- Email: [email protected]
Effects of 17ß-estradiol on endothelium-dependent relaxation induced by acetylcholine in female rat aorta
Life Sciences,Vol. 55, No. 10, pp. PL 187-191, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-32O5/94 $6.00 + .00
Pergamon 0024-3205(94) 00200-2
P H A R M A C O L O G Y LETTERS Accelerated Communication
EFFECTS OF 17fl-ESTRADIOL ON ENDOTHELIUM-DEPENDENT RELAXATION INDUCED BY ACETYLCHOLINE IN FEMALE RAT AORTA David Y. Cheng 1, Chang J. Feng 1, Philip J. Kadowitz I and Carl A. Gruetter2 1Department of Pharmacology, Tulane University, New Orleans, LA and 2Department of Pharmacology, Marshall University, Huntington, WV (Submitted April 26, 1994; accepted May 15, 1994; received in final form June 22, 1994)
Abstract. Estrogens have been postulated to play an important role in modulation of vascular responses to endogenous reactive substances. The effects of chronic in vivo treatment with 17Bestradiol on relaxant responses to acetylcholine were investigated in the rat aorta isolated from prepubertal female rats. The selectivity of effects of 1713-estradiol on acetylcholine-induced relaxation was evaluated using histamine, another endothelium-dependent relaxant in the rat aorta. 175-Estradiol significantly enhanced endothelium-dependent relaxation induced by acetylcholine, but did not alter the vascular responses to acetylcholine in endothelium-denuded aortic rings isolated from prepubertal female rats. In contrast, 171Lestradiol did not change endotheliumdependent relaxation induced by histamine in endothelium-intact aortic rings. The results of the present study demonstrate that 175-estradiol selectively enhances acetylcholine-induced endothelium-dependent relaxation in the rat aorta. Key Words: 17/3-estradiol,acetylcholine,histamine,bethanechol, aorta, relaxation
Introduction It has been demonstrated that estrogens play an important role in modulation of cardiovascular system in response to a variety of endogenous vasoaetive substances (1-6). Differences in the incidence of hypertension and coronary heart diseases between premenopausal female and male or postmenopausal females, and an apparent reduction in cardiovascular diseases with estrogen-replacement therapy in postmenopausal female suggest an important influence of estrogens on the cardiovascular system (1,7). In animal studies, chronic administration of estrogen also attenuates cardiovascular disease profiles in spontaneously hypertensive rats (SHR) and atheroselerotic rabbits and monkeys (7). Decreases in the contractile responses to angiotensin II following chronic treatment with estrogens were observed in in vivo and vitro experiments studied (1,3,6). Recent evidence indicates that estrogens enhance aeetylcholine-indueed relaxation in uterine arteries isolated from virgin guinea pigs (2,8) and in femoral arteries isolated from rabbits (4). However, the effects of estrogens on vascular responses to acetylcholine in isolated rat aorta are unknown. The present study was, therefore, undertaken to investigate the effects of 175estradiol on endothelium-dependent relaxation in response to acetylcholine using aortic rings isolated from prepubertal female rats pretreated chronically with 175-estradiol in vivo. Methods Animals: Prepubertal female Spraque-Dawley rats (50-74 g) were obtained from Taconic Farms Corresponding author: David Y. Cheng, Ph.D., Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112
PL-188
Effects of 17~-Estradiol
Vol. 55, No. 10, 1994
