Estrogen Replacement Suppresses a Prostaglandin H Synthase–Dependent Vasoconstrictor in Rat Mesenteric Arteries

From the Perinatal Research Centre, Departments of Ob/Gyn and Physiology, University of Alberta, Edmonton, AB Canada T6G 2S2.

Correspondence to Sandra T. Davidge, PhD, 220 Heritage Medical Research Centre, University of Alberta, Edmonton, AB Canada T6G 2S2. E-mail sandra.davidge{at}ualberta.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—There is evidence that estrogen can upregulate nitric oxide (NO) synthase expression. There is also evidence that NO increases the activity of prostaglandin H synthase (PGHS). Our initial hypothesis was that removal of ovarian steroids would decrease endothelium/NO-dependent relaxation responses but that estrogen replacement would increase NO and PGHS activity, leading to increased vasodilation. Resistance-sized (<250 µm) mesenteric arteries from ovariectomized Sprague-Dawley rats without and with 17ß-estradiol replacement (0.15 or 0.5 mg/pellet, 60-day release) for 4 weeks were studied in a myograph system. The vasodilator response to methacholine, an endothelium-dependent muscarinic agonist, was reduced in the arteries of the ovariectomized rats compared with estradiol-replaced rats. In the presence of PGHS inhibitors (meclofenamate, valeryl salicylate, and NS-398) or a thromboxane A₂ (TxA₂)/prostaglandin H₂ (PGH₂) receptor blocker (SQ-29548), there was no longer a significant difference among the groups. Contrary to our initial hypothesis, inhibition of the PGHS pathway significantly enhanced the relaxation response in the arteries from the ovariectomized rats, which was similar to the response in the arteries from estradiol-replaced rats, indicating that a PGHS-dependent vasoconstrictor had modified the response to methacholine. Confirming these data, in response to exogenous arachidonic acid, arteries from ovariectomized rats exhibited constriction, whereas the arteries from the estradiol-replaced rats exhibited vasodilation. In the ovariectomized rats, pretreatment with inhibitors of the PGHS pathway reversed the vasoconstriction to a vasodilation. In addition, the vasoconstrictor response to the thromboxane mimetic U-46619 as well as PGH₂ was enhanced in endothelium-denuded arteries from the ovariectomized rats compared with the estradiol-replaced rats. These data demonstrate that removal of ovarian steroids increased endothelium-mediated PGHS-dependent vasoconstriction that was associated with augmented sensitivity of the TxA₂/PGH₂ receptor. Chronic estrogen replacement in the ovariectomized rat suppressed this PGHS-dependent vasoconstrictor response.

Key Words: estrogen, vascular • endothelium • nitric oxide • prostaglandin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is an inherent vascular protection in women that is affected by changes in their hormonal milieu. The incidence of cardiovascular disease is markedly lower in cycling, premenopausal women and estrogen-replaced postmenopausal women than in men or untreated postmenopausal women.¹ Although it is clear that estrogen replacement has a protective effect on vascular function, the biological basis for this is unclear.

A major regulation of vascular tone is through release of endothelium-derived vasoactive agents, such as nitric oxide (NO) and prostaglandin H synthase (PGHS)-dependent products.² NO is a potent vasorelaxant that also inhibits platelet aggregation,^{3 4} whereas PGHS-dependent products act as relaxing and contracting factors as well as regulators of platelet aggregation.^{5 6}

NO is synthesized by oxidation of a guanidino nitrogen of L-arginine in a reaction catalyzed by the enzyme NO synthase.^{3 4} Reports of elevated protein levels of endothelial NO synthase as well as increased NO production in response to 17ß-estradiol have led to the speculation that the beneficial effects of estrogen are probably due to NO.^{7 8} However, the effect of estrogen to be cardioprotective is likely more complex and involves numerous regulatory systems.

PGHS-dependent products are known to have important physiological and pathophysiological roles in vascular function. PGHS is the enzyme that converts arachidonic acid to prostaglandin endoperoxide H₂ (PGH₂). There are 2 known PGHS isoforms, PGHS-1 and PGHS-2, with both being reported in endothelial cells.⁹ Cell-specific isomerization or reduction of PGH₂ then occurs, yielding the major biological eicosanoids, such as prostacyclin and thromboxane (Tx).^{5 6} The functional effect of estrogen regarding the PGHS-dependent pathway is not clear, with reports of no effect^{10 11 12} or of a vasorelaxer component¹³ that modulates adrenergic vasoconstriction. In addition, the influence of NO on the PGHS pathway is still emerging. In cultured endothelial cells, we have reported that NO activates PGHS.¹⁴ Another study has reported an in vivo regulation of PGHS-2 by NO.¹⁵ Consequently, the effect of estrogen on the NO pathway may have a role in increasing PGHS-dependent relaxation, thereby conferring a protective action on the vascular system.

