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Cardiovascular Research 2000 46(1):111-118; doi:10.1016/S0008-6363(99)00424-1
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Copyright © 2000, European Society of Cardiology

Activation of Ca2+-independent nitric oxide synthase by 17β-estradiol in post-ischemic rat heart

Heather Fraser1,a, Sandra T. Davidgeb,c and Alexander S. Clanachana,*

aDepartment of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
bDepartment of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
cDepartment of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

* Corresponding author. Tel.: +1-780-492-0511; fax: +1-780-492-4325 sandy.clanachan{at}ualberta.ca

Received 27 July 1999; accepted 3 December 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Background: Nitric oxide (NO) donors or facilitation of endogenous NO production is cardioprotective. This study sought to determine whether enhanced myocardial NO production might contribute to estrogen-induced cardioprotection. Methods: Ca2+-dependent and Ca2+-independent NOS activities (pmol min–1 mg–1 protein), NOS protein expression (quantitative immunoblot), cGMP content (pmol mg–1 protein) and LV work (Joules) were measured in hearts isolated from ovariectomized rats that were either untreated or treated chronically with 17β-estradiol (0.25 mg, 21 day release formulation). Results: After 14 days, serum levels of 17β-estradiol were 6±1 and 135±16 pg ml–1 in untreated and 17β-estradiol-treated animals, respectively. After 60 min aerobic working mode perfusion, Ca2+-dependent NOS (untreated, 1.47±0 36; 17β-estradiol 1.13±0.25) and Ca2+-independent NOS (untreated, 0.45±0.24; 17β-estradiol, 0.41±0.21) activities, eNOS and iNOS proteins and cGMP content (untreated, 0.64±0.08; 17β-estradiol, 0.76±0.12) were not different in the two groups. After 60 min low-flow (0.5 ml min–1) ischemia and 30 min reperfusion, Ca2+-dependent NOS activities were again similar (untreated, 1.25±0.23; 17β-estradiol, 0.78±0.27). However, after reperfusion, Ca2+-independent NOS activity (untreated, 0.39±0.10; 17β-estradiol, 1.36±0.36) was 3.5-fold higher (P=0.008) and cGMP content (untreated, 0.30±0.03; 17β-estradiol, 0.49±0.07) was 1.6-fold higher (P=0.017) in hearts from 17β-estradiol-treated animals. Although pre-ischemic function was similar, recovery of post-ischemic LV work was 2-fold greater (P=0.024) in the 17β-estradiol group. Conclusion: The ability of ischemia and reperfusion in combination with chronic 17β-estradiol to increase Ca2+-independent NOS activity and cGMP content supports a role for enhanced myocardial NO signaling in 17β-estradiol-induced cardioprotection.

KEYWORDS Hormones; Ischemia; Nitric oxide; Reperfusion; Ventricular function

This article is referred to in the Editorial by M. Barton (pages 20–23) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Heart disease is the primary cause of death in both men and women over the age of 60 in Canada and the US. While premenopausal women have a low incidence of cardiovascular disease compared with men, after menopause mortality rates of women become similar to men [1]. Studies describing improved cholesterol profiles [2], decreased insulin resistance and enhanced glucose tolerance [3] by estrogen replacement therapy (ERT) in menopausal women have provided more direct evidence that estrogen is protective against coronary heart disease. Estrogen also stops the progression of atherosclerosis in cynomolgus monkeys [4] and increases blood flow in many areas of the circulation, including the carotid and coronary arteries [5,6]. In addition, 17β-estradiol reduces myocardial necrosis in rabbits after ischemia and reperfusion [7] and improves recovery of mechanical function following global ischemia in isolated rat hearts [8,9].

Enhanced NO biosignaling has been implicated in many of the cardiovascular changes arising from ERT [10]. Endogenous NO may be generated in the myocardium by various isoforms of nitric oxide synthase (NOS). These are present under basal or pathological conditions in several cell types in the myocardium, including endothelial cells [11] and cardiac myocytes [12]. Although a range of physiological and pharmacological actions has been attributed to endogenous and exogenous NO, several of these may benefit cardiac function and contribute to cardioprotection. These include stimulation of soluble guanylyl cyclase (sGC) leading to elevation of cGMP and coronary smooth muscle dilation [13], and enhanced ventricular relaxation [14–16].

