AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1995-H2001, 2008. First published March 14, 2008; doi:10.1152/ajpheart.01193.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/5/H1995    most recent
01193.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brosnihan, K. B.
Right arrow Articles by Ferrario, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brosnihan, K. B.
Right arrow Articles by Ferrario, C. M.

CALL FOR PAPERS
Sex Steroids and Gender in Cardiovascular-Renal Physiology and Pathophysiology

Estrogen, nitric oxide, and hypertension differentially modulate agonist-induced contractile responses in female transgenic (mRen2)27 hypertensive rats

K. Bridget Brosnihan,1 Ping Li,1 Jorge P. Figueroa,2 Detlev Ganten,3 and Carlos M. Ferrario1

1The Hypertension and Vascular Disease Center and 2Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; and 3Max-Delbruck Center for Molecular Medicine, Berlin-Buch, Germany

Submitted 14 October 2007 ; accepted in final form 14 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17β-estradiol (E2) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E2 or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E2 treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with N{omega}-nitro-L-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E2-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.

angiotensin II; serotonin; phenylephrine

RECENT CLINICAL TRIALS [Heart and Estrogen/Progestin Replacement Study (HERS), Estrogen Replacement and Atherosclerosis (ERA), and Women's Health Initiative (WHI)] (10, 11, 34a) showed that estrogen and/or combined estrogen plus progesterone replacement provides no beneficial cardiovascular effects in postmenopausal women. This is despite the fact that observational studies showed that women in their reproductive age have a much lower incidence of atherosclerosis and hypertension than that of the age-matched men and postmenopausal women (28, 29). In both the observational studies and the clinical trials, little attention was paid to the underlying disease at the time of hormone initiation. Specifically, the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system (RAS) could confound the beneficial effects of estrogen.

Estrogen has both stimulatory and inhibitory effects on components of the RAS, including the downregulation of the angiotensin II (ANG II) type 1 (AT1) receptor (21) and angiotensin I (ANG I)-converting enzyme (ACE) (4) and the upregulation of angiotensinogen (32) and increased circulating levels of ANG II (25). The transgenic (TG) hypertensive rats harboring a single mouse renin (Ren-2) gene [(mRen2)27] provide a genetic model that has many features in common with essential hypertension including low or normal circulating renin (18, 35). The model has an activated tissue RAS (6, 26), which contributes to the fulminant hypertension. This genetic model of hypertension clearly indicates the important role of estrogen in renin-angiotensin-dependent hypertension, since female TG rats have much lower blood pressure than that of age-matched male rats. Studies demonstrated that the in vivo pressor responses to ANG II and phenylephrine (PE) are attenuated in estrogen-treated, ovariectomized TG hypertensive female rats (1). At the same time, chronic estrogen replacement lowers blood pressure, augments the basal contribution of nitric oxide (NO) to vascular tone, and enhances acetylcholine (ACh)-stimulated vascular NO release in TG rats (16). A number of abnormalities in the hypertensive rats were not entirely corrected by estrogen, thus there was a lesser degree of maximal dilation elicited by ACh in TG rats, suggesting the persistence of some degree of endothelial dysfunction (16). Thus the question arose of whether estrogen would modify agonist-induced vasoconstriction and NO mediation of these responses under the preexisting condition of endothelial damage in TG hypertensive rats.

In the present study, we hypothesized that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of NO synthase (NOS) is impaired by hypertension. We investigated the effects of chronic 17β-estradiol (E2) treatment on the vascular contractile responses to the vasoconstrictors ANG II and its precursor ANG I, serotonin (5-HT), and PE and the modulatory role of endothelial NO on each of these vasoconstrictor responses. The variety of agonists selected may uncover whether the amplification of estrogen on the release of NO in endothelial cells is sensitive to modulation under conditions of endothelial damage associated with hypertension (16). Comparisons were conducted between normotensive and TG hypertensive rats to determine whether the effects of estrogen on vascular responsiveness are altered in the animals with angiotensin-dependent hypertension.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparations. Following approval by the Institutional Animal Care and Use Committee, 56 12-wk-old, female heterozygous hypertensive (TG) and Hannover Sprague-Dawley (SD) rats (220–250 g body wt) were obtained from the Hypertension Center Transgenic Animal Research Colony of Wake Forest University School of Medicine. The rats were anesthetized with ketamine (30 mg/kg im) and xylazine (5 mg/kg im) and bilaterally ovariectomized. The animals were allowed free access to water and normal powder chow providing 17 meq of Na+ and 28 meq of K+ per 100 g of solid weight (Rodent Laboratory Chow 5001; Purina Mills, Richmond, IN). After a 2-wk recovery period, the rats were implanted subcutaneously with pellets containing either E2 (5 mg/rat, for 19–21 days release; Innovative Research of America, Sarasota, FL) or placebo (P) for control as previously reported (8, 16). The animals were randomized into four groups: 1) ovariectomized TG/P, 2) ovariectomized TG/E2, 3) ovariectomized SD/P, and 4) ovariectomized SD/E2. The animals were housed individually in a room maintained at 22°C on a 12-h:12-h light-dark cycle. At the end of 3 wk of E2 treatment, the systolic blood pressure of conscious rats was measured by using the tail-cuff method between 9:00 AM and 11:00 AM each experimental day, and the average of three measurements of each rat was used.