Our hypothesis for the present study was that removal of ovarian steroids would decrease endothelium/NO-dependent relaxation responses but that estrogen replacement would increase NO and PGHS activity, leading to increased vasodilation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal Model
Female Sprague-Dawley rats were obtained from Harlan, Indianapolis, Ind, at 10 weeks of age. One week later, ovariectomy was performed. At the time of ovariectomy, 2 groups of rats received a 17ß-estradiol pellet (0.15 or 0.5 mg, 60-day release, Innovative Research of America) subcutaneously. Another group received a placebo (Innovative Research of America). After 4 weeks, the rats were killed under light anesthesia with methohexital sodium (50 mg/kg body wt), and blood was collected by heart puncture for measurement of estradiol levels. 17ß-Estradiol was measured by use of a radioimmunoassay kit (Diagnostic Products Corp). The animal protocols were examined by the University of Alberta Animal Welfare Committee and found to be in compliance with the guidelines issued by the Canada Council on Animal Care.

Vessel Preparation
A section of the mesentery 5 to 10 cm distal to the pylorus was rapidly removed and placed in ice-cold HEPES-buffered PSS (HEPES-PSS). Mesenteric arteries averaging <250 µm in diameter were dissected free from surrounding adipose tissue, cut into 1.8-mm lengths, and threaded onto 20-µm wires. These wires were attached to 2 polyacrylamide blocks in an isometric myograph system (Kent Scientific Corp). The blocks rested in 5-mL glass-jacketed organ baths with HEPES-PSS solution kept at 37°C. Four separate baths were used to study arterial segments simultaneously. Force production was recorded on a data acquisition system (Workbench, Strawberry Tree Inc).

Resting Length-Tension Curve
After mounting, the arteries were stretched to 0.2 mN per millimeter vessel length (1 mN=102 mg) and allowed to equilibrate for 1 hour in HEPES-PSS buffer. The arteries were then given a conditioning stretch of 0.6 mN. A resting length tension curve was generated for each vessel. The arterial circumference that was used to perform the dose-response curves was obtained by using Laplace's law. With this equation, L₁₀₀ is calculated from the exponential curve fit of tension versus circumference. L₁₀₀ is defined as the circumference the vessel would have at a transmural pressure of 100 mm Hg. We have found from our previous studies that the point on the dose-response curve obtained at 0.8 L₁₀₀ provides maximum active force generation with minimum passive tension.

Solutions and Drugs
The HEPES-PSS solution used in these experiments contained (mmol/L) sodium chloride 142, potassium chloride 4.7, magnesium sulfate 1.17, calcium chloride 1.56, potassium phosphate 1.18, HEPES 10, and glucose 5.5. The HEPES-PSS solution was maintained at a pH of 7.4. Stock solutions of phenylephrine (L-phenylephrine hydrochloride), methacholine (acetyl-ß-methylcholine chloride) (both from Sigma Chemical Co), and meclofenamate (Warner Lambert, Ann Arbor) were prepared in water at a concentration of 10 mmol/L for each experiment. Stock solutions of 10 mmol/L SQ-29548, valeryl salicylate, NS-398, U-51605, arachidonic acid, U-46619, prostaglandin E₂ (PGE₂), and PGH₂ (all from Cayman Chemical Co) were prepared in ethanol. Appropriate dilutions of all stocks were obtained using HEPES-PSS.

Experimental Design
Mesenteric arteries were studied from the 3 groups of rats (n=5 or 6 rats per group) for each experimental protocol. Cumulative doses of phenylephrine (0.3 to 10 µmol/L) were administered. The data from the dose-response curves were fitted to the Hill equation, from which a straight line was generated by linear least squares regression analysis. The mean effective concentration that produced a 50% constriction (EC₅₀) was determined from this line for each individual artery. The EC₅₀ of phenylephrine was used to constrict arteries in order to achieve a baseline from which subsequent relaxation responses were measured. After completion of each dose-response curve, a 30-minute recovery period was allowed, during which the baths were changed every 10 minutes with fresh HEPES-PSS. Cumulative doses of the muscarinic agonist, methacholine (0.01 to 1 µmol/L) were administered to assess endothelium-dependent relaxations. To determine whether the responses to methacholine were dependent on the endothelium, one segment of a divided artery was denuded of its endothelium before the methacholine dose-response curve. Endothelium removal was performed mechanically using a human hair threaded through the lumen of the artery and rubbed back and forth.¹⁶