Impairment of NO-mediated biosignaling has been implicated in atherosclerosis [10] and ischemia reperfusion injury [16–18] and enhancement of NO generation reduces atherosclerosis [10] and post-ischemic mechanical dysfunction [16,18–21]. The cardioprotective efficacy of NO donors [21,22] provides direct support for the notion that an enhancement of NO biosignaling, possibly via a myocardial cGMP mechanism, improves the contractility of the post-ischemic heart.

Interestingly, monophosphoryl lipid A, an endotoxin derivative that causes low-level induction of NOS, is also protective in rabbit and canine models of ischemia and reperfusion. It reduces myocardial infarct size by 50 to 70% [23–25] and improves the recovery of post-ischemic mechanical function. A critical role of Ca2+-independent NOS (iNOS) in limiting ischemia-reperfusion is indicated from studies in which cardioprotection associated with the late phase of ischemic preconditioning [26] is inhibited by the iNOS-selective inhibitor, aminoguanidine [27]. Taken together, these data suggest that cardioprotection may arise from a correction in a deficiency in NO biosignaling in the postischemic heart. In contrast, the marked stimulation of iNOS by cytokines is deleterious by increasing atherosclerosis [11] and depressing cardiac contractility [28].

Of particular interest to the present study is the observation that estrogen may enhance NOS expression and/or enzyme activity and so elevate endogenous NO production. Estrogen receptor stimulation induces eNOS [29] and iNOS [30] gene expression. Females possess an enhanced capacity to produce NO that enhances NO-mediated vasodilation and reduces vascular smooth muscle tone [31]. The role of myocardial NO biosignaling in the direct cardioprotective actions of 17β-estradiol has not yet been addressed. Thus, we hypothesized that estrogen may exert its cardioprotective effects by altering NO-mediated biosignaling in cardiac muscle. The present study investigated post-ischemic mechanical function and NO biosignaling in hearts removed from ovariectomized rats treated chronically with 17β-estradiol.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Animal surgery
All rats (Sprague Dawley) were housed and treated according to the standards set by the Canadian Council of Animal Care and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996). Rats received food and water ad libitum and were housed two per cage at room temperature (21°C) under a 12-h light–dark cycle. Post-surgical rats were housed individually. After a 1-week period of acclimatization, rats, that were selected to be within a narrow weight range (275–300 g), were anesthetized with 50 mg/kg Brietal® i.p. and surgical removal of the ovaries was completed using an aseptic technique as described previously [9]. At the time of surgery, treated animals received a 17β-estradiol pellet, which was placed subcutaneously near one incision site. Finally, the skin was stapled shut, and the area cleaned.

2.2 Drug treatment
Rats were divided into two groups: untreated (n=16) or 17β-estradiol-treated (n=14). 17β-Estradiol-treated rats received a time-release pellet subcutaneously for 14 days (17β-estradiol, 0.25 mg, 21-day release formula; Innovative Research of America, Sarasota, Fl). The rats were allowed to recover from surgery for two weeks, at which time they were killed and hearts removed for ex vivo perfusion. In addition, at the time of death, blood was removed from the abdominal aorta for the later determination of serum 17β-estradiol concentrations by radioimmunoassay (Diagnostic Products, Los Angeles, CA) and the uterus was removed and weighed to confirm the effectiveness of 17β-estradiol therapy.

2.3 Heart perfusions
After 14 days of treatment, animals were anesthetized with pentobarbital i.p. and hearts were removed, and perfused in Langendorff mode. After a 10-min Langendorff equilibration period (Fig. 1), the four groups of hearts were switched to working mode and perfused for 60 min under aerobic conditions (Aerobic Groups: Untreated n=8; Treated n=7) or subjected to 60 min of aerobic perfusion followed by 60 min low-flow ischemia (0.5 ml min–1) and 30 min reperfusion, and frozen (Reperfusion Groups: Untreated n=8; Treated n=7). Working hearts were perfused at 37°C under aerobic conditions at a constant left atrial preload (11.5 mm Hg) and aortic afterload (80 mm Hg) and were allowed to beat spontaneously. A modified Krebs-Henseleit perfusate containing both glucose (11 mM) and palmitate (1.2 mM) was used [32] in order to mimic the normal array of energy substrates available for energy production. The high level of fatty acid (1.2 mM) mimics that observed clinically in adults and children during ischemia and reperfusion associated with cardiac surgery [33]. Aortic systolic and diastolic pressures were measured using a Gould P21 pressure transducer connected to the aortic outflow line. Cardiac output, aortic flow and coronary flow (cardiac output minus aortic flow) were measured (ml min–1) using in-line ultrasonic flow probes connected to a Transonic T206 ultrasonic flow meter. Left ventricular minute work (LV work, Joules), calculated as cardiac outputxleft ventricular developed pressure (aortic systolic pressure–preload pressure)x0.133 [34], was used as a continuous index of mechanical function. Coronary vascular conductance (ml min–1 mm Hg–1) was calculated as coronary flowx(aortic pressure)–1.