Vascular ring reactivity. On the next day following the last blood pressure measurements, vascular reactivity was evaluated using isolated aortic rings mounted in organ chambers as previously reported (16). The rats were euthanized by decapitation, and blood was collected for the determination of serum estradiol levels using a commercially available radioimmunoassay kit (Polymedco, Cortlandt Manor, NY). The thoracic aorta was removed and dissected free of connective tissues on ice. The aorta was cut into eight 4-mm-length rings using a tissue cutter (Zivic Miller), and the vascular rings were mounted in 10-ml glass chambers using two stainless steel wire triangles. The chambers were filled with modified Krebs-Henseleit buffer (16); the solution was aerated with 95% O2-5% CO2 at 37°C (pH 7.4) and contained freshly prepared 10 µM indomethacin to prevent the production of vasoactive prostaglandins. The isometric tension was measured continuously by using a polygraph (model 7; Grass Instrument, Quincy, MA). Basic tension was set at 2 g after a 60-min equilibration period. The endothelial cell integrity of vascular rings was tested using ACh (0.1 µM) in rings preconstricted with 1 µM of PE. The functional integrity of the endothelium was determined by the presence of >60% ACh-induced vasodilation. Vessels were discarded if the relaxation induced by ACh was <60%. The aortic rings were stimulated with 80 mM of potassium chloride (KCl) twice to generate the maximal contraction. Cumulative concentration-dependent response curves to PE (10–9–10–4 M) and 5-HT (10–8–10–4 M) were produced in quiescent rings. To evaluate the contribution of vascular NO production to the vasoconstriction induced by different vasoconstrictor agonists, the aortic rings were pretreated with the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 10–4 M) for 30 min, and the contractile responses to PE and 5-HT were then repeated. To prevent tachyphylaxis induced by ANG II in rat vascular rings in vitro (2), each aortic ring was only exposed to one dose of ANG II (10–8–10–6 M) or ANG I (1 µM); separate vessels were studied in the absence or presence of 100 µM L-NAME or 1 µM lisinopril. The inhibition of vascular NO production was confirmed by the absence of ACh-induced relaxation. Pretreatment with L-NAME had no effect on the basal tension of resting aortic rings except a minimal contraction in a few rings. A 60-min equilibration period was allowed between each response curve, and the rings were washed with warm Krebs buffer every 15 min.

Western blot analysis. Endothelial NOS (eNOS) isoform protein expression of the rat aorta was analyzed according to a previously published method (36). Protein concentration was measured with the bicinchoninic acid method using BSA as the standard. The lysate of cultured primary bovine endothelial cells was used as the standard of the eNOS isoform.

RT-PCR assay. The AT1 receptor mRNA was measured by RT-PCR assay as previously published (12). Briefly, 1 µg of RQ1 DNAase-treated, total RNA isolated from the aorta with the TRIzol reagent (GIBCO) was quantified by ultraviolet spectroscopy and RT-PCR assay. Amplication conditions and primers for the AT1 receptor and the control elongation factor-1{alpha} are as previously published (12). Amplifed products were separated on a 6% polyacrylamide gel, visualized using a PhosphorImager, and quantified by computerized densitometry.

Chemicals and drugs. Antibodies for the eNOS isoform were purchased from Transduction Laboratories (Lexington, KY). ANG II and ANG I were obtained from Bachem (Torrance, CA). All other drugs were obtained from Sigma Chemical (St. Louis, MO). Indomethacin was freshly dissolved in 0.2 mol/l Na2CO3 solution in stock and diluted in Krebs buffer. Other agents were prepared in distilled water and stored at –20°C as stock solution. The concentration of drugs reported was expressed as the final molar concentration in the organ chamber.