Inc. (New York). Using a 10-gauge trochar, a 17g-estradiol (5 mg/pellet) or placebo pellet was implanted s.c. at the back of the neck of rats and was left in for 14 days. Levels of plasma estrogens were measured by radioimmunoassay (placebo-treated rats, 21+2.4 pg/ml; 178-estradiol-treated rats, 180_+17.5 pg/ml) (9). Preparation of aortic rin~s: Fourteen days after the treatment, rats were administered 100 mg/kg sodium pentobarbital. After achievement of deep anesthesia, the abdomen was opened and a mesenteric blood vessel was severed for exsanguination and thoracic aortas were isolated. Once isolated, the aortas were placed into Krebs buffer, and the fat and loosely adhering connective tissue was removed. Rings were prepared by transversely cutting vascular segments into 5 mm rings using fine-tipped scissors. To destroy endothelium in selected rings, the intimal surface was rubbed (20-35 s) using needle 10 gauge with lightly scuffed external surface. Measurement of isometric tension: Aortic rings were mounted in 5 or 10 ml jacked tissue baths by suspending them between two L-shaped stainless hooks. The upper hooks were connected to forcedisplacement transducers (FT03C, Grass Instrument Co. Quincy, MA). The bath chambers contained Krebs buffer (37+5°C) constantly bubbled with 95%O2-5%CO2. Isometric force was measured and recorded (Beckman R-611 Dynograph, Sensormedics, Anaheim, CA). After mounting, rings were equilibrated under a 2 g load for 1-2 hr with several adjustments of length until optimal baseline force was maintained. Concentration-response curves for relaxation induced by acetylcholine, histamine and bethanechol were determined cumulatively in aortic rings preeonwaeted with norepinephrine (3x10 -7 M for placebo; 10-7 M for 17B-estradiol) by which tone was raised to 60% of reference contraction induced by 80 mM potassium (3). Drugs and solutions: Norepinephrine hydrochloride, acetylcholine chloride, bethanechol chloride, and histamine dihydrochloride (Sigma Chemical Co. St.. Louis, MO) were dissolved in distill water. 17B-Estradiol (5 rag/pellet) and placebo pellets (Innovative Research of America Toledo, OH) were designed to release 17B-estradiol over a 21 day period. Krebs buffer used in all experiments consisted of (raM): NaCI 118; NaHCO3 25; KC1 4.7; CaC12 1.5; MgC12 1.1; KH2PO4 1.2 and glucose 5.6. Data analvsis: Results were expressed as mean_+SE. Concentration-response curves were compared using Two-Way ANOVA or a paired t-test. A P value of less than 0.05 was used as the criterion for statistical significance. Results
Cumulative concentration-response curves for acetylcholine-induced relaxation in aortic rings with and without endothelium isolated from the prepubertal female rats which were pretreated with 171?,-estradiol or placebo are compared and the data are summarized in Fig. 1. In the presence of endothelium, relaxant responses to acetylcholine were significantly enhanced in doses of 10.8 to 3x10 "6 M in aortic rings isolated from prepubertal female rats pretreated with 17B-estradiol when compared to placebo (Fig. 1, left panel, Two-Way, ANOVA, p<0.05). In contrast, relaxant responses to acetylcholine were diminished after removal of endothelium, and responses to acetylcholine were not different in endothelium-denuded aortic rings isolated from rats pretreated with 171gestradiol when compared to placebo (Fig. 1, right panel, Two-ANOVA, p>0.05). The selectivity of the effects of 171gestradiol on acetylcholine-induced endothelium-dependent relaxation was assessed by investigating the responses to histamine, another endothelium-dependent relaxant agent in the rat aorta, and data are summarized in Fig. 2. The cumulative concentrationresponse curves for histamine-induced relaxation were not different in doses of 10.7 to 3x10"4 M in aortic tings from prepubertal female rats pretreated with 178-estradiol when compared to placebo (Fig. 2, left panel, Two-Way ANOVA, p>0.05). The cumulative concentration-response curves for relaxation induced by bethanechol, an acetylcholine analog, in endothelium-intact aortic tings isolated from prepubertal female rats which were pretreated with 171~estradiol ~ placebo were also compared, and data are summarized in Fig. 2. Relaxant responses to bethanechol in doses of 10.8 to 10"~ M were similar in aortic rings isolated from the prepubertal female rats pretreated with 17Bestradiol when compared to placebo (Fig. 2, right panel, Two-Way ANOVA, p>0.05).