Figure 1
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  		Fig. 1 Diagram of the perfusion protocols used to assess LV mechanical function and NO biosignaling of hearts from untreated and 17β-estradiol (E2)-treated rats. After a 10-min Langendorff equilibration period, hearts were switched to working mode and perfused for 60 min under aerobic conditions (Aerobic Groups) or subjected to 60 min of aerobic perfusion followed by 60 min low-flow ischemia (solid bar, 0.5 ml min–1) and 30 min reperfusion (Reperfusion Groups). At the end of each perfusion protocol, hearts were rapidly frozen (indicated by {downarrow}) for biochemical measurements.

 
2.4 Assay of NOS activities
Heart tissue (150 mg), sampled from frozen and subsequently powdered left ventricle, was homogenized (10 mM Hepes buffer containing sucrose (320 mM), DTT (1 mM), leupeptin (10 µg/ml), soybean trypsin inhibitor (10 µg/ml) and aprotinin (2 µg/ml), pH 7.4) using an ultrasonic processor and centrifuged at 4°C, 10 000 g for 20 min. Supernatant (40 µl) was incubated for 20 min at 37°C with assay solution at pH 7.4 containing final concentrations of KH2PO4 (40 mM), MgCl2 (1 mM), CaCl2 (0.2 mM), valine (45.7 mM), L-citrulline (1 mM), L-arginine (20 µM), dithiothrietol (1 mM), NADPH (0.1 mM), BH4 (0.01 mM), and L-[U-14C]arginine (0.07 µCi). Three samples were prepared; one with assay solution alone, one in the presence of L-NMMA (1 mM) and a third in the presence of EGTA (1 mM, pH 7.2). In this way, Ca2+-dependent NOS and Ca2+-independent NOS activities were determined by the enzymatic conversion of L-[U-14C]arginine to [14C]citrulline and NO. Separation of [14C]citrulline from [14C]arginine was achieved by incubating the samples in an ion exchange resin for 45 min followed by determining the radioactivity counts in 500 µl of solution (no resin) using standard scintillation counting procedures. Protein content of the assay mixtures was determined by the Bradford assay with a standard curve that was prepared using bovine serum albumin. NOS activity was expressed as pmol min–1 mg protein–1.

2.5 Determination of cGMP content
Heart tissue (200 mg), sampled from the frozen and subsequently powdered left ventricle, was homogenized in 500 µl of 2.2% PCA in 0.1 M Hepes buffer (pH 7.4) and 5 mM Na2EDTA. After 15 min, the samples were centrifuged at 10 000 g at 4°C for 2 min. A 250 µl aliquot of supernatant was removed and neutralized with 1.1 M K3PO4 (40 µl), after which samples were centrifuged at 10 000 g at 4°C for 2 min. Myocardial cGMP content was determined using an enzyme immunoassay kit (Cayman Chemical Co). The cGMP content was expressed as pmol mg protein–1.

2.6 Western immunoblot analysis of NOS
Discontinuous SDS polyacrylamide (8% running and 3.5% stacking) gels were prepared and used for electrophoresis of protein. Samples were loaded into wells and run at 120 V constant for 105 min. Protein was transferred to membrane (nylon) using an electroblot system for 2.5 h at 15 V at room temperature. Once transfer was complete, the membrane was blocked in 7% milk solution in Tris-buffered saline (TBS: Tris base, 20 mM; NaCl, 137 mM; HCl, 1 M, pH 7.5) overnight. The next day, the membrane was washed with 0.1% tween-TBS and the primary antibody (mouse monoclonal (1:1000 dilution in TBS-tween) was incubated with the membrane (2 h). After washing, the membrane was incubated with the second antibody (horseradish peroxidase labeled anti-mouse 1:2000 dilution in TBS-tween) at room temperature (1 h), then washed again. Antibody detection was completed using enhanced chemiluminescence (ECL, Amersham LIFE Sciences, Oakville, ON), and then exposed to X-ray film. The positive control (standard) for eNOS was human endothelial cell lysates and for iNOS was lysates from mouse macrophage and from heart removed from rats following induction of septic shock with LPS [35]. Antibodies for eNOS and iNOS were purchased from Transduction Laboratories (Mississauga, ON).