Data analysis and statistics. Vascular constriction was normalized as a percentage of 80 mmol/l KCl-induced maximal contraction (17). The concentration of drugs inducing 50% (EC50) of the maximal constriction was calculated by using a nonlinear regression method (Sigmoid curve fitting program; GraphPad, San Diego, CA). All values are means ± SE. One-way ANOVA and Student's t-test for unpaired observation were used for statistical analysis. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Chronic estrogen replacement reduced the systolic blood pressure of TG female hypertensive rats (189 ± 8 vs. 161 ± 7 mmHg, each group contains 10–12 rats; P < 0.05; P vs. E2). Estrogen did not significantly decrease the systolic blood pressure of SD rats (123 ± 6 vs. 113 ± 5 mmHg, each group includes 8–10 rats; P > 0.05; P vs. E2). The serum levels of E2 were 1,255 ± 104 and 1,231 ± 91 pg/ml for estrogen-treated SD and TG rats, respectively. Both P-treated SD and TG groups had a serum level of E2 <15 pg/ml.

Effect of estrogen treatment on the expression of eNOS in aorta. Figure 1, top, shows a representative expression of eNOS in P- and E2-treated SD and TG rat aortic tissue. Chronic E2 treatment increased vascular eNOS expression by twofold and 1.8-fold in the female ovariectomized normotensive SD and TG hypertensive rat aorta, respectively, compared with the P-treated SD and TG aorta (Fig. 1, bottom; P < 0.05; each group contains 5–7 rats). There was no significant difference of eNOS expression between the E2-treated SD and TG rat aorta.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Effects of chronic 17β-estradiol (E2) treatment on vascular endothelial nitric oxide synthase (eNOS). Top: representative Western blots of eNOS from Sprague-Dawley (SD) and transgenic (TG) rats treated with either placebo (P) or E2. The standard (STD) blot with eNOS is also shown for comparison. C, control. Bottom: average optical density of band of eNOS in P- and estrogen-treated SD and TG rats. *P < 0.05, estrogen vs. P. Each group has 5 to 6 rats.

 
Estrogen treatment reduces ANG II- and ANG I-induced vasoconstriction in rat aorta and AT1 receptor mRNA. Figure 2 shows that chronic E2 treatment reduces AT1 receptor mRNA in the aorta of SD and TG rats. ANG II caused concentration-dependent vasoconstriction in the aortic rings exposed to a single dose of ANG II. There was no difference of ANG II-induced constriction between SD and TG rats. Estrogen reduced ANG II-induced vasoconstriction in both SD and TG rats compared with P-treated control groups (Fig. 3). Pretreatment with L-NAME enhanced the contractile responses to ANG II at doses of 10–7 M in P-treated SD rats and at doses of 10–7 and 10–6 M in P-treated TG rats. With estrogen treatment in SD and TG rats, pretreatment with L-NAME significantly enhanced the vasoconstrictor response to ANG II at the concentration of 10–7 and 10–6 M, reaching levels that were essentially the same as the those of P-treated control groups. However, in the presence of L-NAME, the ANG II-induced contractile response remained significantly attenuated with E2 treatment in both the SD and TG groups compared with the P plus L-NAME groups. ANG I, at 1 µM concentration, caused both SD and TG aortic ring constriction, and estrogen significantly inhibited the ANG I-induced vascular contraction (Fig. 4; P < 0.05). Pretreatment with lisinopril (1 µM) for 10 min nearly abolished the ANG I-induced constriction in both SD and TG rats, indicating that the local vascular tissue ACE converts ANG I to ANG II.


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 2. Effects of chronic E2 treatment on aorta ANG II type 1 (AT1) receptor mRNA. Average levels of aorta AT1 receptor (AT1R) mRNA in P- and estrogen-treated SD and TG rats are shown. Expression values were normalized to elongation factor-1{alpha} (EF-1{alpha}) mRNA. Values are means ± SE. **P < 0.01, estrogen vs. P. Each group has 5–7 rats.