Vol. 55, No. 10, 1994
Effects of 17~-Estradiol
n=7-14 - - o - -
z
100
1
171?,-estradiol
.--o--placebo 10.
+E
PL-189
-Endo
8.
75.
6. 50. 4-
25.
2.
0
0 -9
-8
-7
-6
-5
-9
-8
-7
-6
-5
ACETYLCHOLINE (logM)
Fig. 1 The effects of 17B-estradiol on acetylcholine-induced relaxation in aortic tings with (left panel) and without endothelium (right panel) isolated from prepubertal female rats pretreated with 1713-estradiol or placebo. Each aortic ring precontracted submaximally by norepinephrine was exposed to cumulatively increasing concentrations of acetylcholine. Data were calculated as percentage relaxation of tone induced by norepinephrine, n, indicates number of animals.
+Endo n=7-14 - - o - - 171)-estradiol
- - o - - placebo
100-
100. Z 75O
75
50.
50
25-
0
E -8
HISTAMINE (logM)
-9 -8 -7 -g BETHANECHOL (logM)
Fig. 2 The effects of 17B-eswadiol on relaxation induced by histamine (left panel) and bethanechol (tight panel) in aortic tings with endothelium isolated from prepubertal female rats pretreated with 1713-estradiol or placebo. Each aortic rings precontracted submaximally by norepinephrine was exposed to cumulatively increasing concentrations of histamine and bcthanechol. Data were calculated as percentage relaxation of tone induced by norepinephrine. n, indicates number of animals.
PL-190
Effects of 17~-Estradiol
Vol. 55, No. 10, 1994
Discussion Results from the present study demonstrate that chronic in vivo treatment with 17B-estradiol significantly enhances acetylcholine-induced endothelium-dependent relaxation in aortic rings isolated from prepubertal female rats. Removal of endothelium abolishes acetylcholine-induced relaxation in the aortic rings, and treatment with 17B-estradiol did not alter vascular responses to acetylcholine in the endotheliurn-denuded rat aorta. In contrast to enhancement by estrogens of acctylcholine-indueed relaxation, the data of the present study show that 17~estradiol does not change vascular relaxant responses to histamine, another endothelium-dependent relaxant agent, suggesting that the effect of 17B-cstradiol on endothelium-dependent relaxation induced by acetylcholine is selective in the rat aorta. The observation showing that 17B-estradiol increases acetylcholine-induced endotheliumdependent relaxation is consistent with the results of the previous study in uterine arteries isolated from virgin guinea pigs (3,8) and in femoral arteries from rabbits (4). Plasma concentrations of estrogens have been shown to increase dramatically in normal pregnancy and role for sex hormone in the cardiovascular changes associated with pregnancy has also been suggested (10,11). Contractile responsiveness of vascular tissues to angiotensin II decreases in in vivo and vitro experiments (1,3,10,12,). The results of the present study suggest that estrogens may play an important role in regulation of the vascular tone and may be a useful therapeutic agent for treatment of cardiovascular diseases. The mechanisms by which estrogens increase endothelium-dependent relaxant responses to acetylcholine in blood vessels are uncertain. The acute treatment with 17~estradiol for 24 hr has no effects on contractile responses to angiotensin II and relaxant responses to acetylcholine (data not shown). Functional estrogen receptors have been reported to be present in aortic smooth muscle cells (13) and endothelial cells from rat blood vessels (14). The chronic in vivo treatment with estrogens is more likely related to alteration of the transcription and translation levels of genes. Increases in the number of muscarinic receptors by estrogens have been shown in hypothalamus (5), and enhancement of formation of endothelium-derived nitric oxide (EDNO) (5,8,15) as well as alteration of vascular smooth muscle sensitivity in response to acetylcholine