To determine the role for endogenous vascular prostaglandins in modulating the relaxation responses, methacholine dose-response curves were repeated in the absence or presence of the specific blockers in the PGHS cascade. The studies performed involving the absence and presence of inhibitors were conducted sequentially on the same artery. The reproducibility of repeating curves for these experiments was determined in a preliminary set of experiments designed to test for tachyphylaxis. Meclofenamate (1 µmol/L) was administered to block cyclooxygenase conversion of arachidonic acid. Valeryl salicylate (3 mmol/L) and NS-398 (10 µmol/L) were administered to preferentially inhibit PGHS-1 and PGHS-2, respectively. U-51605 (10 µmol/L) was used to inhibit TxA₂ synthase; SQ-29548 (1 µmol/L) was used to block the TxA₂/PGH₂ receptor. All inhibitors were preincubated with the arteries 15 minutes before the beginning of a dose-response curve.

Another series of experiments was conducted to determine the response of mesenteric arteries to an exogenous source of arachidonic acid. The response to arachidonic acid (0.01 to 5 µmol/L) was obtained after preconstricting the arteries with phenylephrine to 50% of the maximum response. After a 30-minute recovery period, one segment of the divided artery from each rat had the arachidonic acid doses repeated in the presence of specific PGHS inhibitors, valeryl salicylate, and NS-398. The arachidonic response was repeated in the presence or absence of endothelium.

Contractions to increasing concentrations of PGE₂, U-46619 (TxA₂ mimetic), or PGH₂ were obtained in arteries absent of endothelium to determine sensitivity of the response of arteries from ovariectomized compared with estradiol-replaced rats.

Data Analysis
ANOVA was used to determine the statistical difference of the parameters between the control and estradiol-replaced rats shown in the Table. A 2-way ANOVA with repeated measures was used to compare the response to a drug before and after specific inhibitors. Pairwise comparison was then conducted using the Student-Newman-Keuls test. Differences among means were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal Model
As expected, there was a significant dose-dependent increase in plasma 17ß-estradiol levels in the estradiol-replaced rats that were in the physiological range of cycling proestrus and pregnancy levels in the rat. Uterine weight, which provides a biological marker of estrogen replacement, was significantly elevated in the estradiol-replaced group (Table).

Vascular Responses
Before relaxation-response curves were generated, arteries were preconstricted by their EC₅₀ of phenylephrine. There was no significant difference in the EC₅₀ of phenylephrine among the experimental groups. There was a decreased response to methacholine in the ovariectomized rats compared with the estradiol-replaced rats (Figure 1A). Chronic estrogen replacement in the rat dose-dependently improved the relaxation response, as evidenced by a significant decrease in the EC₅₀ to methacholine (Figure 1A, insert). For all groups of rats, relaxation response to methacholine was abolished by endothelium removal. Time-control data indicated that dose-response curves to methacholine could be repeated at least 3 times with a variance of <4%.

View larger version (17K): [in this window] [in a new window]   Figure 1. Concentration-response curves to methacholine in the absence (A) or presence (B) of meclofenamate for mesenteric arteries from ovariectomized (OVX, n=5, ), 0.15 mg/pellet estradiol–replaced (0.15 mg/p, n=5, ), and 0.50 mg/pellet estradiol–replaced (0.5 mg/p, n=6, ) rats. Responses are expressed as a percentage of relaxation from phenylephrine-preconstricted levels. Insert, Bar graphs depicting EC50 values for methacholine among the groups of rats. Data represent mean±SEM. *P<0.05 vs OVX; +P<0.05 vs 0.15 mg/p.

In the presence of meclofenamate, an inhibitor of PGHS activity, there was no longer a significant difference among the groups (Figure 1B). The ovariectomized rats and the 0.15 mg/pellet estradiol–replaced rats shifted the curves to be similar to the 0.5 mg/pellet estradiol–replaced rats, suggesting that a PGHS-dependent vasoconstrictor modified the relaxation responses to methacholine.