 

Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 3. Effects of chronic E2 treatment and acute N{omega}-nitro-L-arginine methyl ester (L-NAME) treatment on the contractile responses to ANG II in aortic rings of SD (top) and TG (bottom) rats. *P < 0.05, compared with P-treated groups of SD and TG rats; {alpha}P < 0.05, compared with P- or E2-treated groups without L-NAME; #P < 0.05, compared with P in the presence of L-NAME. Each group includes 8–10 vascular rings.

 

Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. Effects of chronic estrogen treatment on ANG I-induced aortic ring contraction in both normotensive SD and hypertensive TG rats. Lisinopril (1 µM), an ANG I-converting enzyme (ACE) inhibitor, was added to show the dependency on ACE. *P < 0.05, compared with P-treated SD and TG rats. Each group contains 6–8 aortic rings; #P < 0.05, compared with E2 treatment.

 
Estrogen replacement attenuates 5-HT-mediated vascular constriction. The maximal vascular contractile responses to 5-HT were significantly augmented in the P- and E2-treated TG groups compared with the P- and E2-treated SD groups (Table 1). The EC50 of 5-HT in both the P- and E2-treated TG group was significantly lower compared with that of the P- and E2-treated SD groups. The EC50 of 5-HT in the presence of L-NAME in both the P and E2 of the TG group was significantly lower compared with that of the SD group, even though there was no difference between their maximal contractile responses. E2 treatment attenuated 5-HT-induced maximal responses at higher concentrations in both treated SD and TG groups with and without L-NAME treatment (Fig. 5). L-NAME pretreatment enhanced 5-HT-induced maximal and lowered EC50 contractile responses in both P-treated SD and TG groups. L-NAME pretreatment did not augment the 5-HT-maximal or lower the EC50-induced responses from E2-treated SD and TG rats.


View this table:
[in this window]
[in a new window]

  		Table 1. Effects of chronic estrogen treatment on the serotonin-induced vasoconstriction in SD and TG rat aorta

 

Figure 5
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 5. Effects of E2 and L-NAME treatment on serotonin-induced vascular reactivity in SD (top) and TG (bottom) rats. See Table 1 for statistical comparisons. Each group has 10–15 rings.

 
Effects of estrogen on the PE-induced vasoconstriction. PE-induced contractile responses of aortic rings were not significantly different between SD and TG rats compared with those groups with EC50 and maximal contraction (Fig. 6 and Table 2). Chronic E2 treatment did not alter PE-induced vasoconstriction in both the SD and TG groups (EC50, 91.76 ± 5.74 vs. 83.23 ± 5.75 nM and maximal contraction, 111 ± 13.1% vs. 106 ± 8.7% in E2- and P-treated SD rats; and EC50, 91.27 ± 6.06 vs. 75.56 ± 4.76 nM and maximal contraction, 101 ± 4.8% vs. 113 ± 5.1% in E2- and P-treated TG rats; P > 0.05). Pretreatment with L-NAME significantly augmented the contractile response to PE in both the E2- and P-treated SD and TG rats.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 6. Effects of E2 and L-NAME treatment on phenylephrine-induced vascular reactivity in SD (top) and TG (bottom) rats. See Table 2 for statistical comparisons. Each group includes 10–16 aortic rings.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Effects of chronic estrogen treatment on the phenylephrine-induced vasoconstriction in SD and TG rat aorta

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our studies suggest that estrogen differentially influences both the vascular responses to three vasoconstrictors and the contribution of NO as a modulating influence to each of the agonists in normotensive and hypertensive rats. Although an increase in NOS expression with E2 treatment was found in the aorta of both normotensive and hypertensive animals, the actual participation of NO as a modulator in opposing the vascular responses was observed with ANG II and PE. In the case of 5-HT, there was NO modulation only in the P groups but not the estrogen-treated groups. The persistence of the attenuation of the ANG II response after NOS inhibition with estrogen treatment is consistent with our and others' (21, 33) demonstration of estrogen downregulation of the AT1 receptor. The reduction of ANG I-induced vascular constriction is consistent with the downregulation of the AT1 receptor and inhibition of ACE activity (1, 4) by estrogen. The augmentation of the PE and ANG II responses after NOS inhibition suggests that NO is a primary modulator of adrenergic and ANG II-dependent vasoreactivity responses regardless of the presence or absence of estrogen. On the other hand, only 5-HT elicited an increased responsiveness in hypertensive animals with E2 treatment, attenuating the responses in both normotensive and hypertensive animals and NO playing little or no role with E2 treatment. These studies illustrate the complexities of the regulation of vascular tone when animals possess enhanced cardiovascular risk with hypertension and are undergoing estrogen replacement treatment. The significance of these findings for hypertensive menopausal women receiving hormone replacement therapy (HRT) is threefold. First, there are beneficial effects from HRT increasing NO, which opposes the vasoconstrictor action of specific agonists, namely ANG II and PE. Second, in the case of 5-HT, there was a HRT beneficial effect, but it was not mediated through NO but most likely through the downregulation of the 5-HT receptor. Finally, the preexisting condition of hypertension specifically is detrimental for 5-HT vasoconstrictor effects.