2.7 Data analysis
Data are expressed as the mean±standard error of the mean (S.E.). Comparisons between untreated and 17β-estradiol-treated groups were performed using the unpaired Student's t-test (two-tailed). When sample variances were significantly different (as in the case of serum 17β-estradiol concentrations and uterine weights), a nonparametric test was used (Mann–Whitney U-statistic, unpaired, two-tailed test). Differences were judged to be significant when P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Treatment with 17β-estradiol
As anticipated, there was a significant increase in serum 17β-estradiol concentrations (135±16 vs. 6±1 pg ml–1, P<0.0001) and a lower body weight (303±7 vs. 348±5 g, P<0.001) in the group of estradiol-replaced rats. Serum 17β-estradiol concentrations approximated those associated with transdermal estradiol therapy in post-menopausal women [36]. Uterine weight, which provides a biological marker of estrogen replacement, was also significantly (P<0.001) elevated in the estradiol-treated groups (0.65±0.04 vs. 0.24±0.01 g, P<0.001). As both 17β-estradiol concentrations and uterine weight were increased in each estradiol-treated animal, no hearts had to be removed from this study due to inactive pellets.

3.2 Effects of 17β-estradiol on LV function
During baseline aerobic perfusion, mechanical function (LV work) was stable in hearts from all groups (Table 1). Following 60 min low-flow ischemia, the recovery of LV work in hearts from untreated rats was poor and reached only 15% of pre-ischemic baseline values. In contrast, LV work of hearts from animals treated chronically with 17β-estradiol retained a capacity to resist ischemia-reperfusion damage as evinced by their significantly higher recovery of LV work (2-fold) during reperfusion compared with hearts from the untreated group. This occurred in the absence of any changes in coronary vascular conductance (Fig. 2).


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  		Table 1 Baseline characteristics of the four perfusion groupsa

 

Figure 2
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  		Fig. 2 LV work and coronary vascular conductance measured during reperfusion of isolated rat hearts. Values are means±S.E. for hearts from Untreated and E2-treated animals. * Indicates a significant difference (P<0.05) between Untreated and E2-treated groups.

 
3.3 Effects of 17β-estradiol on NO biosignaling
To determine whether changes in NO biosignaling were involved in the improved recovery of post-ischemic mechanical function, Ca2+-dependent NOS and Ca2+-independent NOS activities were determined at the end of aerobic perfusion and following 60 min of reperfusion. After 60-min of aerobic perfusion, Ca2+-dependent NOS activity exceeded Ca2+-independent NOS activity by 3.3-fold and 2.7-fold in hearts from untreated and 17β-estradiol-treated rats, respectively (Figs. 3 and 4Go). Neither Ca2+-dependent NOS nor Ca2+-independent NOS activity was altered in hearts from 17β-estradiol-treated rats during aerobic perfusion (Figs. 3 and 4Go). Ca2+-dependent NOS activity was also similar between 17β-estradiol and untreated groups at the end of reperfusion (Fig. 3). However, in hearts from 17β-estradiol-treated animals, Ca2+-independent NOS activity was increased 3.5-fold compared with hearts from untreated animals (Fig. 4). Furthermore, cGMP content was significantly increased in reperfused hearts from the 17β-estradiol group (Fig. 5), whereas cGMP content was similar in aerobic hearts from both groups.


Figure 3
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  		Fig. 3 Ca2+-dependent NOS activity (citrulline assay) and eNOS protein (quantitative Western immunoblot) in hearts from untreated and 17β-estradiol-treated rats. Values are means±S.E. for hearts frozen after Aerobic perfusion and after Reperfusion. Also shown are representative immunoblots of eNOS for hearts from Untreated (U) and E2-treated hearts after 60 min aerobic perfusion. Bands were in the 155 kDa range (identified by a range of molecular weight markers) and co-localized with an endothelial eNOS standard (S).