Figure 2 depicts the EC₅₀ values for the relaxation response to methacholine alone and methacholine in the presence of specific inhibitors to the PGHS pathway. In the ovariectomized rats (Figure 2A), valeryl salicylate (PGHS-1 inhibitor), NS-398 (PGHS-2 inhibitor), or SQ-29548 (TxA₂ synthase inhibitor) significantly reduced the EC₅₀ to methacholine (enhanced the relaxation response), whereas U-51605 (TxA₂ synthase inhibitor) had no effect. Similar results were obtained for the 0.15 mg/pellet estradiol–replaced rats (Figure 2B). In the 0.5 mg/pellet estradiol–replaced rats (Figure 2C), the PGHS pathway inhibitors did not have a significant effect on the relaxation response to methacholine. The PGHS pathway inhibitors normalized the EC₅₀ of the ovariectomized rats and the 0.15 mg/pellet estradiol–replaced rats to be similar to the EC₅₀ of the 0.5 mg/pellet estradiol–replaced rats.

View larger version (24K): [in this window] [in a new window]   Figure 2. Bar graphs showing EC50 values for methacholine alone (open bars) and methacholine in the presence of PGHS pathway inhibitors (hatched bars) in mesenteric arteries from ovariectomized rats (n=5) (A), 0.15 mg/pellet estradiol–replaced (n=6) (B), and 0.50 mg/pellet estradiol–replaced (n=6) (C) rats. Valeryl salicylate is a PGHS-1 inhibitor; NS-398, a PGHS-2 inhibitor; SQ-29548, a TxA2/PGH2 receptor blocker; and U-51605, a TxA2 synthase inhibitor. Bars represent mean±SEM. *P<0.05 vs methacholine alone.

Arachidonic Acid Response
Arteries from ovariectomized rats resulted in vasoconstriction in response to exogenous arachidonic acid, whereas the arteries from the estradiol-replaced rats exhibited vasodilation (Figure 3). There was no vascular response to arachidonic acid in the absence of endothelium from any of the groups of rats (data not shown). Pretreatment with valeryl salicylate or NS-398 (inhibitors of PGHS-1 and PGHS-2, respectively) reversed the vasoconstriction in the ovariectomized rats to a vasodilation (Figure 4A) but caused a further relaxation in the 0.15 mg/pellet estradiol–replaced rats (Figure 4B). The PGHS inhibitors had no effect on the arachidonic acid responses in the arteries of the 0.5 mg/pellet estradiol–replaced rats (Figure 4C).

View larger version (13K): [in this window] [in a new window]   Figure 3. Concentration-response curves to arachidonic acid for mesenteric arteries from ovariectomized (n=5, ), 0.15 mg/pellet estradiol–replaced (n=6, ), and 0.50 mg/pellet estradiol–replaced (n=6, ) rats. Responses are expressed as a percentage of relaxation from phenylephrine-preconstricted levels. Data represent mean±SEM.

View larger version (23K): [in this window] [in a new window]   Figure 4. Bar graphs showing contractile responses to arachidonic acid (1 µmol/L) alone (open bar) and arachidonic acid in the presence of valeryl salicylate (PGHS-1 inhibitor) or NS-398 (PGHS-2 inhibitor) (hatched bars) in mesenteric arteries from ovariectomized (n=5) (A), 0.15 mg/pellet estradiol–replaced (n=6) (B), and 0.50 mg/pellet estradiol–replaced (n=6) (C) rats. Bars represent mean±SEM. *P<0.05 vs arachidonic acid alone.

PGE₂, U-46619, and PGH₂ Vasoconstrictor Responses
PGE₂ induced similar vasoconstriction in endothelium-denuded arteries from ovariectomized and estradiol-replaced rats (Figure 5A). U-46619 and PGH₂ evoked greater vasoconstrictor responses in the arteries from ovariectomized compared with estradiol-replaced rats (Figure 5, panels B and 5C, respectively). U-46619, but not PGH₂, also induced a significantly greater vasoconstriction from arteries of the 0.15 mg/pellet estradiol–replaced rats compared with the 0.5 mg/pellet estradiol–replaced rats.

