AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H1169-H1180, 2002; doi:10.1152/ajpheart.00397.2000
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 HighWire
Right arrow Citing Articles via ISI Web of Science (27)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mershon, J. L.
Right arrow Articles by Clark, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mershon, J. L.
Right arrow Articles by Clark, K. E.
Vol. 283, Issue 3, H1169-H1180, September 2002

Estrogen increases iNOS expression in the ovine coronary artery

John L. Mershon, R. Scott Baker, and Kenneth E. Clark

Department of Obstetrics and Gynecology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0526


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen is believed to protect postmenopausal women from coronary vascular disease, in part by increasing production of nitric oxide (NO). In this study, we investigated the possibility that transcriptional activation of inducible NO synthase (iNOS) is responsible for a component of the estrogen-induced increase in coronary blood flow. Twenty-two ewes were instrumented with Doppler flow probes on their left circumflex coronary and pulmonary arteries. Nine ewes received 17beta -estradiol (1 µg/kg), and the coronary vascular response was followed for 16 h. Estradiol significantly increased coronary blood flow by 22 ± 4% over baseline and the peak response occurred at 2 h (P < 0.01). To examine the effect of estrogen on NOS expression in the ovine coronary artery, 17 noninstrumented animals were killed 2 h after administration of estradiol or vehicle. Coronary arteries were analyzed for ovine iNOS and endothelial NOS (eNOS) expression by semiquantitative RT-PCR. PCR primers were based on partial cDNA clones for ovine eNOS and iNOS isolated as part of this study. The expression of iNOS was significantly increased (27-fold) by the administration of estradiol, whereas the expression of eNOS was much weaker (2-fold). To confirm these effects in vivo, additional instrumented animals received either the estrogen receptor (ER) antagonist ICI-182,780 (n = 5), the iNOS antagonist dexamethasone (n = 5), or pyrrolidine dithiocarbamic acid, an inhibitor of nuclear factor-kappa B (n = 5). All three antagonists inhibited estrogen-induced increases in coronary blood flow and increases in cardiac output by over 85%. These results strongly support the hypothesis that 17beta -estradiol increases coronary blood flow in the unanesthetized nonpregnant ewe via an ER-dependent mechanism that results in an increase in both eNOS and iNOS expression.

nitric oxide synthase; blood flow; hormone; coronary disease


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CORONARY ARTERY HEART DISEASE is a leading cause of death in women after menopause, both natural and surgical (5). There is considerable evidence, based on in vitro studies, animal models, and clinical trials, that estrogen has a beneficial effect on the cardiovascular system (36) and reduces the incidence of the disease in women (1). For example, the largest study to date by Stampfer et al. (52) has shown that postmenopausal women receiving hormone replacement therapy had a 50% lower rate of coronary heart disease compared with women not receiving therapy. This lower rate of coronary heart disease appears to be independent of other risk factors. Recently, several studies have questioned the beneficial effect of estrogen on the cardiovascular system. The Heart Estrogen/Progestin Replacement Study, a randomized clinical trial of estrogen replacement therapy (ERT) versus placebo in women with established heart disease, failed to show a reduction in coronary events (18). The estrogen replacement and atherosclerosis study, utilizing quantitative coronary angiography, demonstrated that ERT did not slow the progression of atherosclerosis in postmenopausal women (17). The Women's Estrogen for Stroke Trial showed no reduction with ERT use in the incidence of recurrent stroke in women with prior stroke or transient ischemic attack (57). One possible explanation for these contrasting findings is the relationship of estrogen therapy with an increase in inflammatory markers of cardiovascular disease, such as C-reactive protein (48).

The presence of specific receptors for estrogen and progesterone in nonuterine vascular tissue has been documented in myocardium, coronary vessels, and the aorta (24, 47). These receptors appear to be functional, and stimulation leads to increased nitric oxide (NO) production in vessels (35). Previous studies from our laboratory have shown that estradiol can induce a significant and time-dependent increase in coronary artery blood flow in unanesthetized ovariectomized sheep and supported a role for NO in mediating this effect (29).

A short-lived free radical, NO is produced by a family of enzymes known as NO synthases (NOS), which includes endothelial (eNOS), inducible (iNOS), and neuronal NOS (nNOS) (41). A considerable body of evidence now exists in support of the role of eNOS in mediating estrogen-induced vasodilation in the coronary artery (14). In vitro studies have demonstrated an estrogen-induced increase in eNOS at many levels, including enzyme translocation (15) and activity (26), protein expression (64), mRNA expression (31), and promoter activity (27). The majority, but not all, of these studies has shown that this effect on eNOS is rapid (within minutes), short lived, estrogen receptor (ER) dependent, and calcium dependent.

Although eNOS is well known to play a role in the vascular actions of estrogen, there is evidence that eNOS activation does not explain the entire NO response. For example, some studies have demonstrated that the coronary response to estrogen is endothelium independent (11, 40) or directly involves the vascular smooth muscle cell (38). Clearly expressed in vascular smooth muscle (13) and to a lesser extent in endothelial cells (22), we hypothesized that iNOS is a strong candidate for mediating these effects. Importantly, the activity of iNOS is controlled at the level of transcription, independent of calcium, and is known to be regulated by steroid hormones (37). These properties support a possible role for iNOS in mediating a portion of estrogen-induced NO production through a well-defined, ER-dependent, genomic pathway. Thus the present study was undertaken to determine the potential role that iNOS might play in mediating estrogen-induced increases in coronary blood flow and to compare the magnitude of its expression in the coronary vasculature with that of eNOS in the nonpregnant sheep.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Procedures

Thirty-nine nonpregnant ewes were purchased from a commercial supplier (weighing between 40 and 60 kg), and twenty-two ewes underwent a thoracotomy 5 days after delivery to the facility. Ewes were fasted and water was withheld for 24 h before surgery. On the day of surgery, ewes were sedated with intravenous pentobarbital sodium (15 mg/kg), intubated, and placed on a ventilator with 2-3% isoflurane and oxygen to maintain anesthesia. Animals were placed on their right side and underwent a left lateral thoracotomy exposing the left circumflex coronary artery and the pulmonary trunk. Each vessel was fitted with a transit-time Doppler flow probe (3-4 and 24 mm respectively, Transonic Systems; Ithaca, NY) for determination of coronary blood flow and cardiac output as well as for calculation of coronary and systemic vascular resistance. The incision was closed in planes, and a large caliber intercostal catheter supplemented by suction was used for 48 h to reduce pneumothorax. Cables were tethered to the skin and placed in a tightly closed plastic bag within a canvas storage pouch attached to the ewe's side. Postoperatively, all ewes were housed in mobile carts with ad libitum access to commercial sheep feed and water. Ewes received penicillin G the day before, the day of, and 3 days after surgery and buprenorphine (Reckitt and Colman; Richmond, VA) for analgesia as needed for the first 3 days postoperatively.