The inhibition of ANG II-induced contraction following chronic estrogen treatment is consistent with the reports in ovariectomized rats (2) and sheep (22) treated with estrogen. These findings are consistent with our finding of a downregulation of the AT1 receptor gene expression by estrogen in the aorta from SD and TG rats and similar findings reported by Nickenig et al. (21). Cheng and Gruetter (2) studied the ANG II response in rat aortic vessels with and without the endothelium and showed that in the presence or the absence of the endothelium there were markedly attenuated ANG II responses with the magnitude of difference being greater in the endothelium-denuded vessels. In our study, we used NOS inhibition to remove the NO modulation. Similar to the studies of Cheng and Gruetter (2), we found that chronic E2 treatment reduced the ANG II responses, and we were able to show that this occurred with or without the blockade of NOS. The increase of the ANG II response after NOS inhibition reveals the contribution of NO as a counterbalancing modulator. In the ovariectomized, P-treated SD rats, the modulation by NO of the ANG II response was not observed (at ANG II concentrations of 10–8 and 10–6 M), suggesting little or no role for NO modulation in ANG II-mediated responses when estrogen is absent. These studies reveal that there are at least two components contributing to the depression of the ANG II response with E2 treatment, i.e., the downregulation of the AT1 receptor and increased contribution of NO. It is likely that the increase in NO originated from the upregulation of eNOS by estrogen; however, our studies do not eliminate a possible contribution of inducible NOS, which has been shown to be present in endothelial and vascular smooth muscle cells (13) and would be inhibited by L-NAME and regulated by estrogen (13, 20). The contribution of other endothelial factors that may be upregulated and contribute to the attenuated ANG II response was not tested in these studies. In our study, the presence of hypertension did not significantly alter the pattern or magnitude of ANG II responses. Previous studies from our group (4) and others (24) reported that chronic estrogen treatment decreases serum and tissue ACE activity and downregulates tissue ACE mRNA. In the present study, we showed that E2 treatment suppressed ANG I-induced vascular constriction. Because the ANG I response is mediated by the AT1 receptor after its conversion to ANG II, both the downregulation of the AT1 receptor and the NO modulation of this response are components of this response. ACE inhibition of ANG I, however, acts more proximally to the ANG II response and illustrates that the downregulation of ACE by estrogen may contribute to blood pressure reduction.

Reports on estrogen effects on the responses to PE or norepinephrine have been somewhat controversial. Estrogen has been reported to downregulate, upregulate, and cause no change of adrenergic receptor-mediated responses (2, 7, 30, 37). Whether estrogen attenuates or potentiates the pressor/vasoconstrictor response to PE/norepinephrine may be dependent on the vascular beds studied and the duration and magnitude of estrogen treatment. In our study, we observed that the in vitro response to PE in rat aortic vessels was not influenced by the E2 treatment in SD or TG hypertensive rats. Similar findings to PE were reported in spontaneously hypertensive rats (33). The blockade of NOS revealed that NO played a major role in depressing the PE response since the maximal responses were enhanced in both SD and TG rats. Zhang and Davidge (37) found that N{omega}-monomethyl-l-arginine significantly increased the sensitivity to PE in mesenteric vessels from both ovariectomized and estrogen-replaced SD rats. Because there was no difference in the L-NAME-treated vessels whether estrogen was present or not, our data would suggest that the {alpha}-adrenergic responses in aortic vessels comprised a strong opposing modulatory influence by NO, regardless of the presence or absence of estrogen.