View larger version (15K): [in this window] [in a new window]   Figure 5. Concentration-response curves to PGE2 (A), TxA2 mimetic U-46619 (B), or PGH2 (C) for endothelium-denuded mesenteric arteries from ovariectomized (n=5, ), 0.15 mg/pellet estradiol–replaced (n=6, ), and 0.50 mg/pellet estradiol–replaced (n=6, ) rats. Data represent mean±SEM. *P<0.05 vs ovariectomized rats; +P<0.05 vs 0.15 mg/pellet estradiol–replaced rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experiments were designed to test the hypothesis that removal of ovarian steroids would decrease endothelial/NO-dependent relaxation responses but that estrogen replacement would increase NO and PGHS activity, leading to increased vasodilation. Contrary to our hypothesis, the principal effect of estrogen replacement was to suppress PGHS-dependent vasoconstriction. The present experiments demonstrate that ovariectomy in the rat altered endothelium-dependent function as a result of an increased PGHS-dependent vasoconstriction that was associated with an augmented sensitivity of the PGH₂/TxA₂ receptor. Estrogen replacement prevented this increased vasoconstrictor response.

Many studies, including our own, indicate that the beneficial effect of estrogen is attributed, in part, to a direct effect on endothelium function.^{10 11 12 13 17 18 19 20} There are a number of reports suggesting that NO production and thus vasodilation is increased due to estrogen.^{10 13 17 18 19 20} In our study design, methacholine (an acetylcholine analogue) was used as an endothelium/NO-dependent relaxing agent to evaluate vascular function. We observed an increased relaxation response to methacholine in the mesenteric arteries of the estrogen-replaced rats compared with the ovariectomized rats in the absence of PGHS inhibition. These data could have been interpreted to reflect an increased endothelium/NO-dependent relaxation response. However, when PGHS activity was inhibited, the relaxation response in the arteries from the ovariectomized animals was enhanced. The extent of PGHS-dependent vasoconstriction was reduced in a dose-dependent manner with chronic estradiol replacement in the rat. These data indicate that estrogen did not increase NO-dependent vascular relaxation but that the principal effect of estrogen was to suppress a PGHS-dependent vasoconstriction.

In agreement with our data, a recent study reported that gender differences in endothelium-dependent relaxations of porcine coronary arteries did not involve the NO pathway but that PGHS-dependent vasoconstriction modified the endothelium-dependent response.²¹ These data provide support that PGHS-dependent vasoconstriction is important in endothelium-dependent function. However, unlike the present study, the lack of estrogen was not primarily responsible for the gender differences, since the male pigs had higher concentrations of plasma estradiol than the females. Since there are many differences in males compared with females, many studies use an ovariectomized animal model without and with estrogen replacement. Some of these previous estrogen replacement studies have also incorporated inhibition of the PGHS pathway for in vitro vascular studies. In contrast to our findings, others have observed in estrogen-replaced animals either no effect in response to PGHS inhibition^{10 11 12} or a response indicating an increased prostacyclin component modifying adrenergic vasoconstriction.¹³ In the present study, we observed in estradiol-replaced rats an enhanced endothelium-dependent relaxation response to methacholine due to a suppressed PGHS-dependent vasoconstriction. The disparity of the effect of estradiol on vascular responses may be due to the different species studied, the concentration and duration of estrogen replacement, and the vascular bed studied. In the present study, we chronically (4 weeks) treated rats with 2 doses of physiological relevant (cycling and pregnancy) levels of estrogen for rats or alternatively with the higher dose of estrogen replacement equivalent to that of hormone replacement levels in humans.

In the present study, pretreatment with valeryl salicylate or NS-398, to inhibit PGHS-1 and PGHS-2, respectively, increased the relaxation response in the ovariectomized rats, indicating that both PGHS-1 and PGHS-2 are important contributors to the PGHS-dependent vasoconstriction. Little is know regarding the regulation of activity or expression PGHS-1 and PGHS-2 by estrogen. In agreement with our vascular function data, it has been described that ovariectomy enhances but that estrogen replacement inhibits PGHS-2 expression and activity through bone marrow factors (primarily interleukin-1).²² In the endometrial tissue of ewes treated with sex steroids, estrogen lowered PGHS mRNA but may have also increased the stability of the enzyme.²³ In contrast, however, Wu et al²⁴ recently reported that estradiol increases PGHS-2 mRNA and protein in the myometrium of nonpregnant sheep,²⁴ suggesting a complexity of estrogen regulation depending on the tissue bed, animal model, and experimental design of the study.