One week later, the 22 ewes underwent a second surgical procedure utilizing the same preoperative, anesthetic, and postoperative procedures described above. The animals were secured in the supine position. The abdomen and left flank were cleansed with germicidal soap and draped aseptically. The femoral artery and vein were dissected through a 3- to 4-cm incision over the left groin. The femoral artery and vein were tied distally and cannulated with polyvinyl catheters (0.050 × 0.090 in.) to the level of the distal abdominal aorta or vena cava. The femoral artery catheter was used to measure arterial blood pressure, whereas the femoral vein catheter was used for systemic administration of compounds under study. The abdominal cavity was opened via a 10- to 15-cm midline incision to expose the uterus. All animals were bilaterally oophorectomized to prevent cyclic fluctuations of estrogen. The abdominal incision was closed in layers. Catheters were passed through a subcutaneous tunnel and then through a small skin incision on the ewe's left flank and tethered to the skin. The stopcock of each catheter was wrapped in an alcohol-soaked sponge and placed in a tightly closed plastic bag within a canvas storage pouch attached to the ewe's side. Ewes received penicillin G the day before, the day of, and 3 days after surgery.

With the use of the same preoperative, anesthetic, and postoperative procedures described above, 17 additional animals under went femoral artery and vein catheterization plus ovariectomy. These animals were used for hormone treatment and euthanized for tissue to determine the effects of 17beta -estradiol on iNOS and eNOS expression. After surgery, all ewes were housed in movable carts with free access to commercial sheep feed and water. All vascular catheters were flushed with heparin solution (1,000 U/ml) daily to maintain patency. In instrumented animals, cardiovascular measurements were begun 3 days after the abdominal surgery, whereas pharmacological studies were performed no earlier than the seventh postoperative day to ensure full recovery from anesthesia and surgical stress. All procedures described were performed in a completely accredited American Association for Accreditation of Laboratory Animal Care facility and were approved by the Institutional Animal Care and Use Committee.

Monitoring of Physiological Parameters

Systemic arterial blood pressure was monitored during all experiments using Micron MP-15 blood pressure transducers (Micron; Simi Valley, CA), and heart rate was recorded continuously by a SensorMedic cardiotachometer (Sensor Medics; Yorba Linda, CA), which is triggered by the pressure pulse wave. Cardiac output (pulmonary artery blood flow) and left circumflex coronary artery blood flow were monitored continuously by Transonic flowmeters. All parameters were continuously recorded on a SensorMedics R-612 physiological recorder. Before the studies, animals, which remained in the laboratory continuously, were connected to the recorder for a period of at least 1 h before estrogen administration to provide a stable baseline.

eNOS and iNOS Expression Studies

Seventeen animals that were ovariectomized but not instrumented were randomly divided into three groups. Eleven animals received 17beta -estradiol (1 µg · kg-1 · day-1 iv) for 7 days. On the seventh day, all animals received 17beta -estradiol (1 µg/kg iv), and 6 of 11 animals received dexamethasone (4 mg) 15 min after estrogen. Six animals in the third group received an equivalent vehicle. On the seventh day, 2 h after the animals received their final dose of estrogen, estrogen and dexamethasone, or vehicle, they were euthanized. Coronary artery tissues were rapidly removed, placed in liquid nitrogen, and stored at -70°C until studied.

Cloning of Ovine eNOS and iNOS

Two PCR primers, based on two small partial cDNA sequences for bovine iNOS in Genbank, were designed and synthesized in the University of Cincinnati Medical Center DNA Core Facility. They are designated BNOS 1 and BNOS 2 (see Table 1). The predicted product size, based on the human iNOS sequence, is ~3,000 bp. With the use of these primers, RT-PCR was performed using total RNA extracted from ovine white blood cells. The PCR product was subcloned into a plasmid vector (Stratagene) and sequenced in the DNA Core Laboratory. A similar procedure was carried out for eNOS, using primers based on the full-length bovine eNOS sequence in Genbank (Accession No. M89952), and designated BNOS 3 and 4 (Table 1). The predicted product size is ~1,100 bp. A RT-PCR reaction was carried out using the coronary artery as the tissue source. A PCR product of the appropriate size was obtained, subcloned, and sequenced as above described.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   PCR primers

Comparison of the ovine iNOS cDNA sequence with the human iNOS cDNA revealed three gaps in the sequence. Reference of these gaps to the human iNOS gene revealed the missing sequence to correspond exactly to exon 7 (91 bp), exon 18 (79 bp), and exon 21 (6, 7). Additional PCR primers were then designed to span these exons: ONOS 1 and INOS343R to span exon 7 and primers INOS 1444F and INOS1949R to span both exons 18 and 21 (see Table 1). These primers were used to perform RT-PCR, using the ovine coronary artery as the source of RNA. The major PCR product obtained was the intact cDNA (data not shown). PCR products corresponding to the missing exons were either not seen or were a very minor part of the PCR product. This suggests that the "exon skipping" seen in the initial clone was a minor species but selected for based on the size of the clone. These products were also subcloned and sequenced to complete the partial ovine iNOS cDNA.

RT-PCR for Ovine eNOS and iNOS

These studies were performed using primers specific for ovine eNOS and iNOS as determined above (see Table 1). Coronary arteries were removed from the six treated animals, snap-frozen in liquid nitrogen, and stored at -70°C until use. Approximately 5 µg total cellular RNA was used for reverse transcription (Superscript, GIBCO-BRL; Gaithersburg, MD), primed with oligo-dT in a reaction volume of 50 µl. Five microliters of the RT reaction were used for PCR using Pfu Turbo polymerase according to their protocol. Oligonucleotide primers were synthesized, based on the sequence for ovine iNOS. Thirty-five cycles of PCR were performed with annealing at 60°C for 15 s, extension at 72°C for 1 min, and denaturation at 95°C for 30 s. Quantitative analysis of the iNOS PCR product (n = 4) at different numbers of cycles (32-44) revealed a direct exponential increase through 41 cycles (Fig. 1). The reaction products were purified. The fragments were then electrophoresed on a 1% agarose gel stained with ethidium bromide. The relative amounts of the PCR products were quantified by computerized densitometry (ImageQuant). To confirm the PCR reactions, products were transferred to a nitrocellulose membrane and hybridized to a 32P-labeled cDNA probe specific for ovine iNOS.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1.   Increase in the inducible nitric oxide (NO) synthase (iNOS) PCR product with increasing numbers of cycles. The RT-PCR reaction for iNOS was performed as described. A: an aliquot of the reaction was removed every 3 cycles from 20 to 50 cycles and resolved on an agarose gel. B: density of the bands plotted against the cycle number. The iNOS PCR product increased in a linear fashion through 35 cycles.