have been suggested (4). Although potential mechanisms by which acetylcholine-induced endothelium-dependent relaxation was enhanced by 17B-estradiol were not directly determined in the present study, observation that estrogens had no significant effects on relaxation induced by histamine, another endotheliumdependent relaxant, suggests that the treatment with 17B-estradiol does not alter production of EDNO or sensitivity of vascular smooth muscle in response to acetylcholine in the rat aorta. The observation showing that estrogens do not alter the sensitivity of vascular smooth muscle to El)NO in the rat aorta is in agreement with results of the previous study in isolated rabbit aorta in which treatment with 1713estradiol for 4 days had no significant effects on relaxant responses to calcium ionophore, A23187 (4). It has been interestingly noticed that 17g-estradiol dose not alter endothelium-dependent relaxation induced by bethanechol, an acetylcholine analog, resistant to hydrolysis by acetylcholinesterase. The data suggest that the chronic in vivo treatment with estrogen may down regulate activity of acetylcholinesterase that inactivates acetylcholine in the action site of blood vessels and increased levels of acetylcholine may enhance the endothelium-dependent relaxation in the rat aorta. If estrogens augment the number of muscarinic receptors to a significant extent, acetylcholineand bethanechol-indueed endothelium-dependent relaxation would be enhanced in a similar manner in the rat aorta. The finding in the present study does not support the hypothesis that enhancement of estrogens on endothelium-dependent relaxation induced by acetylcholine is due to increases in the muscarinic receptors (4). Future experiments should be designed to investigate mechanisms by which responses to acetylcholine are mediated and the effects of estrogens on acetylcholinesterase activity need to be further characterized in isolated endothelial cells from the rat aorta. In conclusion, the results of the present study demonstrate that 17B-cstradiol selectively enhances aeetylcholine-induced endothelium=dependent relaxation and has no effects on vascular smooth muscle in response to aeetyleholine in the isolated rat aorta. The data suggest that enhancement by 17B-estradiol of acetylcholine-induced endothelium-dependent relaxation may be involved in down regulation of acetylcholinesterase activity in the rat aorta.
Vol. 55, No. 10, 1994
Effects of 17/3-Estradiol
PL-191
Acknowledmnents This work was supported in part by grants from the U.S. Public Health Service (HL35711) and American Heart Association, West Virginia Affdiate. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
R. ABDUL-KARIM and N.S. ASSALI, Am. J. Obstet. Gynecol. 82 266-272 (1961). C. BELL, Br. J. Pharmacol. 49 595-601 (1973). D.Y. CHENG and C.A. GRUE'I'I~R, Eur. L Pharmacol. 215 171-176 (1992). V. GISCLARD, V.M. MILLER and P.M. VANHOUTTE, J. Phannacol, Exp. Ther. 244 19-22 (1987). T.C. RAINBOW, V. DEGROFF, V.H. LUINE and B.S. McEWAN, Brain Res. 198 239-243 (1980). R.C. ROSENFELD and M.G. JACKSON, Am. J. Obst. Gynecol. 148 429-435 (1984). J. HOGE, M. WILLIS and M.H. WEINHERGER, Am. J. Physiol. 2 H369H374 (1977). C. BELL and C.J. COFFEY, Repro. Fertil. 66 133-137 (1982). I. HASHIMOTO, M. KAWAMINAMI, K. MOTOH and H. WAKURI, Excerpta Medica International Congress Series 672 628-635 (1984). R.C. ROSENFELD, F.H. MORRIS, F.C. Jr.BATI'AGLIA, E.L.,MAKOWSKI and G. MESCHIA, Am. J. Obstet. Gynecol. 124, 618-629 (1976). K. UCLAND, and J.T. PARER, Am. J. Obstet. Gynecol. 96 400-412 (1966). M.S. PALLER, Am. J. Kidney. Dis. 9 308-315 (1984). A.L. LIN and S.A. SYDNEY, Arteriosclerosis 5 688-692 (1985). P. COLBURN and V. BUONASSISI, Science 201 817-819 (1978). R.F. FURCHGO'Vr, Circ. Res. 53 557-573 (1983).