Previous studies have suggested that abnormal hyperresponsive vascular function can be detected relatively easily with the use of 5-HT under conditions of atherosclerosis, hypertension, and endothelial damage (9, 15). Further confirmation of this statement is observed in our study, where TG rats had an augmented maximal vasoconstrictor response to 5-HT compared with that of normotensive rats. The findings between the SD and TG groups with P and E2 treatment were confirmed when the EC50 was used, but its use additionally showed that TG animals treated with L-NAME had reduced EC50 compared with that of the SD animals. Overall, our results when comparing the maximal responses and the EC50 are similar; however, it is known that the EC50 value is more sensitive in demonstrating effects. An endothelial component to the 5-HT response has been suggested by studies that demonstrate an enhanced contraction to 5-HT in denuded vessels (23) and in carotid arteries from female eNOS knockout mice compared with wild-type mice (14). Another possibility is that the changes in vascular reactivity in the (mRen2)27 rat are due to changes in reactive oxygen species production, which have been demonstrated to be elevated in the (mRen2)27 (34). In our study in aortic vessels from P-treated SD and TG rats, there was NO modulation of the 5-HT response, consistent with endothelial NO attenuating the 5-HT response. Yet with the estrogen treatment, there was no evidence of NO modulation of the 5-HT response in either the SD or TG rats. This lack of an effect of NO modulation with estrogen treatment was unexpected, especially in light of the increase in NOS expression with E2 treatment and the presence of NO modulation of 5-HT in the P-treated animals. Estrogen treatment, however, attenuated the constrictor response to 5-HT in the presence and absence of NO inhibition. The attenuation of the 5-HT response with estrogen treatment has been previously described in rabbit basilar artery segments (3, 27), human internal mammary arteries (19), and porcine coronary arteries (31). Taken together, these latter findings would be consistent with estrogen downregulating the 5-HT receptor, altering 5-HT signal transduction, or augmenting NO release, although the contribution of other endothelium-derived relaxing factors has not been eliminated.

In these studies, one would have anticipated that since HRT elevated NOS, its effects on all of the responses of the agonists would have been comparable. Yet this was not the case, and this may be due in part to the multiple steps modifying any agonist-mediated response. For example, it appears that HRT can downregulate the receptor of the agonist, which was demonstrated for the AT1 receptor and is consistent with our 5-HT responses. In the case of ANG II, HRT acts through multiple pathways in both NOS upregulation and downregulation of its receptor to achieve its beneficial effects. It is unclear why the preexisitng condition of hypertension, which entails long-term vascular compliance changes and endothelial damage, only augmented the 5-HT responses. Overall, the specific modification of each agonist response indicates why it is difficult to predict whether HRT will be beneficial or detrimental to hypertensive postmenopausal women.

One limitation of the studies conducted is that aortic vessels were used rather than resistance vessels such as the mesenteric and femoral artery. It is entirely possible that the prehypertensive effects of the agonists and their interaction with estrogen and NO may more readily be observed at the level of the resistance vessels. Finally, the dose of estrogen used in these studies is within the range that would be found with pregnancy or hormone replacement rather than the lower levels found during the normal estrous cycle.