Pharmacological Studies

Once animals had recovered from surgery and reproducible responses to 1 µg/kg 17beta -estradiol were established, studies were undertaken to determine the potential mechanism of estrogen-induced coronary vasodilation.

Studies with the ER antagonist ICI-182,780. To determine whether 17beta -estradiol produces its response via a classical ER, the ER antagonist ICI-182,780 (ICI; Tocris; Ballwin, MO) was administered. ICI was dissolved in a 30% ethanol and saline solution and infused intravenously at 10 µg · kg-1 · min-1 over a period of 10 min beginning 5 min before the administration of 17beta -estradiol (1 µg/kg). All physiological parameters were recorded continuously as above.

Dexamethasone studies. We investigated the role that iNOS might play in mediating estradiol-induced coronary vasodilation by giving 4 mg dexamethasone (Elkins-Sinn; Cherry Hill, NC) intravenously 15 min after estrogen administration. Because of theoretical concerns that dexamethasone might compete for binding to ER, it was administered after estradiol. All physiological parameters were recorded continuously as above. An additional 11 animals were exposed to either 17beta -estradiol (n = 5) or 17beta -estradiol and 4 mg dexamethasone (n = 6), and the animals were euthanized 2 h after estrogen administration to validate inhibition of iNOS expression by dexamethasone.

Nuclear factor-kappa B inhibitor studies. To determine the involvement of nuclear factor (NF)-kappa B in mediating estradiol-induced coronary vasodilation, a dose-response curve of 1, 2, and 3 mg/min pyrrolidine dithiocarbamic acid (PDTC; Alexis Biochemicals; San Diego, CA) (50) dissolved in saline was infused intravenously for 100 min immediately after 17beta -estradiol (1 µg/kg) administration in two animals as a pilot study. A total of five animals then received the 2 mg/min study dose as a control and in the presence of 17beta -estradiol (1 µg/kg). All physiological parameters were recorded continuously as described above.

Statistical Analysis

The data were analyzed using ANOVA with significance declared at P < 0.05. Where appropriate, the paired Student's t-test was used with significance declared at the P < 0.05 level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen-Induced Increases in Coronary Blood Flow

Administration of 17beta -estradiol (1 µg/kg) in nine sheep was associated with a significant increase in coronary blood flow (Fig. 2 and Table 2; P < 0.01 by ANOVA). After administration of 17beta -estradiol, coronary blood flow showed a small but nonsignificant increase at 5 min and then returned to baseline. This was followed by a much larger and significant increase in coronary blood flow, which began at ~35 min after estrogen administration and peaked at ~2 h, reaching an average increase of 22 ± 4% over baseline. This increase was sustained over the next 8-12 h before a slow return to baseline flow by 24 h (Fig. 2).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of estrogen on coronary blood flow (CBF) in the ovariectomized ewe (n = 9). At time 0 (arrow), 17beta -estradiol (E2; 1 µg/kg) was administered intravenously. Shown here is the percent change in CBF versus time (in h). Estrogen significantly increases CBF, beginning after a delay of 35-45 min. The peak response was seen at 2 h, lasted for several hours before returning to baseline by 12 h, and was highly significant (P < 0.001 by ANOVA).


                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Actual cardiovascular values

Cloning of Ovine iNOS and eNOS

Because only very small partial cDNA sequences of ovine iNOS and eNOS are described in Genbank, initial studies were undertaken to obtain larger partial cDNA sequences for the expression studies. With the use of a RT-PCR approach based on the bovine eNOS sequence, a partial ovine eNOS cDNA clone of 1,101 bp was isolated and sequenced (Genbank Accession No. AF223471). The cDNA has an open reading frame encoding 367 amino acids. The ovine eNOS clone has strong homology with the published human eNOS sequence (20, 34), 91% at the nucleotide level and 94.5% at the amino acid level. The predicted amino acid sequence for this ovine eNOS clone contains many of the cofactor binding sites, including flavin adenine dinucleotide (FAD)-pyrophosphate, FAD-isoalloxazine, NADPH ribose, and a partial binding site for NADPH-adenine at the amino terminal end (Fig. 3).


View larger version (51K):
[in this window]
[in a new window]
  		Fig. 3.   Nucleotide sequences of the ovine endothelial NO synthase (eNOS) partial cDNAs. The predicted amino acid translation is shown below the nucleotide sequence. Consensus binding sites for the cofactors FAD-pyrophosphate, FAD-isoalloxazine, NADPH-ribose, and NADPH-adenine are shown in the boxes.

A similar approach was used to obtain an ovine iNOS cDNA of 2,838 nucleotides. This cDNA has an open reading frame encoding 946 amino acids (Genbank Accession No. AF223942). The ovine iNOS cDNA displays 88.7% homology to the human sequence, whereas the predicted ovine iNOS protein is 88.8% homologous to the human protein. Examination of the predicted ovine iNOS protein demonstrates binding sites for calmodulin, FAD, flavin mononucleotide (FMN), NADPH-ribose, and NADPH-adenine (Fig. 4). In these regions, there was a very high degree of homology with other species.


View larger version (72K):
[in this window]
[in a new window]
  		Fig. 4.   Nucleotide sequences for the partial cDNAs of ovine iNOS obtained in this study. The predicted amino acid translation is shown below the nucleotide sequence. Consensus binding sites for calmodulin and the cofactors FMN, FAD-pyrophosphate, FAD-isoalloxazine, NADPH-ribose, and NADPH-adenine are shown in the boxes.