Pergamon 0024-3205(94) 00200-2
P H A R M A C O L O G Y LETTERS Accelerated Communication
EFFECTS OF 17fl-ESTRADIOL ON ENDOTHELIUM-DEPENDENT RELAXATION INDUCED BY ACETYLCHOLINE IN FEMALE RAT AORTA David Y. Cheng 1, Chang J. Feng 1, Philip J. Kadowitz I and Carl A. Gruetter2 1Department of Pharmacology, Tulane University, New Orleans, LA and 2Department of Pharmacology, Marshall University, Huntington, WV (Submitted April 26, 1994; accepted May 15, 1994; received in final form June 22, 1994)
Abstract. Estrogens have been postulated to play an important role in modulation of vascular responses to endogenous reactive substances. The effects of chronic in vivo treatment with 17Bestradiol on relaxant responses to acetylcholine were investigated in the rat aorta isolated from prepubertal female rats. The selectivity of effects of 1713-estradiol on acetylcholine-induced relaxation was evaluated using histamine, another endothelium-dependent relaxant in the rat aorta. 175-Estradiol significantly enhanced endothelium-dependent relaxation induced by acetylcholine, but did not alter the vascular responses to acetylcholine in endothelium-denuded aortic rings isolated from prepubertal female rats. In contrast, 171Lestradiol did not change endotheliumdependent relaxation induced by histamine in endothelium-intact aortic rings. The results of the present study demonstrate that 175-estradiol selectively enhances acetylcholine-induced endothelium-dependent relaxation in the rat aorta. Key Words: 17/3-estradiol,acetylcholine,histamine,bethanechol, aorta, relaxation
Introduction It has been demonstrated that estrogens play an important role in modulation of cardiovascular system in response to a variety of endogenous vasoaetive substances (1-6). Differences in the incidence of hypertension and coronary heart diseases between premenopausal female and male or postmenopausal females, and an apparent reduction in cardiovascular diseases with estrogen-replacement therapy in postmenopausal female suggest an important influence of estrogens on the cardiovascular system (1,7). In animal studies, chronic administration of estrogen also attenuates cardiovascular disease profiles in spontaneously hypertensive rats (SHR) and atheroselerotic rabbits and monkeys (7). Decreases in the contractile responses to angiotensin II following chronic treatment with estrogens were observed in in vivo and vitro experiments studied (1,3,6). Recent evidence indicates that estrogens enhance aeetylcholine-indueed relaxation in uterine arteries isolated from virgin guinea pigs (2,8) and in femoral arteries isolated from rabbits (4). However, the effects of estrogens on vascular responses to acetylcholine in isolated rat aorta are unknown. The present study was, therefore, undertaken to investigate the effects of 175estradiol on endothelium-dependent relaxation in response to acetylcholine using aortic rings isolated from prepubertal female rats pretreated chronically with 175-estradiol in vivo. Methods Animals: Prepubertal female Spraque-Dawley rats (50-74 g) were obtained from Taconic Farms Corresponding author: David Y. Cheng, Ph.D., Department of Pharmacology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112
PL-188
Effects of 17~-Estradiol
Vol. 55, No. 10, 1994
Inc. (New York). Using a 10-gauge trochar, a 17g-estradiol (5 mg/pellet) or placebo pellet was implanted s.c. at the back of the neck of rats and was left in for 14 days. Levels of plasma estrogens were measured by radioimmunoassay (placebo-treated rats, 21+2.4 pg/ml; 178-estradiol-treated rats, 180_+17.5 pg/ml) (9). Preparation of aortic rin~s: Fourteen days after the treatment, rats were administered 100 mg/kg sodium pentobarbital. After achievement of deep anesthesia, the abdomen was opened and a mesenteric blood vessel was severed for exsanguination and thoracic aortas were isolated. Once