In conclusion, these studies demonstrated that only 5-HT vasoreactive responses are markedly augmented by the preexisting condition of hypertension. In addition, although the studies confirmed that NOS is upregulated with estrogen treatment, they revealed that the contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor agonists and/or the presence of hypertension. In the case of PE and ANG II, the NO contribution to the attenuated response appeared to be unaffected by estrogen treatment, whereas the NO contribution to 5-HT was only found with P treatment. Estrogen attenuated the 5-HT response without a significant contribution of NO. These studies illustrate the complexities of regulation of vascular tone when animals possess enhanced cardiovascular risk with hypertension and are undergoing estrogen replacement treatment.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We gratefully acknowledge grant support in part provided by Unifi (Greensboro, NC) and the Farley-Hudson Foundation (Jacksonville, NC). This work was supported in part by National Heart, Lung, and Blood Institute Grant P01-HL-51952.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. B. Brosnihan, Hypertension and Vascular Disease Center, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-0132 (e-mail: bbrosnih{at}wfubmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Brosnihan KB, Li P, Ganten D, Ferrario CM. Estrogen protects transgenic hypertensive rats by shifting the vasoconstrictor-vasodilator balance of RAS. Am J Physiol Regul Integr Comp Physiol 273: R1908–R1915, 1997.[Abstract/Free Full Text]
  2. Cheng DY, Gruetter CA. Chronic estrogen alters contractile responsiveness to angiotensin II and norepinephrine in female rat aorta. Eur J Pharmacol 215: 171–176, 1992.[CrossRef][Web of Science][Medline]
  3. Futo J, Shay J, Block S, Holt J, Beach M, Moss J. Estrogen and progesterone withdrawal increases cerebral vasoreactivity to serotonin in rabbit basilar artery. Life Sci 50: 1165–1172, 1992.[CrossRef][Web of Science][Medline]
  4. Gallagher PE, Li P, Lenhart JR, Chappell MC, Brosnihan KB. Estrogen regulation of angiotensin-converting enzyme mRNA. Hypertension 33: 323–328, 1999.[Abstract/Free Full Text]
  5. Ganten D, Hermann K, Unger T, Lang RE. The tissue renin angiotensin systems: focus on brain angiotensin, adrenal gland and arterial wall. Clin Exp Hypertens A5: 1099–1118, 1983.[CrossRef][Web of Science]
  6. Gisclard V, Miller VM, Vanhoutte PM. Effect of 17β-estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther 244: 19–22, 1988.[Abstract/Free Full Text]
  7. Goetz RM, Morano I, Calovini T, Studer R, Holtz J. Increased expression of endothelial constitutive nitric oxide synthase in rat aorta during pregnancy. Biochem Biophys Res Commun 205: 905–910, 1994.[CrossRef][Web of Science][Medline]
  8. Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL. Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys. Circ Res 54: 711–718, 1984.[Abstract/Free Full Text]
  9. Herrington DM, Reboussin DM, Brosnihan KB, Sharp PC, Shumaker SA, Snyder TE, Furberg CD, Kowalchuk GJ, Stuckey TD, Rogers WJ, Givens DH, Waters D. Effects of estrogen replacement on the progression of coronary-artery atherosclerosis. N Engl J Med 343: 522–529, 2000.[Abstract/Free Full Text]
  10. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E, Research Group HERS. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in post menopausal women. JAMA 280: 605–613, 1998.[Abstract/Free Full Text]
  11. Ishiyama Y, Gallagher PE, Averill DB, Tallant EA, Brosnihan KB, Ferrario CM. Upregulation of angiotensin-converting enyzme 2 after myocardial infarction by blockade of angiotensin II receptors. Hypertension 43: 970–976, 2004.[Abstract/Free Full Text]
  12. Kleinert H, Schwarz PM, Forstermann U. Regulation of the expression of inducible nitric oxide synthase. Biol Chem 384: 1343–1364, 2003.[CrossRef][Web of Science][Medline]
  13. Lamping KG, Faraci FM. Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin. Arterioscler Thromb Vasc Biol 21: 523–528, 2001.[Abstract/Free Full Text]
  14. Lamping KG, Piegors DJ, Benzuly KH, Armstrong ML, Heistad DD. Enhanced coronary vasoconstrictive response to serotonin subsides after removal of dietary cholesterol in atherosclerotic monkeys. Arterioscler Thromb 14: 951–957, 1994.[Abstract/Free Full Text]
  15. Li P, Ferrario CM, Ganten D, Brosnihan KB. Chronic estrogen treatment in female transgenic (mRen2)27 hypertensive rats augments endothelium-derived nitric oxide release. Am J Hypertens 10: 662–670, 1997.[CrossRef][Web of Science][Medline]
  16. Li P, Tong C, Eisenach JC. Pregnancy and ephedrine increase the release of nitric oxide in ovine uterine arteries. Anesth Analg 82: 288–293, 1996.[Abstract]