The initial ovine iNOS PCR reaction yielded a product of 2,461 bp, much smaller than the predicted size. Comparison of this sequence with the human iNOS cDNA revealed that the ovine PCR product contained three gaps of 91, 79, and 207 bp, which correspond exactly to exons 7, 18, and 21 of the human gene (7), suggesting that this RNA resulted from exon skipping. Translation of the first two splice variants would result in a frame shift to yield a nonfunctional product. The product of the exon 21(-) mutation could result in a protein with 69 amino acids deleted. This deletion would result in the removal of the FAD-isoalloxazine binding site (6). When the ovine coronary artery was analyzed to determine the extent to which these mutants were expressed, no exon 7(-) was detected (data not shown). The other mutants were seen but to a very minor extent, suggesting that these mutants represent artifacts of the extremely sensitive RT-PCR technique.

RT-PCR for Ovine eNOS and iNOS

Effects of 17beta -estradiol on eNOS expression. Six ovariectomized animals were used in this study. Three animals received estrogen vehicle, and three animals received 17beta -estradiol (1 µg/kg) given intravenously for 7 days. The coronary arteries were removed and snap-frozen at the 2-h peak. eNOS expression was assessed by RT-PCR as described in METHODS. Figure 5, top, shows the ethidium bromide-stained agarose gel of the PCR products. GAPDH served as a control (Fig. 5, bottom). The results show an increase in eNOS mRNA in response to estradiol. The level of expression varied among the different animals, and one animal did not show an increase in eNOS expression with estrogen. Computerized scanning densitometry with normalization to GAPDH showed a control level of 0.52 ± 0.09. This increased significantly to 0.88 ± 0.08 in the estrogen-treated animals, or a 1.7-fold increase (P < 0.01). We did not observe an increase in the expression of GAPDH with estrogen.


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of estradiol on eNOS expression. Six ovariectomized animals were used in this study. Three animals received saline (C) and three animals received 17beta -estradiol (1 µg/kg) intravenously (E). The coronary artery was removed and snap-frozen. eNOS expression was assessed by RT-PCR as described. Top: agarose gel of the PCR products. GAPDH served as a control (bottom). The results show an increase in eNOS mRNA in response to estradiol (~2 fold). We did not observe an increase in the expression of GAPDH.

Effects of 17beta -estradiol on iNOS expression. The same six ovariectomized sheep used in the eNOS study were utilized to determine the effect of 17beta -estradiol on iNOS expression in the ovine coronary artery. These results are shown in Fig. 6. Figure 6, top, shows the ethidium bromide-stained agarose gel of the PCR products. GAPDH served as a control (Fig. 6, middle). The results of Southern blot transfer and hybridization to a specific ovine iNOS cDNA are shown in Fig. 6, bottom. The data reveal a dramatic increase in iNOS expression in the estrogen-treated animals. In two of the controls, essentially no basal expression of iNOS is seen, as expected. A very low degree of expression is seen in the third animal. Analysis with computerized scanning densitometry and normalization to GAPDH revealed an average of 0.22 ± 0.07 in the controls, increasing significantly to 6.09 ± 0.03 with estrogen treatment (Fig. 7, P < 0.008). This is a 27-fold increase in expression of iNOS with estrogen.


View larger version (69K):
[in this window]
[in a new window]
  		Fig. 6.   Effects of estradiol on iNOS expression. Six ovariectomized animals were used in this study. Three animals received saline (C) and three animals received estradiol (1 µg/kg iv) (E). iNOS expression was assessed by RT-PCR as described. Top: agarose gel of the PCR products. GAPDH served as a control (middle). Bottom: gels were then transferred and hybridized to a specific iNOS cDNA. The results show a dramatic increase in iNOS expression in the estrogen-treated animals.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of estradiol on eNOS and iNOS expression shown as normalized units after computerized densitometry. The eNOS in control animals was 0.52 ± 0.09 and was increased by estrogen to 0.88 ± 0.08 (1.7-fold increase, **P < 0.01). The iNOS in controls was 0.22 ± 0.07 and increased to 6.09 ± 0.03 (27-fold increase, P < 0.008).

Pharmacological Studies

Effect of ICI. To determine whether the estrogen-induced increase in coronary blood flow and cardiac output occurred via an interaction with ER, the antagonist ICI was administered to five animals at the rate of 10 µg · kg-1 · min-1 into the femoral artery given 5 min before and continued for 5 min after 17beta -estradiol administration. This concentration of ICI was able to abolish the 17beta -estradiol-induced increase in coronary blood flow with estrogen-treated animals having a 16 ± 2% increase in coronary blood flow and animals receiving estrogen and ICI showing a nonsignificant decrease in coronary blood flow (-3 ± 2%, P < 0.001; Fig. 8). Estrogen administration was associated with a significant decrease in coronary vascular resistance (Table 3), which was blocked by ICI treatment. Estrogen treatment produced a significant increase in cardiac output of 15 ± 6%, which was reduced to 5 ± 2% in the presence of ICI. Systemic vascular resistance was reduced by 12 ± 5% with estrogen, and this was prevented by pretreatment with ICI (Table 3). ICI administered by itself had no effect on any of the parameters measured (n = 4).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of the estrogen receptor antagonist ICI-182,780 (ICI) on estrogen-induced increases in CBF and cardiac output (CO). The coadministration of estrogen and ICI completely blocked the CBF response to estrogen (**P < 0.01, n = 5) and attenuated the CO response (*P < 0.05, n = 5). A reduction in CO from baseline was observed when ICI was administered by itself (n = 4), but that response was not different from time controls (n = 5).


                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Calculated vascular resistance

Effect of dexamethasone. To determine the potential role of iNOS in mediating estrogen effects on coronary blood flow, animals received a 4 mg iv dose of dexamethasone given 15 min after the estrogen administration. The results are shown in Fig. 9. Compared with the control response, in which estrogen increased coronary blood flow by 19 ± 6% over baseline, the addition of dexamethasone significantly decreased this response to 3 ± 4% (P < 0.01 versus the estradiol response). 17beta -Estradiol produced a significant increase in cardiac output of 16 ± 9%, which was significantly reduced to 4 ± 6% in the presence of dexamethasone. Administration of estrogen significantly decreased systemic vascular resistance by 19 ± 6%, which was completely abolished by pretreatment with dexamethasone (Table 3). Administration of dexamethasone alone had no effect on any of the parameters measured (n = 4). An additional 11 animals were exposed to either 17beta -estradiol (n = 5) or 17beta -estradiol and 4 mg dexamethasone (n = 6), and the animals were euthanized 2 h after estrogen administration to validate inhibition of iNOS expression by dexamethasone. Dexamethasone produced a significant (P < 0.01) reduction in iNOS expression at the dose used in the present study (Fig. 10).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 9.   Effect of the dexamethasone (Dex) on estrogen-induced increases in CBF and CO. The coadministration of Dex and estradiol inhibited the CBF estrogen response by 85% (**P < 0.01, n = 6). The increase in CO was inhibited by 80% (*P < 0.05, n = 5). A reduction in CO from baseline was observed when Dex was administered by itself (n = 4), but that response was not different from time controls (n = 5).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 10.   Effect of Dex on estrogen-induced increases in iNOS expression. A: agarose gel of the PCR products for iNOS in response to 17beta -estradiol (n = 5) and 17beta -estradiol and Dex (n = 6). B: significant reduction in iNOS message expression in the presence of Dex (**P < 0.01). Values shown are normalized for individual GAPDH values for each assay.

