP-treated (5 ug/Kg once a day per two days) (0). Data are expressed as percentage of the response to norepinephrine and shown as means + S.E.M., n=7 in each group. No differences were observed between the three groups.
Effect of E2 treatment on PGI2 release The release of PGl2, measured as its stable metabolite 6-ketoPGF,,, was evaluated after incubation of aortic rings at 37°C in Krebs solution for 5 minutes. The results are shown in Fig. 5: in OVX E2-treated rats, the levels of PGls, released into the incubation medium were significantly higher than those observed in the preparations obtained from untreated animals (195.8k17.4 vs. 132.2+-9.6 pg/mg dry tissue, p
22%
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8
9
6
SNP, -Log Im0l.l
Fig. 4 Percent of relaxation to SNP of aortic rings obtained from male (0 ), female OVX E2-treated (0 ) (5pg/Kg once a day per two days), and female OVX oiltreated rats (0). Aortic rings were submaximally contracted with norepinephrine before obtaining cumulative response to SNP. Values are the mean f S.E.M. of 5 separate experiments. Responses were similar in all three groups. 250
** ............................ ............ . ........................... .....................
50
Fig. 5 6-keto-PGFlo released into the incubation medium by aortic rings obtained from female OVX oil-treated (white column) and OVX E2-treated (black column) (5 ug/Kg once a day per two days) rats. Each value is the mean fS.E.M. of 7 experiments. Statistical significance: **PcO.Ol. shown in Figs. 6 and 7, such a treatment resulted in a significant decrease in the responses to both SOD and L-NMA. Furthermore, the concentration-dependent relaxation to either ACh or SNP was affected by the treatment with EE at this high dose. Indeed, the endothelium-dependent (ACh) and the endothelium-independent (SNP) relaxations were lower in aortic rings from EE-treated compared to untreated animals (Figs 8 and 9).
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Ii1
;
10
loo
SOD,Ulnd
Fig. 6 Concentration-response curves to SOD in aortic rings obtained from intact females rats treated with high doses of EE ( l) (100 pg/Kg daily for one month) or oil (0). Values are expressed as percentage of the response to norepinephrine and are the mean + S.E.M. of 7 separate experiments. Statistical significance: ’ P< 0.05. so
40
i” jm 10
0:
II
Fig. 7 Concentration-response curves to L-NMA in aortic rings obtained from intact females treated with high doses (100 pg/Kg daily for one month) of EE (a) or oil (0). Data are expressed as percentage of the response to norepinephrine and are the mean f S.E.M. of 6 separate experiments. Statistical significance: P< 0.05. l
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5
ACh, -Logfmo~ Fig. 8 Concentration-response curves to ACh cumulatively added to aortic rings obtained from intact female treated with high doses (100 pg/Kg daily for one month) of EE ( l) or oil (0). Data are expressed as percentage of the response to norepinephrine and are the mean f S.E.M. of 6 separate experiments. Statistical significance:* P< 0.05.
9
6
7
6
SNP, -Log knol.l Fig. 9 Percent of relaxation of aortic rings obtained from intact female treated with high doses (100 pg/Kg daily for one month) of EE (0) or oil (0). Aortic rings were submaximally contracted with norepinephrine before obtaining cumulative response to SNP. Values are the mean f S.E.M. of 6 separate experiments. Statistical significance: P< 0.05. l
We also evaluated the effect of the treatment with EE on the release of PGl2 by aortic tissue. After 5 min incubation, the release of 6-keto-PGF1, (the stable product of PGls catabolism) underwent a statistically significant decrease in aortic rings obtained from EE-treated females compared to controls (247.4k38.7 vs. 414.4k60.5 pg/mg dry tissue, peO.01) (Fig.1 0).
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Fig. 10 6-keto-PGF,, released into the incubation medium by aortic rings obtained from intact female rats treated with high doses (100 pg/Kg daily for a month) of EE (black column) or oil (white column). Each value is the mean + S.E.M. of 7 experiments. Statistical significance: **PC 0.01.