  17. Moriguchi A, Brosnihan KB, Kumagai H, Ganten D, Ferrario CM. Mechanisms of hypertension in transgenic rats expressing the mouse Ren-2 gene. Am J Physiol Regul Integr Comp Physiol 266: R1273–R1278, 1994.[Abstract/Free Full Text]
  18. Mugge A, Barton M, Fieguth HG, Riedel M. Contractile responses to histamine, serotonin, and angiotensin II are impaired by 17β-estradiol in human internal mammary arteries in vitro. Pharmacology 54: 162–168, 1997.[Web of Science][Medline]
  19. Nawate S, Fukao M, Sakuma I, Soma T, Nagai K, Takikawa O, Miwa S, Kitabatake A. Reciprocal changes in endothelium-derived hyperpolarizing factor- and nitric oxide-system in the mesenteric artery of adult female rats following ovariectomy. Br J Pharmacol 144: 178–189, 2005.[CrossRef][Web of Science][Medline]
  20. Nickenig G, Baumer AT, Grohe C, Kahlert S, Strehlow K, Rosenkranz S, Stablein A, Beckers F, Smits JF, Daemen MJ, Vetter H, Bohm M. Estrogen modulates AT1 receptor gene expression in vitro and in vivo. Circulation 97: 2197–2201, 1998.[Abstract/Free Full Text]
  21. Rosenfeld CR, Jackson GM. Estrogen-induced refractoriness to the pressor effects of infused angiotensin II. Am J Obstet Gynecol 148: 429–435, 1984.[Web of Science][Medline]
  22. Russell A, Watts S. Vascular reactivity of isolated thoracic aorta of the C57BL/6J mouse. J Pharmacol Exp Ther 294: 598–604, 2000.[Abstract/Free Full Text]
  23. Schunkert H, Jan Danser AH, Hense HW, Derkx FHM, Kurzinger S, Riegger GAJ. Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women. Circulation 95: 39–45, 1997.[Abstract/Free Full Text]
  24. Seely EW, Brosnihan KB, Jeunemaitre X, Okamura K, Williams GH, Hollenberg NK, Herrington DM. Effects of conjugated oestrogen and droloxifene on the renin-angiotensin system, blood pressure and renal blood flow in postmenopausal women. Clin Endocrinol (Oxf) 60: 315–321, 2004.[CrossRef][Medline]
  25. Senanayake PS, Smeby RR, Martins AS, Moriguchi A, Kumagai H, Ganten D, Brosnihan KB. Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats. Peptides 19: 1685–1694, 1998.[CrossRef][Web of Science][Medline]
  26. Shay J, Futo J, Badrov N, Moss J. Estrogen withdrawal selectively increases serotonin reactivity in rabbit basilar artery. Life Sci 55: 1071–1081, 1994.[CrossRef][Web of Science][Medline]
  27. Stamper MJ, Colditz GA, Willett WC, Manson JAE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the nurses' health study. N Engl J Med 325: 756–762, 1991.[Abstract]
  28. Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI. Hormone replacement therapy and the cardiovascular system. Drugs 47: 35–41, 1994.
  29. Sudhir K, Esler MD, Jennings GL, Komesaroff PA. Estrogen supplementation decreases norepinephrine-induced vasoconstriction and total body norepinephrine spillover in perimenopausal women. Hypertension 30: 1538–1543, 1997.[Abstract/Free Full Text]
  30. Teoh H, Quan A, Leung SWS, Man RYK. Differential effects of 17β-estradiol and testosterone on the contractile responses of porcine coronary arteries. Br J Pharmacol 129: 1301–1308, 2000.[CrossRef][Web of Science][Medline]
  31. Tewksbury DA. Angiotensinogen—biochemistry and molecular biology. In: Hypertension: Pathophysiology, Diagnosis and Management, edited by Laragh JH and Brenner BM. New York: Raven, 1990, p. 1197–1216.
  32. Wassmann S, Baumer AT, Strehlow K, van Eickels M, Grohe C, Ahlbory K, Rosen R, Bohm M, Nickenig G. Endothelial dysfunction and oxidative stress during estrogen deficiency in spontaneously hypertensive rats. Circulation 103: 435–441, 2001.[Abstract/Free Full Text]
  33. Whaley-Connell A, Habibi J, Nistala R, Cooper SA, Karuparthi PR, Hayden MR, Rehmer N, DeMarco VG, Andresen BT, Wei Y, Ferrario C, Sowers JR. Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment. Hypertension 51: 474–480, 2008.[Abstract/Free Full Text]
  34. Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. JAMA 288: 321–333, 2002.[Abstract/Free Full Text]
  35. Wong PC, Zimmerman BG. Comparison of intrarenal and extrarenal converting enzyme inhibition by SQ20881 on canine renal blood flow. Clin Sci 61: 553–557, 1981.[Web of Science][Medline]
  36. Zhang J, Massmann GA, Mirabile CP, Figueroa JP. Nonpregnant sheep uterine type I and type III nitric oxide synthase expression is differentially regulated by estrogen. Biol Reprod 60: 1198–1203, 1999.[Abstract/Free Full Text]
  37. Zhang Y, Davidge ST. Effect of estrogen replacement on vasoconstrictor responses in rat mesenteric arteries. Hypertension 34: 1117–1122, 1999.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/5/H1995    most recent
01193.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brosnihan, K. B.
Right arrow Articles by Ferrario, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brosnihan, K. B.
Right arrow Articles by Ferrario, C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.