Effect of the NF-kappa B inhibitor PDTC. To determine the potential role of NF-kappa B in mediating estradiol-induced coronary blood flow, an NF-kappa B inhibitor, PDTC, was used. Animals received an intravenous infusion of PDTC of 1, 2, or 3 mg/min, beginning at the time of estradiol administration. The results are shown in Fig. 11. Compared with the control response, in which estradiol increased coronary blood flow by 21 ± 2% over baseline, the addition of PDTC blocked estrogen-induced increases in coronary blood flow in a dose-related fashion, with doses >2 mg/min totally inhibiting this response to -2 ± 3% (P < 0.01 versus the estradiol response, n = 5). Administration of PDTC alone (n = 3) had no effect on any of the parameters measured and was not different from time controls (Fig. 11).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 11.   Effect of pyrrolidine dithiocarbamic acid (PDTC), a nuclear factor-kappa B inhibitor, on estrogen-induced increases in CBF (n = 5). PDTC at the 1-mg dose attenuated estrogen-induced CBF; the addition of 2 or 3 mg PDTC completely inhibited the estrogen response (**P < 0.01). PDTC alone (n = 3) had no effect on CBF compared with controls (n = 5, data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen is now known to have a wide variety of physiological actions on numerous tissues and cell types. Many of these actions are considered to have a favorable effect on the complex process of atherosclerosis and cardiovascular disease. It is now clear that one of the most important of these diverse actions is the ability of estrogen to increase production of NO in vascular tissues (16). Not only is NO a potent coronary vasodilator, but NO suppresses several key processes in the development of atherosclerosis: inhibition of leukocyte attraction and adhesion (4), decreased proliferation of vascular smooth muscle (39), and decreased platelet aggregation (9). Other beneficial effects of estrogen are recognized. For example, estrogen itself can act as an antioxidant (51) and thus prevent oxidative damage to the coronary endothelium. Estrogen has been shown to increase the expression of vascular endothelial growth factor in endothelial cells, which also acts to increase blood flow and stimulate endothelial cell growth (45).

In the present study, we examined the ability of estrogen to increase coronary blood flow in conscious, unrestrained sheep, an effect we have previously demonstrated to involve the synthesis of NO (29). Here, we extend previous observations by examining the effect of 17beta -estradiol on eNOS and iNOS gene expression in the ovine coronary artery. We demonstrated that, as others have shown, estrogen does increase eNOS RNA expression, but only to a limited extent (twofold). On the other hand, estrogen administration resulted in a 27-fold increase in iNOS mRNA expression compared with controls.

To determine whether the 17beta -estradiol-induced increase in coronary blood flow and cardiac output was dependent on ER stimulation, we utilized the pure ER antagonist ICI. This compound was able to block the 17beta -estradiol-induced increases in coronary blood flow and cardiac output by over 85%. To determine whether iNOS was responsible for the majority of this physiological response, animals were treated with dexamethasone, a corticosteroid hormone well known to inhibit iNOS but not eNOS activity (46). Dexamethasone was able to significantly inhibit estrogen-induced increases in coronary blood flow and cardiac output (Fig. 9) and significantly reduce iNOS expression (Fig. 10)

The ability of PDTC, an inhibitor of NF-kappa B (50), to completely block the estrogen response further supports a role for iNOS in mediating this effect. The human iNOS promoter contains several binding sites for NF-kappa B, one of which has been shown to be required for cytokine induction of the iNOS gene. Dithiocarbamates have been shown to potently inhibit the activation of NF-kappa B without affecting other DNA binding or cell transduction pathways such as the induction of activator protein-1 (AP-1) by phorbol ester (50). When administered to laboratory animals in vivo, dithiocarbamates are potent inhibitors of the inflammatory process, including the expression of iNOS (10). Gene transcription of eNOS, on the other hand, does not appear to be affected by NF-kappa B but by SP-1 and members of the Ets family (23). These findings suggest that the majority of the estrogen-induced increase in NO production was from the iNOS isoform. Taken together, these results strongly support a role for estrogen, acting through its ER, to stimulate iNOS production in the coronary and systemic vasculature of the nonpregnant sheep.

iNOS is present in many tissues, including vascular smooth muscle (24). Unlike eNOS, iNOS enzyme is not expressed under basal conditions, but once synthesized it is active, independent of intracellular calcium, and produces large sustained amounts of NO (62). The primary mechanism of regulation of iNOS is at the transcriptional level. Mediators of inflammation, including cytokines and toxins such as lipopolysaccharide (LPS), significantly increase the expression of iNOS (58). Of considerable importance, it has been shown that steroid hormones such as glucocorticoids (28, 46) and progesterone (37) are regulators of iNOS gene expression. This transcriptional regulation of iNOS suggests that it is an excellent candidate for regulation by estrogen, a transcriptional activator. Indeed, there is evidence that estrogen-induced vasodilation is mediated by increased expression of iNOS. For example, in human (40) and rabbit (21) coronary artery rings in vitro, estradiol exposure resulted in a significant relaxation that was independent of endothelium. Binko et al. (2, 3) have shown a significant increase in iNOS protein in the rat aorta with estrogen administration. In addition, Zhu et al. (65) have recently shown that estrogen selectively increases iNOS in the ER-alpha knockout mouse aorta (no increase in eNOS or nNOS), suggesting that the ER-beta receptor may mediate the increases in iNOS. Estrogen has been shown to increase iNOS expression in other tissues as well, including cardiac myocytes (43) and kidney (42) and uterine leukocytes (19). There is clearly some disagreement in the field, however, because other investigators have not found an increased expression of iNOS by estrogen (25, 53, 63) or that vasoprotective actions of estrogen may be independent of iNOS (54).