In the endothelium, there is limited and contradictory data regarding estrogen and the PGHS pathway. In cell culture, estradiol has been shown to either have no effect²⁵ or increase²⁶ prostacyclin production in cultured human vascular endothelial cells. In vivo treatment of female rabbits with the synthetic estrogen, ethinyl estradiol, results in diminished in vivo vascular production of prostacyclin.²⁷ These data could reflect a decrease in PGHS activity due to estrogen, which is similar to our findings. In our study, it was not possible to measure eicosanoid production released into the organ baths from the mesenteric arteries because of their size (<250 µm in diameter). We did, however, administer exogenous arachidonic acid to substantiate that the arteries from the ovariectomized rats had an increased capacity for a PGHS-dependent vasoconstriction. Furthermore, the addition of exogenous arachidonic acid should also indicate whether vascular differences in the groups of rats is due to differences in phospholipase activity that regulates the availability of arachidonic acid or to differences in cyclooxygenase activity. Previous reports on the vascular effects of exogenous arachidonic acid have been in conflict, depending on species, tissue, or the experimental model of the disease studied.^{28 29 30} Specific to estrogen replacement, arachidonic acid has been shown to augment endothelium-dependent contractions in rabbit aorta.³¹ In contrast, our data demonstrated a dose-dependent vasoconstriction only in arteries from ovariectomized rats and a vasorelaxation in the mesenteric arteries of estradiol-treated rats. The presence of a PGHS inhibitor eliminated the difference among the groups. These data further indicate that the increased vasoconstrictor response was not due to alterations of arachidonic acid availability but rather an effect at the level of PGHS-dependent products.

Comparable to the ovariectomized rat, an increase of PGHS-dependent vasoconstriction has been observed in animal models of hypertension,^{32 33 34 35 36} diabetes³⁷ and oxidative stress.³⁸ Although some reports have suggested that a deficiency of NO contributes to the pathogenesis of hypertension,^{39 40} there are data indicating that the release of NO is normal and that a concomitant release of a PGHS-dependent endothelium-derived contracting factor accounts for the impaired relaxation.^{32 33 34 35} Relevant to our study, in female compared with male spontaneously hypertensive rats, there is both an increased NO-dependent response as well as decreased PGHS-dependent vasoconstriction.³⁶ It is possible that estrogen has a beneficial role to slow the progression of hypertension through suppression of PGHS-dependent vasoconstriction.

In the spontaneously hypertensive rat model, it has been suggested that the endothelium-derived PGHS-dependent vasoconstrictor is likely to be PGH₂.^{33 35} Our data for the ovariectomized rats agree with the finding of a concomitant release of a PGHS-dependent endothelium-derived contracting factor. The presence of a TxA₂/PGH₂ receptor antagonist resulting in a response similar to PGHS inhibition indicated that the PGHS-dependent vasoconstrictor was eliciting a response through this receptor. Since TxA₂ and PGH₂ bind to the same receptor, the PGHS-dependent vasoconstrictor could be either TxA₂ or PGH₂. Inhibition of TxA₂ synthase with U-51605 did not alter the relaxation response to methacholine in the ovariectomized rats, suggesting that TxA₂ may not be responsible for the PGHS-dependent vasoconstriction. However, inhibition of a specific synthase may shunt the pathway to other eicosanoids produced through isomerization or reduction of PGH₂.

Along with the observed increase of PGHS-dependent vasoconstriction in the ovariectomized rats, there appeared to be enhanced sensitivity of the PGH₂/TxA₂ receptor. The vascular constriction response to both PGH₂ and the TxA₂ mimetic U-46619 was enhanced, whereas there was no difference in the vascular response to PGE₂ in the ovariectomized rats compared with the estradiol-replaced rats. The increased sensitivity to PGH₂ in the ovariectomized rats is similar to the PGH₂ hypersensitivity observed in the aortas of spontaneously hypertensive rats.³⁵ Furthermore, a recent study reported a decrease in contractility of guinea pig coronary arteries to the TxA₂ mimetic U-46619 in estrogen-replaced guinea pigs; however, unlike our data, their data indicated that NO mediated the reduced contractility.¹⁸ In our study, using endothelium-denuded mesenteric arteries from Sprague-Dawley rats, both PGH₂ and U-46619 induced a vasoconstriction that was greater in ovariectomized rats compared with estradiol-replaced rats. These data suggest that estrogen has the potential to suppress a PGHS-dependent vasoconstrictor response via the PGH₂/TxA₂ receptor.

In summary, estrogen-replacement in an ovariectomized rat suppressed PGHS-dependent vasoconstriction. The present results provide a novel observation that may explain, in part, the vascular physiology in postmenopausal women. Furthermore, these data provide another potential mechanism for the protective effect of estrogen that should be further studied for development of alternative therapeutic approaches for cardiovascular disease in men and women.