Discussion Several epidemiological studies pointed out the protective role of estrogens against the occurence of CHD, as indicated by the low CHD rates in premenopausal women, the narrowing of the gender gap in CHD mortality after menopause, and the decreased incidence of CHD in women undergoing estrogen replacement therapy after menopause (2,5,20,21). On the other hand, the significant increase in the risk of thromboembolic disease in oral contraceptive users has been ascribed to the estrogenic component (4,22). It can be speculated, therefore, that estrogens can induce remarkably different effects on the biology of the vascular wall depending on the physiological status, the duration of the treatment and the level of circulating hormones. In view of this hypothesis, we measured the vascular responses mediated by NO, as well as the release of PGl2, in aortic rings obtained from female rats following low- or high-dose estrogen treatment. The basal release of NO was indirectly evaluated by utilyzing SOD, an enzyme able to scavenge superoxide anions, and L-NMA, an L-arginine analogue which competes for NO synthase and inhibits NO formation (23). E2 replacement administration to OVX female rats induced a greater relaxant response to SOD and a more marked contractile response to L-NMA than that observed in tissue of untreated OVX females or males. No differences among the three experimental groups, however, were detected when either the ACh-stimulated release of NO from endothelial cells or the responsiveness of smooth muscle cells to SNP, a stable NO donor (24), was evaluated. These findings are in agreement with those previously reported by other investigators (10,25), suggesting that the gender differences in NO basal release are related to an increased release of NO by endothelial cells and not to an increased sensitivity of vascular smooth muscle to the released NO. Indeed, the relaxant responses elicited by endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilators were not affected by sex hormone treatment. In contrast, previous studies reported that estrogen treatment
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enhances endothelium-dependent relaxation of precontracted vascular preparations. Gisclard et al. (26) observed this effect in femoral artery tissue, whereas ParedesCarbajal et al. (15) reported enhanced carbachol-induced relaxation of 8x viva phenilephrine-precontracted aortic rings following one day estrogen administration. Cheng et al., (27) explained the enhanced ACh-induced responses after a 1Cday treatment with 17-fl-estradiol with a depressed acetylcolinesterase activity. The discrepancy with our results could be explained by differences in the activity of acetylcholinesterase and in the responsiveness to released NO among vascular tissues, as well as by the regional variation in the ACh-stimulated release of NO along the aorta (26), the use of carbachol, which is resistant to acetylcholinesterase and the different protocols of estrogen administration. In contrast to the low-dose E2 treatment, however, prolonged EE administration resulted not only in a reduced basal release of NO, but also in an impairement of endothelial function and/or smooth muscle responsiveness to NO, as suggested by the decreased relaxation to ACh and SNP of aortic rings excised from EE-treated animals. Taken together, our results would suggest that estrogens affect the vessel wall functionality at two levels. A relatively rapid vasodilatory effect might account for the observed differences in the basal release of NO, whereas a later-onset effect might eventually lead to alterations in endothelial functions and smooth muscle responsiveness to NO. The latter component is likely to be mediated by estrogen receptor-dependent alterations in NO synthase gene expression (29,30). Moreover, the contrasting effects of short- and long-term estrogen administration on the NO release pathway and PG12production in aortic tissue could be accounted for by the down regulation or desensitization of estrogen receptors following prolonged exposure to unphysiolqical levels of hormone in the vessel wall. Alternatively, changes in ACh receptor characteristics might occur. As far as the effects of estrogens on PGl2 release are concerned, in vitro (31,32) and in viva studies (33) produced highly inconsistent results, probably on the account of the variable experimental conditions used by different investigators. In our hands, low-dose E2 treatment induced a rise in PGl2generation in OVX female, in comparison with untreated controls (34) whereas EE-treated intact females produces less PG12than controls (35). Interestingly, the aortic release of PG12by untreated OVX female turned out to be significantly lower than that observed in untreated intact females (compare Figs. 5 and lo), thereby supporting the concept of a reduced availability of vasodilating agents in estrogen deficiency. Our results suggest that the production of PGl2, as well as that of NO, is stimulated by physiological levels of estrogens impaired by higher hormone concentrations, consistently with the general impairement of the relaxant vascular responses under the latter conditions. The possibility that E2-treatment also increases the synthesis and release of vasocontricting cyclooxygenase products (15,36,37), which could further modulate vascular responses, cannot be rouled out. In addition, an intriguing link between NO and the cyclooxygenase pathway has been recently described (36) and we cannot exclude that such an interaction is relevant also under our experimental conditions. In conclusion, the protective effects exerted by physiological levels of estrogen are related to the enhanced availability of vasodilating agents (NO, PG12) within the vessel wall. Such a protection is dimmed when plasma estrogen concentrations are either below (OVX animals) or above (long-term EE treatment) the physiological range, thereby suggesting the existance of regulatory mechanisms (possibly mediated by specific receptors) which appear to be tightly controlled by the level of circulating estrogens.
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References 1. 2. 3. 4. 5. 6.
;: 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19. 20.
21. 22. 23. 24.
25. 26. 27.