Although it has been shown that the coronary artery expresses ER (47), analysis of the iNOS promoter in several species has not revealed evidence of a classical estrogen response element. Nevertheless, transcriptional activation may be mediated through a direct interaction of ER with an imperfect estrogen response element in the iNOS promoter (3). Another possibility is that estrogen may act through other response elements, as has been shown with the eNOS promoter (27). We propose that a good candidate is an AP-1 site (59), which is present in the iNOS promoter and has been demonstrated to be required for cytokine induction (33). An example of this type of regulation is the collagenase gene, in which estrogen stimulates, whereas glucocorticoids and progestins inhibit, transcription from this AP-1-containing promoter (55).

eNOS, found predominantly if not exclusively in endothelial cells, has a well-characterized role in the modulation of coronary blood flow, both in the basal state and in response to a variety of vasoactive substances, including estrogen. A considerable body of literature, including in vivo and in vitro studies from a number of species, now exists to support the ability of estrogen to upregulate eNOS. In vivo studies in the human (16), macaque (61), sheep (49), and guinea pig (60) have demonstrated that estrogen administration results in vasodilation that is dependent on the activation of NOS. A plausible mechanism for these properties arising from a steroid hormone that traditionally acts as a transcription factor was not, until recently, proposed (8). Chen and co-workers (8) have recently demonstrated that this rapid response of eNOS to estrogen is ER dependent but nongenomic, mediated by an activation of mitogen-activated protein kinase (8). In the present study, we noticed a biphasic response to 17beta -estradiol with a small nonsignificant increase in coronary blood flow at 15 min after estrogen administration, followed by a much greater and longer response that lasts for hours. It is not clear at this time, but the early increase in flow (15 min) may represent this nongenomic component, which has been reported by others. In addition to these rapid, short-term actions of estrogen, there is evidence that estrogen can also increase eNOS over much longer periods of time (56). For example, Vagnoni et al. (56) demonstrated a progressive increase in eNOS protein in the uterine artery endothelium through 10 days of exposure to estradiol in ovariectomized ewes (56). Others have shown that estrogen stimulates eNOS mRNA (31) and promoter activity (27), supporting a genomic action of estrogen.

The time course of the estrogen-induced coronary vasodilation response, as seen in these studies, is also consistent with a genomic mechanism of action. The second phase of the response, which is much greater in magnitude and duration than the brief rapid response, is delayed for 35-45 min. Peaking at 2 h, the response plateaus for the next 3-4 h, and then slowly returns to baseline over the next 10 h. Several investigators (30, 32) have demonstrated that the genomic response of iNOS to stimulation occurs within hours. Liu et al. (30) examined the time course of the iNOS response to induction with a single stimulus of LPS in vivo in rats. They showed that iNOS mRNA levels increased significantly within 40 min, peaked from 4 to 8 h, and returned too much lower levels by 24 h. The effect was markedly attenuated with the coadministration of dexamethasone (30). This response bears a striking resemblance to the coronary blood flow response we showed in sheep. Taken together with the ER dependence and the estrogen-induced increase in iNOS expression, we believe these results strongly support a genomic activation of the iNOS gene by estradiol in the ovine coronary artery.

We obtained a partial cDNA clone spanning the majority of the coding region of the ovine iNOS sequence. The cDNA shows a high degree of homology to other iNOS cDNA clones reported to date. An ovine iNOS cDNA has not to our knowledge been reported previously. A very small partial cDNA for ovine iNOS (245 bp) is found in Genbank (Accession No. AF097486) but does not overlap our clone. The binding sites for all of the major cofactors, including calmodulin, FAD, FMN, and NADPH, are present in the predicted ovine iNOS protein. Our finding of several mutant clones representing exon skipping suggests that not only is the sequence highly conserved across species, but the basic structure of the gene is as well. None of the deletion mutants described here are in frame, and are expressed at very low levels, so it is unlikely that they are of physiological importance. Others have also demonstrated alternative splicing of the iNOS transcript, including exon skipping as we have seen. Park and colleagues (44) described a deletion mutant involving exon 19. Eissa et al. (12) described several iNOS deletion mutants, including deletions of exons 5, exons 8 and 9, exons 9-11, and exons 15 and 16. While several of these mutations are in frame, it has not been shown that they are translated into protein; thus their functional significance remains unclear.

In summary, we provide strong evidence that the iNOS isoform mediates a significant component of the estrogen-induced vasodilation response in the ovine coronary artery. Physiological studies in this whole animal model demonstrate that 85% of the coronary response is blocked by dexamethasone, an inhibitor of iNOS. The time course of the estrogen response, characterized by an initial delay followed by a sustained vasodilation occurring over several hours, is supportive of a genomic interaction. This was further supported by our findings of a strong induction of iNOS mRNA with estrogen administration. Partial cDNA clones of ovine eNOS and iNOS are reported in this study. These clones show strong homology with previously reported sequences in other species. We also provide data on several splice variants of the iNOS sequence, due to exon skipping, suggesting that the basic gene structure of the ovine iNOS gene is conserved. Although it is well known that the eNOS isoform is involved in estrogen-induced vasodilation, we believe these data provide compelling evidence that the iNOS isoform is also involved in this response, particularly in the coronary artery. This work supports a role for iNOS in mediating certain physiological functions in the organism in addition to its well-recognized role in the inflammatory response.


    ACKNOWLEDGEMENTS

The authors acknowledge Jeanne Hirth and Angela Friedman for excellent technical assistance.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-49901, HL-51051, and HL-62490.

Address for reprint requests and other correspondence: K. E. Clark, Dept. of Obstetrics and Gynecology, PO Box 670526, Univ. of Cincinnati College of Medicine, 231 Sabin Way, Cincinnati, OH 45267-0526 (E-mail: Kenneth.Clark{at}uc.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.

10.1152/ajpheart.00397.2000

Received 5 May 2000; accepted in final form 20 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barrett-Connor, E, and Grady D. Hormone replacement therapy, heart disease, and other considerations. Annu Rev Public Health 19: 55-72, 1998[ISI][Medline].

2.   Binko, J, and Majewski H. 17beta -Estradiol reduces vasoconstriction in endothelium-denuded rat aortas through inducible NOS. Am J Physiol Heart Circ Physiol 274: H853-H859, 1998[Abstract/Free Full Text].

3.   Binko, J, Murphy T, and Majewski H. 17beta -Oestradiol enhances nitric oxide synthase activity in endothelium-denuded rat aorta. Clin Exp Pharmacol Physiol 25: 120-127, 1998[ISI][Medline].