 

Figure 4
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  		Fig. 4 Ca-independent NOS activity (citrulline assay) and iNOS protein (quantitative Western immunoblot) in hearts from untreated and 17β-estradiol-treated rats. Values are means±S.E. for hearts frozen after Aerobic perfusion and after Reperfusion. Also shown are representative immunoblots of iNOS for hearts from Untreated (U) and E2-treated hearts after 60 min aerobic perfusion. Bands were in the 130 kDa range (identified by a range of molecular weight markers) and co-localized with a macrophage iNOS standard (S) and with a major band in hearts from LPS-treated rats (L). * Indicates a significant difference (P<0.05) between Untreated and E2-treated groups.

 

Figure 5
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  		Fig. 5 Content of cGMP in hearts from untreated and 17β-estradiol-treated rats. Values are means±S.E. for hearts frozen after Aerobic perfusion and after Reperfusion. * Indicates a significant difference (P<0.05) between Untreated and E2-treated groups.

 
Immunoblots for eNOS and iNOS standards were in the 155 kDa and 130 kDa regions, respectively. Quantification of the immunoblots for myocardial NOS protein showed no change in eNOS protein expression after 60 min of aerobic perfusion (Fig. 3). Although iNOS protein in gels containing standards (hearts from LPS-treated rats and macrophages from mouse) was marked, iNOS expression in hearts from both untreated and 17β-estradiol-treated ovariectomized rats was very low compared with expression of eNOS. There were no measurable differences in eNOS or iNOS protein expression between these two groups, either after aerobic perfusion or after post-ischemic reperfusion (Figs. 3 and 4Go).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The objective of this study was to investigate potential mechanisms of 17β-estradiol-induced cardioprotection by testing the hypothesis that 17β-estradiol alters myocardial NO biosignaling. The study has shown that chronic 17β-estradiol treatment was indeed cardioprotective and increased the recovery of post-ischemic mechanical function two-fold compared with hearts from untreated animals. Activity of Ca2+-independent NOS, measured after post-ischemic reperfusion, was elevated in hearts from 17β-estradiol-treated animals. Activation of Ca2+-independent NOS occurred as a result of 17β-estradiol treatment in combination with ischemia as activity measured prior to ischemia was not elevated. Activities of Ca2+-dependent NOS were similar after aerobic perfusion and reperfusion in both untreated and 17β-estradiol-treated groups. Further, enhanced NO biosignaling in hearts from 17β-estradiol-treated rats was supported by the finding of elevated myocardial content of cGMP at the end of reperfusion, but not at the end of aerobic perfusion.

Previous studies have implicated an upregulation of NOS in 17β-estradiol-induced alteration of vascular function and that such an effect may contribute to cardioprotection. In endothelium, estrogen receptor activation results in an upregulation of eNOS [29,37]. Ligand binding data, that indicate that estrogen receptors are present in the heart [38], are supported by additional studies that confirm the functional integrity of estrogen receptors in cardiomyocytes by the demonstration of estrogen-mediated gene activation [30]. The ability of estrogen to upregulate both eNOS [29,39] and iNOS [30] in cardiomyocytes identify the heart as a potential target for the direct effects of estrogen, but the role of estrogen in modulating NO biosignaling and its contribution to cardioprotection have not been determined. Furthermore, impaired bioavailability of endothelial NO and coronary vasomotor dysfunction have been implicated in ischemia reperfusion injury [17,18].

Two types of evidence are apparent from the present experiments for enhanced NO biosignaling in hearts from 17β-estradiol-treated rats. First, in hearts frozen at the end of reperfusion following 60 min of low-flow ischemia, Ca2+-independent NOS activity was enhanced 3.5-fold by 17β-estradiol treatment whereas Ca2+-dependent NOS activity was unchanged. Second, cGMP content, a marker of NO-mediated stimulation of soluble guanylyl cyclase (sGC), was also elevated in hearts from 17β-estradiol-treated animals. Thus, it appears that estrogen treatment may have resulted in an enhanced NOS activity in post-ischemic heart and thereby elicited a cardioprotective effect. Definite proof that enhanced NO biosignaling is the cause of the beneficial actions of estrogen would require additional experiments with inhibitors of NOS and/or sGC.