isolated, the aortas were placed into Krebs buffer, and the fat and loosely adhering connective tissue was removed. Rings were prepared by transversely cutting vascular segments into 5 mm rings using fine-tipped scissors. To destroy endothelium in selected rings, the intimal surface was rubbed (20-35 s) using needle 10 gauge with lightly scuffed external surface. Measurement of isometric tension: Aortic rings were mounted in 5 or 10 ml jacked tissue baths by suspending them between two L-shaped stainless hooks. The upper hooks were connected to forcedisplacement transducers (FT03C, Grass Instrument Co. Quincy, MA). The bath chambers contained Krebs buffer (37+5°C) constantly bubbled with 95%O2-5%CO2. Isometric force was measured and recorded (Beckman R-611 Dynograph, Sensormedics, Anaheim, CA). After mounting, rings were equilibrated under a 2 g load for 1-2 hr with several adjustments of length until optimal baseline force was maintained. Concentration-response curves for relaxation induced by acetylcholine, histamine and bethanechol were determined cumulatively in aortic rings preeonwaeted with norepinephrine (3x10 -7 M for placebo; 10-7 M for 17B-estradiol) by which tone was raised to 60% of reference contraction induced by 80 mM potassium (3). Drugs and solutions: Norepinephrine hydrochloride, acetylcholine chloride, bethanechol chloride, and histamine dihydrochloride (Sigma Chemical Co. St.. Louis, MO) were dissolved in distill water. 17B-Estradiol (5 rag/pellet) and placebo pellets (Innovative Research of America Toledo, OH) were designed to release 17B-estradiol over a 21 day period. Krebs buffer used in all experiments consisted of (raM): NaCI 118; NaHCO3 25; KC1 4.7; CaC12 1.5; MgC12 1.1; KH2PO4 1.2 and glucose 5.6. Data analvsis: Results were expressed as mean_+SE. Concentration-response curves were compared using Two-Way ANOVA or a paired t-test. A P value of less than 0.05 was used as the criterion for statistical significance. Results
Cumulative concentration-response curves for acetylcholine-induced relaxation in aortic rings with and without endothelium isolated from the prepubertal female rats which were pretreated with 171?,-estradiol or placebo are compared and the data are summarized in Fig. 1. In the presence of endothelium, relaxant responses to acetylcholine were significantly enhanced in doses of 10.8 to 3x10 "6 M in aortic rings isolated from prepubertal female rats pretreated with 17B-estradiol when compared to placebo (Fig. 1, left panel, Two-Way, ANOVA, p<0.05). In contrast, relaxant responses to acetylcholine were diminished after removal of endothelium, and responses to acetylcholine were not different in endothelium-denuded aortic rings isolated from rats pretreated with 171gestradiol when compared to placebo (Fig. 1, right panel, Two-ANOVA, p>0.05). The selectivity of the effects of 171gestradiol on acetylcholine-induced endothelium-dependent relaxation was assessed by investigating the responses to histamine, another endothelium-dependent relaxant agent in the rat aorta, and data are summarized in Fig. 2. The cumulative concentrationresponse curves for histamine-induced relaxation were not different in doses of 10.7 to 3x10"4 M in aortic tings from prepubertal female rats pretreated with 178-estradiol when compared to placebo (Fig. 2, left panel, Two-Way ANOVA, p>0.05). The cumulative concentration-response curves for relaxation induced by bethanechol, an acetylcholine analog, in endothelium-intact aortic tings isolated from prepubertal female rats which were pretreated with 171~estradiol ~ placebo were also compared, and data are summarized in Fig. 2. Relaxant responses to bethanechol in doses of 10.8 to 10"~ M were similar in aortic rings isolated from the prepubertal female rats pretreated with 17Bestradiol when compared to placebo (Fig. 2, right panel, Two-Way ANOVA, p>0.05).