4.   Bult, H. Nitric oxide and atherosclerosis: possible implications for therapy. Mol Med Today 2: 510-518, 1996[ISI][Medline].

5.   Bush, TL. Evidence for primary and secondary prevention of coronary artery disease in women taking oestrogen replacement therapy. Eur Heart J 17: 9-14, 1996[Free Full Text].

6.   Charles, IG, Palmer RM, Hickery MS, Bayliss MT, Chubb AP, Hall VX, Moss DW, and Moncada S. Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte. Proc Natl Acad Sci USA 90: 11419-11423, 1993[Abstract/Free Full Text].

7.   Chartrain, NA, Geller DA, Koty PP, Sitrin NF, Nussler AK, Hoffman EP, Billiar TR, Hutchinson NI, and Mudgett JS. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J Biol Chem 269: 6765-6772, 1994[Abstract/Free Full Text].

8.   Chen, Z, Yuhanna I, Galcheva-Gargova Z, Karas R, Mendelsohn M, and Shaul P. Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103: 401-406, 1999[ISI][Medline].

9.   Cooke, JP, and Dzau VJ. Nitric oxide synthase: role in the genesis of vascular disease. Annu Rev Med 48: 489-509, 1997[ISI][Medline].

10.   Cuzzocrea, S, Chatterjee PK, Mazzon E, Dugo L, Serraino I, Britti D, Mazzullo G, Caputi AP, and Thiemermann C. Pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation. Br J Pharmacol 135: 496-510, 2002[ISI][Medline].

11.   Darkow, DJ, Lu L, and White RE. Estrogen relaxation of coronary artery smooth muscle is mediated by nitric oxide and cGMP. Am J Physiol Heart Circ Physiol 272: H2765-H2773, 1997[Abstract/Free Full Text].

12.   Eissa, NT, Strauss AJ, Haggerty CM, Choo EK, Chu SC, and Moss J. Alternative splicing of human inducible nitric-oxide synthase mRNA. Tissue-specific regulation and induction by cytokines. J Biol Chem 271: 27184-27187, 1996[Abstract/Free Full Text].

13.   Geng, Y, Almqvist M, and Hansson GK. cDNA cloning and expression of inducible nitric oxide synthase from rat vascular smooth muscle cells. Biochim Biophys Acta 1218: 421-424, 1994[Medline].

14.   Gilligan, DM, Quyyumi AA, and Cannon RO, III. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation 89: 2545-2551, 1994[Abstract/Free Full Text].

15.   Goetz, RM, Thatte HS, Prabhakar P, Cho MR, Michel T, and Golan DE. Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96: 2788-2793, 1999[Abstract/Free Full Text].

16.   Guetta, V, Quyyumi AA, Prasad A, Panza J, Waclawiw M, and Cannon R. The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women. Circulation 96: 2795-2801, 1997[Abstract/Free Full Text].

17.   Herrington, DM, Reboussin DM, Brosnihan KB, Sharp PC, Shumaker SA, Snyder TE, Furberg CD, Kowalchuk GJ, Stuckey TD, Rogers WJ, Givens DH, and 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].

18.   Hulley, S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, and Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/Progestin Replacement Study (HERS) Research Group. JAMA 280: 605-613, 1998[Abstract/Free Full Text].

19.   Hunt, JS, Miller L, Roby KF, Huang J, Platt JS, and DeBrot BL. Female steroid hormones regulate production of pro-inflammatory molecules in uterine leukocytes. J Reprod Immunol 35: 87-99, 1997[ISI][Medline].

20.   Janssens, SP, Shimouchi A, Quertermous T, Bloch DB, and Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 267: 14519-14522, 1992[Abstract/Free Full Text].

21.   Jiang, CW, Sarrel PM, Lindsay DC, Poole-Wilson PA, and Collins P. Endothelium-independent relaxation of rabbit coronary artery by 17beta -oestradiol in vitro. Br J Pharmacol 104: 1033-1037, 1991[ISI][Medline].

22.   Kaku, Y, Nanri H, Sakimura T, Ejima K, Kuroiwa A, and Ikeda M. Differential induction of constitutive and inducible nitric oxide synthases by distinct inflammatory stimuli in bovine aortic endothelial cells. Biochim Biophys Acta 1356: 43-52, 1997[Medline].

23.   Karantzoulis-Fegaras, F, Antoniou H, Lai SL, Kulkarni G, D'Abreo C, Wong GK, Miller TL, Chan Y, Atkins J, Wang Y, and Marsden PA. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem 274: 3076-3093, 1999[Abstract/Free Full Text].

24.   Karas, RH, Patterson BL, and Mendelsohn ME. Human vascular smooth muscle cells contain functional estrogen receptor. Circulation 89: 1943-1950, 1994[Abstract/Free Full Text].

25.   Kauser, K, Sonnenberg D, Diel P, and Rubanyi GM. Effect of 17beta -oestradiol on cytokine-induced nitric oxide produciton in rat isolated aorta. Br J Pharmacol 123: 1089-1096, 1998[ISI][Medline].

26.   Kirsch, E, Yuhanna I, Chen Z, German Z, Sherman T, and Shaul P. Estrogen acutely stimulates endotheial nitric oxide synthase in H441 human airway epithelial. Am J Respir Cell Mol Biol 20: 658-666, 1999[Abstract/Free Full Text].

27.   Kleinert, H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, and Forstermann U. Estrogens increase transcription of the human endothelial NO synthase gene. Hypertension 31: 582-588, 1998[Abstract/Free Full Text].

28.   Kunz, D, Walker G, Eberhardt W, and Pfeilschifter J. Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1beta -stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation. Proc Natl Acad Sci USA 93: 255-259, 1996[Abstract/Free Full Text].

29.   Lang, U, Baker RS, and Clark K. Estrogen-induced increases in coronary blood flow are antagonized by inhibitors of nitric oxide synthesis. Eur J Obstet Gynecol Reprod Biol 74: 229-235, 1997[ISI][Medline].

30.   Liu, SF, Barnes PJ, and Evans TW. Time course and cellular localization of lipopolysaccharide-induced inducible nitric oxide synthase messenger RNA expression in the rat in vivo. Crit Care Med 25: 512-518, 1997[ISI][Medline].

31.   MacRitchie, AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS, and Shaul PW. Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res 81: 355-362, 1997[Abstract/Free Full Text].