After 60 min of aerobic perfusion, neither Ca2+-independent NOS activities, Ca2+-dependent NOS activities nor cGMP content were different between groups. The activation of Ca2+-independent NOS was only apparent in hearts from rats treated chronically with 17β-estradiol and that had undergone reperfusion following 60-min of low-flow ischemia. However, there were no differences between the levels of expression of the NOS isoforms in the two groups. This finding contrasts with an earlier demonstration that estrogen increases eNOS, but not iNOS, expression [39]. The increase in Ca2+-independent NOS activity, in the absence of changes in iNOS protein, suggests that Ca2+-independent NOS activity was altered as a result of post-translational events occurring during ischemia and/or reperfusion. Indeed, the activity of Ca2+-independent NOS is controlled by dimerization [40] as well as by phosphorylation by tyrosine kinase [41]. Interestingly, there appears to be a specificity for the observed change in Ca2+-independent NOS activity with estrogen. In hearts where post-ischemic function was improved by the adenosine A1 receptor agonist, N6-cyclohexyladenosine [42], myocardial Ca2+-dependent NOS and Ca2+-independent NOS activities are unaltered. Thus, it appears that induction of Ca2+-independent NOS is analogous to the mechanism of protection observed following chronic therapy with monophosphoryl lipid A [20] and in association with the late phase or second window of preconditioning [27].

Induction of NOS has also been cited as the mechanism for many of the beneficial actions of 17β-estradiol in the vasculature. A recent study evaluating the vascular responsiveness of endothelium-denuded rat aortas to constrictor substances supports the involvement of an iNOS mechanism [43]. Furthermore, iNOS upregulation has been associated with other beneficial actions of 17β-estradiol, including prevention of platelet aggregation at sites of vascular injury [44]. Although data presented here suggest that activation of iNOS plays a role in the cardioprotective actions of 17β-estradiol, the notion that iNOS can be beneficial is controversial. Detrimental effects may occur in response to higher concentrations of NO. These include inhibition of Ca2+ entry during cardiac failure [45] or generation of hydroxyl radicals via the interaction of NO with superoxide and the formation of peroxynitrite [12,46] that, in turn, impairs cardiac mechanical function and cardiac efficiency [47]. The formation of peroxynitrite undoubtedly occurs when free radical scavengers can not deal with, and inactivate, increased NO production. While eNOS is considered to release small amounts of NO in response to increases in cytosolic Ca2+, iNOS, once activated, is able to release large amounts of NO and/or superoxide that contribute to tissue damage. However, increases in iNOS activity, if of a limited magnitude, may not result in excess NO production, but rather mimic the beneficial actions of exogenous NO donors. NO, in low concentrations, is beneficial to the vasculature [43] as well as the heart [20,27].

NO, produced from either eNOS or iNOS, is a potent stimulator of sGC that results in the conversion of GTP to cGMP. While a number of factors may stimulate sGC activity, the NO-sGC-cGMP transduction system is involved in mediating numerous physiological effects of NO including vascular and non-vascular smooth muscle relaxation. Inhibition of Ca2+ activated K+ channels [48], inhibition of L-type Ca2+ channels [49] or activation of large-conductance K+ channels [50] by a cGMP-mediated mechanism all result in vasorelaxation. Moreover, correction of the deficiency in NO biosignaling in post-ischemic hearts enhances ventricular compliance and improves diastolic relaxation [14–16]. Thus, these results provide further evidence for the involvement of the NO-sGC-cGMP transduction system in estrogen-mediated cardioprotective effects.

Time for primary review 30 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
HF was a recipient of an Alberta Heritage Foundation for Medical Research (AHFMR) Studentship and is now an AHFMR Postdoctoral Fellow in the Division of Cardiology, Johns Hopkins University. STD is an AHFMR and Heart and Stroke Foundation of Canada Scholar. This study was supported by a grant from the Heart and Stroke Foundation of Alberta. The authors wish to thank Barbara Zielnik-Drabik and Yunlong Zhang for their technical assistance.


   Notes
 
1 Present address: Division of Cardiology, Johns Hopkins University, 720 Rutland Avenue, Ross 844, Baltimore, MD 21205, USA. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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