	Acknowledgments

 
This study was supported by the Medical Research Council of Canada. Dr S.T. Davidge is a scholar of the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research.

Received January 20, 1998; accepted May 21, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Stampfer MJ, Colditz GA, Willet WC, Manson JE, 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. 1991;325:756–762.[Abstract]
2. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990;323:27–36.[Medline] [Order article via Infotrieve]
3. Hecker M, Mulsch A, Bassemge E, Forstermann R, Buss R. Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications. Biochem J. 1994;299:247–252.
4. Michel T, Smith TW. Nitric oxide synthases and cardiovascular signaling. Am J Cardiol. 1993;72:33C–38C.[Medline] [Order article via Infotrieve]
5. Smith WL, Marnett LJ, DeWitt DL. Prostaglandin and thromboxane biosynthesis. Pharmacol Ther. 1991;49:153–179.[Medline] [Order article via Infotrieve]
6. DeWitt DL. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim Biophys Acta. 1991;1083:121–134.[Medline] [Order article via Infotrieve]
7. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T. Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett. 1995;360:291–293.[Medline] [Order article via Infotrieve]
8. Weiner CP, Lizasoain I, Baylis S, Knowles RG, Charles L, Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci U S A. 1994;91:5212–5216.[Abstract/Free Full Text]
9. Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A. 1992;89:7384–7388.[Abstract/Free Full Text]
10. Rosenfeld CR, Cox BE, Roy T, Magness RR. Nitric oxide contributes to estrogen-induced vasodilation of the ovine uterine circulation. J Clin Invest. 1996;98:2158–2166.[Medline] [Order article via Infotrieve]
11. Gisclard V, Miller VM, Vanhoutte PM. Effect of 17ß-estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther. 1988;244:19–22.[Abstract/Free Full Text]
12. Vedernikov YP, Liao Q-P, Jain V, Saade GR, Chwalisz K, Garfield RE. Effect of chronic treatment with 17ß-estradiol and progesterone on endothelium-dependent and endothelium-independent relaxation in isolated aortic rings from ovariectomized rats. Am J Obstet Gynecol. 1997;176:603–608.[Medline] [Order article via Infotrieve]
13. Meyer MC, Cummings K, Osol G. Estrogen replacement attenuates resistance artery adrenergic sensitivity via endothelial vasodilators. Am J Physiol. 1997;272:H2264–H2270.[Abstract/Free Full Text]
14. Davidge ST, Baker PN, McLaughlin MK, Roberts JM. Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. Circ Res. 1995;77:274–283.[Abstract/Free Full Text]
15. Salvemini D, Settle SL, Masferrer JL, Seibert K, Currie MG, Needleman P. Regulation of prostaglandin production by nitric oxide: an in vivo analysis. Br J Pharmacol. 1995;114:1171–1178.[Medline] [Order article via Infotrieve]
16. Osol G, Cipolla M, Knutson S. A new method for mechanically denuding the endothelium of small (50–150 µm) arteries with a human hair. Blood Vessels. 1989;26:320–324.[Medline] [Order article via Infotrieve]
17. Wellman GC, Bonev AD, Nelson MT, Brayden JE. Gender differences in coronary artery diameter involve estrogen, nitric oxide, and Ca²⁺-dependent K⁺ channels. Circ Res. 1996;79:1024–1030.[Abstract/Free Full Text]
18. Thompson LP, Weiner CP. Long-term estradiol replacement decreases contractility of guinea pig coronary arteries to the thromboxane mimetic U46619. Circulation. 1997;95:709–714.[Abstract/Free Full Text]
19. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A. 1992;89:11259–11263.[Abstract/Free Full Text]
20. Gilligan DM, Quyyumi AA, Cannon RO. Effects of physiological levels of coronary vasomotor function in postmenopausal women. Circulation. 1994;89:2545–2551.[Abstract/Free Full Text]
21. Barber DA, Miller VM. Gender differences in endothelium-dependent relaxations do not involve NO in porcine coronary arteries. Am J Physiol. 1997;273:H2325–H2332.