32.   Maier, R, Bilbe G, Rediske J, and Lotz M. Inducible nitric oxide synthase from human articular chondrosytes: cDNA cloning and analysis of mRNA expression. Biochim Biophys Acta 1208: 145-150, 1994[Medline].

33.   Marks-Konczalik, J, Chu SC, and Moss J. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappa B-binding sites. J Biol Chem 273: 2201-2208, 1998.

34.   Marsden, PA, Schappert KT, Chen HS, Flowers M, Sundell CL, Wilcox JN, Lamas S, and Michel T. Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS Lett 307: 287-293, 1992[ISI][Medline].

35.   Mendelsohn, ME. Mechanisms of estrogen action in the cardiovascular system. J Steroid Biochem Mol Biol 74: 337-343, 2000[ISI][Medline].

36.   Mendelsohn, ME, and Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med 340: 1801-1811, 1999[Free Full Text].

37.   Miller, L, Alley EW, Murphy WJ, Russell SW, and Hunt JS. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol 59: 442-450, 1996[Abstract].

38.   Minshall, RD, Miyagawa K, Chadwick CC, Novy MJ, and Hermsmeyer K. In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones. FASEB J 12: 1419-1429, 1998[Abstract/Free Full Text].

39.   Morey, A, Pedram A, Razandi M, Prins B, Hu RM, Biesiada E, and Levin E. Estrogen and progesterone inhibit vascular smooth muscle proliferation. Endocrinology 138: 3330-3339, 1997[Abstract/Free Full Text].

40.   Mugge, A, Riedel M, Barton M, Kuhn M, and Lichtlen PR. Endothelium independent relaxation of human coronary arteries by 17beta -oestradiol in vitro. Cardiovasc Res 27: 1939-1942, 1993[ISI][Medline].

41.   Nathan, C, and Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915-918, 1994[ISI][Medline].

42.   Neugarten, J, Ding Q, Friedman A, Lei J, and Silbiger S. Sex hormones and renal nitric oxide synthetase. J Am Soc Nephrol 8: 1240-1246, 1997[Abstract].

43.   Nuedling, S, Kahlert S, Loebbert K, Doevendans PA, Meyer R, Vetter H, and Grohe C. 17beta -Estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo. Cardiovasc Res 43: 666-674, 1999[Abstract/Free Full Text].

44.   Park, CS, Park R, and Krishna G. Constitutive expression and structural diversity of inducible isoform of nitric oxide synthase in human tissues. Life Sci 59: 219-225, 1996[ISI][Medline].

45.   Perrot-Applanat, M, Ancelin M, Buteau-Lozano H, Meduri G, and Bausero P. Ovarian steroids in endometrial angiogenesis. Steroids 65: 599-603, 2000[ISI][Medline].

46.   Radomski, MW, Palmer RMJ, and Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci USA 87: 10043-10047, 1990[Abstract/Free Full Text].

47.   Register, TC, and Adams MR. Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor beta. J Steroid Biochem Mol Biol 64: 187-191, 1998[ISI][Medline].

48.   Ridker, PM, Hennekens CH, Buring JE, and Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 342: 836-843, 2000[Abstract/Free Full Text].

49.   Rosenfeld, CR, Cox BE, Roy T, and Magness RR. Nitric oxide contributes to estrogen-induced vasodilation of the ovine uterine circulation. J Clin Invest 98: 2158-2166, 1996[ISI][Medline].

50.   Schreck, R, Meier B, Mannel DN, Droge W, and Baeuerle PA. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med 175: 1181-1194, 1992[Abstract/Free Full Text].

51.   Shwaery, G, Vita J, and Keaney J, Jr. Antioxidant protection of LDL by physiological concentrations of 17beta -estradiol. Circulation 95: 1378-1385, 1997[Abstract/Free Full Text].

52.   Stampfer, MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, and Hennekens CH. Post menopausal estrogen therapy and cardiovascular disease: ten year follow-up from the nurse's health study. N Engl J Med 325: 756-762, 1991[Abstract].

53.   Tamura, K, Yamaguchi K, and Kogo H. 17beta -Estradiol inhibits ovariectomy-induced expression of inducible nitric oxide synthase in rat aorta in vivo. Life Sci 66: PL259-PL264, 2000.

54.   Tolbert, T, Thompson JA, Bouchard P, and Oparil S. Estrogen-induced vasoprotection is independent of inducible nitric oxide synthase expression: evidence from the mouse carotid artery ligation model. Circulation 104: 2740-2745, 2001[Abstract/Free Full Text].

55.   Uht, RM, Anderson CM, Webb P, and Kushner PJ. Transcriptional activities of estrogen and glucocorticoid receptors are functionally integrated at the AP-1 response element. Endocrinology 138: 2900-2908, 1997[Abstract/Free Full Text].

56.   Vagnoni, KE, Shaw C, Phernetton T, Meglin B, Bird I, and Magness R. Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase. Am J Physiol Heart Circ Physiol 275: H1845-H1856, 1998[Abstract/Free Full Text].

57.   Viscoli, CM, Brass LM, Kernan WN, Sarrel PM, Suissa S, and Horwitz RI. A clinical trial of estrogen-replacement therapy after ischemic stroke. N Engl J Med 345: 1243-1249, 2001[Abstract/Free Full Text].

58.   Wang, Y, and Marsden PA. Nitric oxide synthases: gene structure and regulation. Adv Pharmacol 34: 71-90, 1995[Medline].

59.   Webb, P, Lopez GN, Uht RM, and Kushner PJ. Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin of the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9: 443-456, 1995[Abstract].

60.   Weiner, CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, and Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91: 5212-5216, 1994[Abstract/Free Full Text].

61.   Williams, JK, Delansorne R, and Paris J. Estrogens, progestins, and coronary artery reactivity in atherosclerotic monkeys. J Steroid Biochem Mol Biol 65: 219-224, 1998[ISI][Medline].

62.   Xie, Q, and Nathan C. The high-output nitric oxide pathway: role and regulation. J Leukoc Biol 56: 576-582, 1994[Abstract].

63.   Zancan, V, Santagati S, Bolego C, Vegeto E, Maggi A, and Puglisi L. 17Beta-estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology 140: 2004-2009, 1999[Abstract/Free Full Text].

64.   Zhang, J, Massmann GA, Mirabile CP, and Figueroa JP. Nonpregnant sheep uterine type I and type III nitric oxide synthase expression is differentially regulated by estrogen. Biol Rep