N.K. WENGER, L. SPEROFF and B. PACKARD, N. Engl. J. Med. m 247-256 (1993). M.J. STAMPFER and G.A. COLDITZ, Prev. Med. 2Q 47-63 (1991). T.L. BUSH and E. BARRET-CONNOR, Epidemiol. Rev. Z 89-104 (1985). P.D. STOLLEY, J.A. TONASCIA, M.S. TOCKMAN, P.E. SARTWELL, A.H. RUTLEDGE and M.P. JACOBS, Am. J. Epidem. 142 197-201 (1975). D. GRADY, S.M. RUBIN, D.B. PETITTI, C.S. FOX, B. ETTINGER, U.L. ERNSTER and S.R. CUMMINGS, Ann. Inter. Med. 1z 1016-l 037 (1992). J.B. LABAT, S.D. BERGMANN and J.H. ZAVORAL, Cardiovasc. Risk Fact. 4 5159 (1994). R. BOTTING and J.R. VANE, Arch. Mal. Coer. 82 11-4 (1989). S.W. WERNS, J.A. WALTON, H.H. HSAI, E.G. NABEL, M.L. SANZ and B. PITT, Circulation z9 287-291 (1989). H. SHIMOKAWA and P.M. VANHOUlTE, J. Clin. Invest. 64 900-914 (1989). T. HAYASHI, J.M. FUKUTO, L.J. IGNARRO and G. CHAUNDURI, Procl. Natl. Acad. Sci. USA 89 11259-l 1263 (1992). W. CHANG, J. NAKAO, H. ORIMO and S. MUROTA, Biochim. Biophys. Acta m 107-l 18 (1989). C. SEILLAN, C. ODY, F. RUSSO-MARIE and D. DUVAL, Prostaglandins 263-12 (1983). V. GISCLARD, N.A. FLAVAHAN and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther. 24p 466-470 (1987). V.M. MILLER and M. VANHOUTTE, Am. J. Physiol. 261 R1022-1027 (1993). M.C. PAREDES-CARBAJAL, M.A. JUAREZ-OROPEZA, CM. ORTIZ-MENDOZA and D. MASHER, Life Sci. =473-486 (1995). J.K. WILLIAMS, M.R. ADAMS and H.S. KLOPFENSTEIN, Circulation 81 16801687 (1990). P. COLLINS, G.M.C. ROSANO, P.M. SARREL, L. ULRICH, S. ADAMOPOULOS, C.M. BEALE, J.G. MCNEILL and P.A. POOLEWILSON, Circulation 92 24-30 (1995). R.E. WATSON, G.E. HOFFMAN and S.J. WIEGAND, Brain Res. m 157-163 (1986). J.K.H. LU, P.S. LAPOLT, T.E. NASS, D.W. MAlT and H.L. JUDD, Endocrinology 116 1953-i 959 (1985). E.D. EAKER and W.P. CASTELLI, Coronary heart disease in women. E. Eaker, B. Packard, N.K. Wenger, T.B. Clarkson and H.A. Tyroler (Eds), 122-132, Haymarket Doyma, New York (1987). S.A. SAAMAN and M.H. CRAWFORD, J. Am. Coil. Cardiol. al403-1410 (1995). W.H.W. INMAN, M.P. VESSEY, B. WESTERHOLM and A. ENGELUND, Brit. Med. J. 2 203-208 (1970). D.D. REES, R.M.J. PALMER and S. MONCADA, Proc. Natl. Acad. Sci. USA u 3375-3378 (1989). L.J. IGNARRO, H. LIPTON, J.C. EDWARDS, W.H. BARICOS, A.L. HYMAN, P.J. KADOWITZ and C.A. GRUETTER, J. Pharmacol. Exp. Ther. 2LB 739-749 (1981). T. HAYASHI, J.M. FUKUTO, L.J. IGNARRO and G. CHADHURI, J. Cardiovasc. Pharmacol. 26792-802 (1995). V. GISCLARD, V. MILLER and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther.244 19-22 (1988). D.Y. CHENG, C.J. FENG, P. KADOWITZ and C.A. GRUETTER, Life Sci. ti 187-191
(1994).
2302
28. 29. 30. 31. 32. 33. 34 35. 36. 37. 38.
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A.R. GREGG. L.P. THOMPSON. J.E. HERRIG and C.P. WEINER, J. Vast. Res. x 106-l 11 (l.995). C.P. WEINER. I. LIZASOAIN. S.A. BAYLIS. R.G. KNOWLES, LG. CHARLES and S. MONCADA, x5212-521 6 (1994). T. HAYASHI, K. YAMADA, T. ESAKI, M. KUZUYA, S. SATAKE, T. ISHIKAWA and A. IGUCHI, Biophys. Res. Commun.m 847-655 (1995). K. POMERANTZ, Y. MADDOX, F. MAGGI, E. RAMEY and P.W. RAMWELL, Life Sci. z1233-1236 (1980). M. DAVID, A. GRIESMACHER and M.M. MUELLER, Prostaglandins a431 -438 (1989). R.W. HULL, J.A. HASBARGEN, S. FALL and T.P. O’BARR, Chest. al 11611 19 (1991). T. MIKKOLA, P. TURUNEN, K. AVELA, A. ORPANA, L. VIINIKKA and 0. YLIKORKALA, J. Clin. Endocrinol. Metab. 8Q1832-1836 (1995). L.N. BERGE, J.B. HANSEN, B. SVENSSON, V. LYNGMO and Y. NORDO, Haemostasis 2Q 313-320 (1990). V.M. MILLER and P.M. VANHOUTTE, Am. J. Physiol. mR1502-1507 (1990). B. ZAMORANO, M.E. BRUZZONE and J.L. MARTINEZ, Gen. Pharmacol. 26 1613-1618 (1995). D. SALVEMINI, T.P; MISKO, J.L. MASFERRER, K. SEIBERT, M.G. CURRIE and P. NEEDLEMAN, Proc. Natl. Acad. Sci. USA Qe7240-7244 (1993).