Vol. 55, No. 10, 1994
Effects of 17~-Estradiol
n=7-14 - - o - -
z
100
1
171?,-estradiol
.--o--placebo 10.
+E
PL-189
-Endo
8.
75.
6. 50. 4-
25.
2.
0
0 -9
-8
-7
-6
-5
-9
-8
-7
-6
-5
ACETYLCHOLINE (logM)
Fig. 1 The effects of 17B-estradiol on acetylcholine-induced relaxation in aortic tings with (left panel) and without endothelium (right panel) isolated from prepubertal female rats pretreated with 1713-estradiol or placebo. Each aortic ring precontracted submaximally by norepinephrine was exposed to cumulatively increasing concentrations of acetylcholine. Data were calculated as percentage relaxation of tone induced by norepinephrine, n, indicates number of animals.
+Endo n=7-14 - - o - - 171)-estradiol
- - o - - placebo
100-
100. Z 75O
75
50.
50
25-
0
E -8
HISTAMINE (logM)
-9 -8 -7 -g BETHANECHOL (logM)
Fig. 2 The effects of 17B-eswadiol on relaxation induced by histamine (left panel) and bethanechol (tight panel) in aortic tings with endothelium isolated from prepubertal female rats pretreated with 1713-estradiol or placebo. Each aortic rings precontracted submaximally by norepinephrine was exposed to cumulatively increasing concentrations of histamine and bcthanechol. Data were calculated as percentage relaxation of tone induced by norepinephrine. n, indicates number of animals.
PL-190
Effects of 17~-Estradiol
Vol. 55, No. 10, 1994
Discussion Results from the present study demonstrate that chronic in vivo treatment with 17B-estradiol significantly enhances acetylcholine-induced endothelium-dependent relaxation in aortic rings isolated from prepubertal female rats. Removal of endothelium abolishes acetylcholine-induced relaxation in the aortic rings, and treatment with 17B-estradiol did not alter vascular responses to acetylcholine in the endotheliurn-denuded rat aorta. In contrast to enhancement by estrogens of acctylcholine-indueed relaxation, the data of the present study show that 17~estradiol does not change vascular relaxant responses to histamine, another endothelium-dependent relaxant agent, suggesting that the effect of 17B-cstradiol on endothelium-dependent relaxation induced by acetylcholine is selective in the rat aorta. The observation showing that 17B-estradiol increases acetylcholine-induced endotheliumdependent relaxation is consistent with the results of the previous study in uterine arteries isolated from virgin guinea pigs (3,8) and in femoral arteries from rabbits (4). Plasma concentrations of estrogens have been shown to increase dramatically in normal pregnancy and role for sex hormone in the cardiovascular changes associated with pregnancy has also been suggested (10,11). Contractile responsiveness of vascular tissues to angiotensin II decreases in in vivo and vitro experiments (1,3,10,12,). The results of the present study suggest that estrogens may play an important role in regulation of the vascular tone and may be a useful therapeutic agent for treatment of cardiovascular diseases. The mechanisms by which estrogens increase endothelium-dependent relaxant responses to acetylcholine in blood vessels are uncertain. The acute treatment with 17~estradiol for 24 hr has no effects on contractile responses to angiotensin II and relaxant responses to acetylcholine (data not shown). Functional estrogen receptors have been reported to be present in aortic smooth muscle cells (13) and endothelial cells from rat blood vessels (14). The chronic in vivo treatment with estrogens is more likely related to alteration of the transcription and translation levels of genes. Increases in the number of muscarinic receptors by estrogens have been shown in hypothalamus (5), and enhancement of formation of endothelium-derived nitric oxide (EDNO) (5,8,15) as well as alteration of vascular smooth muscle sensitivity in response to acetylcholine