22. Kawaguchi M, Pilbeam CC, Vargas SJ, Morse EE, Lorenzo JA, Raisz LG. Ovariectomy enhances and estrogen replacement inhibits the activity of bone marrow factors that stimulate prostaglandin production in cultured mouse calvariae. J Clin Invest. 1995;96:539–548.
23. Salamonsen LA, Hampton AL, Clements JA, Findlay JK. Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus. J Reprod Fertil. 1991;92:393–406.[Abstract]
24. Wu WX, Ma XH, Zhang Q, Buchwalder L, Nathanielsz PW. Regulation of prostaglandin endoperoxide H synthase 1 and 2 by estradiol and progesterone in nonpregnant ovine myometrium and endometrium in vivo. Endocrinology. 1997;138:4005–4012.[Abstract/Free Full Text]
25. Corvazier E, Dupuy E, Dosne AM, Maclouf J. Minimal effect of estrogens on endothelial growth and production of prostacyclin. Thromb Res. 1984;34:303–310.[Medline] [Order article via Infotrieve]
26. Mikkola T, Turunen P, Avela K, Orpana A, Viinikka L, Ylikorkala O. 17ß-Estradiol stimulates prostacyclin, but not endothelin-1, production in human vascular endothelial cells. J Clin Endocrinol Metab. 1995;80:1832–1836.[Abstract]
27. Elam MB, Lipscomb GE, Chesney CM, Terragno DA, Terragno NA. Effect of synthetic estrogen on platelet aggregation and vascular release of PGI₂-like material in the rabbit. Prostaglandins. 1980;20:1039–1051.[Medline] [Order article via Infotrieve]
28. Pagano PJ, Lin L, Sessa WC, Nasjletti A. Arachidonic acid elicits endothelium-dependent release from the rabbit aorta of a constrictor prostanoid resembling prostaglandin endoperoxides. Circ Res. 1991;69:396–405.[Abstract/Free Full Text]
29. Messina EJ, Sun D, Koller A, Wolin MS, Kaley G. Role of endothelium-derived prostaglandins in hypoxia-elicited arteriolar dilation in rat skeletal muscle. Circ Res. 1992;71:790–796.[Abstract/Free Full Text]
30. Oyekan AO, McGiff JC, Quilley J. Cytochrome P-450–dependent vasodilator responses to arachidonic acid in the isolated, perfused kidney of the rat. Circ Res. 1991;68:958–965.[Abstract/Free Full Text]
31. Miller VM, Vanhoutte PM. 17ß-Estradiol augments endothelium-dependent contractions to arachidonic acid in rabbit aorta. Am J Physiol. 1990;258:R1502–R1507.[Abstract/Free Full Text]
32. Lüscher TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension. 1986;8:344–348.[Abstract/Free Full Text]
33. Diederich D, Yang Z, Bühler FR, Lüscher TF. Impaired endothelium-dependent relaxations in hypertensive resistance arteries involve cyclooxygenase pathway. Am J Physiol. 1990;258:H445–H451.[Abstract/Free Full Text]
34. Noll G, Lang MG, Tschudi MR, Ganten D, Luscher TF. Endothelial vasoconstrictor prostanoids modulate contractions to acetylcholine and ANG II in Ren-2 rats. Am J Physiol. 1997;272:H493–H500.[Abstract/Free Full Text]
35. Ge T, Hughes H, Junquero DC, Wu KK, Vanhoutte PM, Boulanger CM. Endothelium-dependent contractions are associated with both augmented expression of prostaglandin H synthase-1 and hypersensitivity to prostaglandin H₂ in the SHR aorta. Circ Res. 1995;76:1003–1010.[Abstract/Free Full Text]
36. Kauser K, Rubanyi GM. Gender difference in endothelial dysfunction in the aorta of spontaneously hypertensive rats. Hypertension. 1195;25:517–523.[Abstract/Free Full Text]
37. Tesfamariam B, Jakubowski JA, Cohen RA. Contraction of diabetic rabbit aorta caused by endothelium-derived PGH₂-TxA₂. Am J Physiol. 1989;275:H1327–H1333.
38. Davidge ST, Hubel CA, McLaughlin MK. Cyclooxygenase-dependent vasoconstrictor alters vascular function in the vitamin E–deprived rat. Circ Res. 1993;73:70–88.
39. Gryglewski RJ, Botting RM, Vane JR. Mediators produced by the endothelial cell. Hypertension. 1988;12:530–548.[Abstract/Free Full Text]
40. Vanhoutte PM. Role of calcium and endothelium in hypertension, cardiovascular disease, and subsequent vascular events. J Cardiovasc Pharmacol. 1992;19:S6–S10.