have been suggested (4). Although potential mechanisms by which acetylcholine-induced endothelium-dependent relaxation was enhanced by 17B-estradiol were not directly determined in the present study, observation that estrogens had no significant effects on relaxation induced by histamine, another endotheliumdependent relaxant, suggests that the treatment with 17B-estradiol does not alter production of EDNO or sensitivity of vascular smooth muscle in response to acetylcholine in the rat aorta. The observation showing that estrogens do not alter the sensitivity of vascular smooth muscle to El)NO in the rat aorta is in agreement with results of the previous study in isolated rabbit aorta in which treatment with 1713estradiol for 4 days had no significant effects on relaxant responses to calcium ionophore, A23187 (4). It has been interestingly noticed that 17g-estradiol dose not alter endothelium-dependent relaxation induced by bethanechol, an acetylcholine analog, resistant to hydrolysis by acetylcholinesterase. The data suggest that the chronic in vivo treatment with estrogen may down regulate activity of acetylcholinesterase that inactivates acetylcholine in the action site of blood vessels and increased levels of acetylcholine may enhance the endothelium-dependent relaxation in the rat aorta. If estrogens augment the number of muscarinic receptors to a significant extent, acetylcholineand bethanechol-indueed endothelium-dependent relaxation would be enhanced in a similar manner in the rat aorta. The finding in the present study does not support the hypothesis that enhancement of estrogens on endothelium-dependent relaxation induced by acetylcholine is due to increases in the muscarinic receptors (4). Future experiments should be designed to investigate mechanisms by which responses to acetylcholine are mediated and the effects of estrogens on acetylcholinesterase activity need to be further characterized in isolated endothelial cells from the rat aorta. In conclusion, the results of the present study demonstrate that 17B-cstradiol selectively enhances aeetylcholine-induced endothelium=dependent relaxation and has no effects on vascular smooth muscle in response to aeetyleholine in the isolated rat aorta. The data suggest that enhancement by 17B-estradiol of acetylcholine-induced endothelium-dependent relaxation may be involved in down regulation of acetylcholinesterase activity in the rat aorta.
Vol. 55, No. 10, 1994
Effects of 17/3-Estradiol
PL-191
Acknowledmnents This work was supported in part by grants from the U.S. Public Health Service (HL35711) and American Heart Association, West Virginia Affdiate. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
R. ABDUL-KARIM and N.S. ASSALI, Am. J. Obstet. Gynecol. 82 266-272 (1961). C. BELL, Br. J. Pharmacol. 49 595-601 (1973). D.Y. CHENG and C.A. GRUE'I'I~R, Eur. L Pharmacol. 215 171-176 (1992). V. GISCLARD, V.M. MILLER and P.M. VANHOUTTE, J. Phannacol, Exp. Ther. 244 19-22 (1987). T.C. RAINBOW, V. DEGROFF, V.H. LUINE and B.S. McEWAN, Brain Res. 198 239-243 (1980). R.C. ROSENFELD and M.G. JACKSON, Am. J. Obst. Gynecol. 148 429-435 (1984). J. HOGE, M. WILLIS and M.H. WEINHERGER, Am. J. Physiol. 2 H369H374 (1977). C. BELL and C.J. COFFEY, Repro. Fertil. 66 133-137 (1982). I. HASHIMOTO, M. KAWAMINAMI, K. MOTOH and H. WAKURI, Excerpta Medica International Congress Series 672 628-635 (1984). R.C. ROSENFELD, F.H. MORRIS, F.C. Jr.BATI'AGLIA, E.L.,MAKOWSKI and G. MESCHIA, Am. J. Obstet. Gynecol. 124, 618-629 (1976). K. UCLAND, and J.T. PARER, Am. J. Obstet. Gynecol. 96 400-412 (1966). M.S. PALLER, Am. J. Kidney. Dis. 9 308-315 (1984). A.L. LIN and S.A. SYDNEY, Arteriosclerosis 5 688-692 (1985). P. COLBURN and V. BUONASSISI, Science 201 817-819 (1978). R.F. FURCHGO'Vr, Circ. Res. 53 557-573 (1983).